BEHAVIOR, FUNCTIONAL MORPHOLOGY, AND ECOLOGY RELATED TO

FEEDING IN AQUATIC INSECTS WITH PARTICULAR REFERENCE TO

STENACRON INTERPUNCTATUM, RHITHROGENA PELLUCIDA

(EPHEMEROPTERA: HEPTAGENIIDAE), AND

EPHEMERELLA NEEDHAMI (EPHEMEROPTERA: EPHEMERELLIDAE)

A Thesis Submitted to the Faculty of

Purdue University

by

Dave McShaffrey

In Partial Fulfillment of the Requirements for the Degree

of

Doctor of Philosophy

August 1988

TABLE OF CONTENTS

List of Figures

Preface to the HTML Edition

ABSTRACT

CHAPTER I - INTRODUCTION

CHAPTER II - METHODS

  1. Field observations
  2. Videomacroscopy
  3. Electronics
  4. Optics
  5. Observational arenas
  6. Documentation
  7. Behavioral analysis
  8. Scanning electron microscopy
  9. Gut content analysis
  10. Synthesis

CHAPTER III - FEEDING BEHAVIOR OF STENACRON INTERPUNCTATUM

  1. Methods
  2. Results
    1. Field observations
    2. Effects of experimental conditions on behavior
    3. Stereotypic feeding behavior
    4. Resting position
    5. Brushing cycle
    6. Gathering cycle
    7. Filtering cycle
    8. Food processing
    9. Gut content analysis
  3. Discussion
  4. Acknowledgments

CHAPTER IV - FEEDING BEHAVIOR OF RHITHROGENA PELLUCIDA

  1. Methods
  2. Results
    1. Field observations
    2. Effects of experimental conditions on behavior
    3. Stereotypic feeding behavior
    4. Resting position
    5. Feeding cycles
    6. Labial brushing cycle
    7. Maxillary scraping cycle
    8. Food processing
    9. Gut content analysis
  3. Discussion
  4. Acknowledgments

CHAPTER V - COMPARATIVE FUNCTIONAL MORPHOLOGY OF STENACRON INTERPUNCTATUM AND RHITHROGENA PELLUCIDA, WITH COMPARISONS TO OTHER HEPTAGENIIDAE

  1. Methods
  2. Results and Discussion
    1. Glossary of setae
    2. Morphological and functional descriptions: Stenacron interpunctatum  
      1. Labrum
      2. Mandibles
      3. Maxillae
      4. Hypopharynx
      5. Labium
    3. Morphological and functional descriptions: Rhithrogena pellucida
      1. Labrum
      2. Mandibles
      3. Maxillae  
      4. Hypopharynx  
      5. Labium  
    4. Comparative functional morphology of Stenacron interpunctatum and Rhithrogena pellucida
    5. General trends

CHAPTER VI - FEEDING BEHAVIOR AND RELATED FUNCTIONAL MORPHOLOGY OF EPHEMERELLA NEEDHAMI

  1. Methods
  2. Results
    1. Effects of experimental conditions on behavior
    2. Stereotypic feeding behavior
    3. Resting position
    4. Feeding cycles
    5. Maxillary brushing cycle
    6. Mandibular biting cycle
    7. Predatory feeding
    8. Food processing
    9. Diet
  3. Discussion
  4. Acknowledgments

CHAPTER VII - ECOLOGICAL ASSOCIATION OF EPHEMERELLA NEEDHAMI AND THE GREEN ALGA CLADOPHORA

  1. Methods
  2. Results
    1. Field observations
    2. Food availability and diet
    3. Stereotypic feeding behavior
  3. Discussion
  4. Acknowledgments

CHAPTER VIII - MISCELLANEOUS FEEDING OBSERVATIONS

  1. Gastropoda
  2. Amphipoda
  3. Ephemeroptera  
    1. Callibaetis sp.  
    2. Isonychia sp.  
    3. Potamanthus sp.  
    4. Heptagenia flavescens  
    5. Stenonema sp.  
    6. Caenis sp.  
    7. Ephoron sp.
  4. Trichoptera
    1. Macrostemmum zebratum  
  5. Coleoptera  
    1. Psephenus herricki
  6. Diptera  
    1. Eusimulium spp.  
    2. Cricotopus sp.  

CHAPTER IX - CONCLUSIONS AND HORIZONS  

LIST OF REFERENCES  

VITA  

Dedication

Acknowledgements (Global)

LIST OF FIGURES

  1. Figure 1. Areas of investigation
  2. Figure 2. Observation cell
  3. Figure 3. Ventral view of the mouthparts of Stenacron interpunctatum
  4. Figure 4. Diagrammatic cross-section of mouthparts of Stenacron interpunctatum
  5. Figures 5-9. Stenacron interpunctatum mouthparts
    1. 5. Labium of S. interpunctatum.
    2. 6. Maxillae of S. interpunctatum.
    3. 7. Hypopharynx of S. interpunctatum.
    4. 8. Mandibles of S. interpunctatum.
    5. 9. Labrum of S. interpunctatum.
  6. Figures 10-15. Stenacron interpunctatum, brushing feeding cycle
    1. 10. Labium and Maxillae, resting position.
    2. 11. Labium and Maxillae, Stage 1 of initial cycle.
    3. 12. Labium and Maxillae, Stage 2.
    4. 13. Labium and Maxillae, Stage 3.
    5. 14. Maxillae and Mandibles, Stage 3.
    6. 15. Maxillae and Hypopharynx, Stage 1 of subsequent cycle.
  7. Figure 16. Tank cell used in feeding experiments
  8. Figure 17. Plan view, surface of glass microscope slide
  9. Figure 18. Oblique view, surface of glass microscope slide
  10. Figure 19. Ventral view of mouthparts of Rhithrogena pellucida
  11. Figure 20. Diagrammatic cross section of mouthparts of Rhithrogena pellucida
  12. Figures 21-25. Rhithrogena pellucida mouthparts
    1. 21. Labium of R. pellucida.
    2. 22. Maxillae of R. pellucida.
    3. 23. Hypopharynx of R. pellucida.
    4. 24. Mandibles of R. pellucida.
    5. 25. Labrum of R. pellucida.
  13. Figures 26-32. Rhithrogena pellucida, labial brushing cycle
    1. 26. Resting Position.
    2. 27. Stage 1 of first cycle.
    3. 28. Stage 2 of first cycle.
    4. 29. Intermediate position of Stage 3 of first cycle.
    5. 30. Final position of Stage 3 of first cycle: Labium.
    6. 31. Final position of Stage 3 of first cycle: Maxilla.
    7. 32. Stage 1 of second and all subsequent cycles.
  14. Figure 33. Pectinate-stout setae, Rhithrogena pellucida .
  15. Figure 34. Mandibular molae and epipharynx of Rhithrogena pellucida
  16. Figure 35. Lateral view of heptageniid larva in feeding position
  17. Figures 36-41. Setal types, Simple to Pinnate
    1. 36. (2536) Simple-hairlike seta.
    2. 37. (2434) Simple-triangular seta.
    3. 38. (2921) Pilose seta.
    4. 39. (2535) Pinnate-hairlike setae.
    5. 40. (3085) Pinnate-hairlike setae.
    6. 41. (2365) Pinnate-hairlike setae.
  18. Figures 42-47. Setal types, Pinnate to Bipectinate  
    1. 42. (2648) Pinnate-plumose setae.
    2. 43. (3016) Pinnate-plumose setae.
    3. 44. (3055) Pinnate-lanceolate setae.
    4. 45. (2533) Pectinate-hairlike setae.
    5. 46. (2423) Bipectinate-stout setae.
    6. 47. (2853) Bipectinate-hairlike setae, Types I and II.
  19. Figures 48-52. Setal types, Bipectinate to Palmate  
    1. 48. (2880) Bipectinate hairlike seta, Type III.
    2. 49. (2426) Furcate-stout setae.
    3. 50. (2918) Bifurcate setae.
    4. 51. (2917) Pinnate-plumose setae.
    5. 52. (2428) Palmate setae.
  20. Figures 53-58. Stenacron interpunctatum mouthparts, Labrum to Mandibles
    1. 53. (2526) Labrum and maxillae.
    2. 54. (2529) Diatom on Simple-hairlike seta.
    3. 55. (5456) Right mandible.
    4. 56. (5457) Left mandible.
    5. 57. (5465) Left mandible, apical projections.
    6. 58. (5458) Left mandible, mola.
  21. Figures 59-64. Stenacron interpunctatum mouthparts, Mandibles
    1. 59. (5459) Left mandible, mola.
    2. 60. (5460) Left mandible.
    3. 61. (5466) Right mandible, apical projections.
    4. 62. (5463) Right mandible, mola.
    5. 63. (5461) Right mandible, mola.
    6. 64. (5464) Right mandible, mola.
  22. Figures 65-70. Stenacron interpunctatum mouthparts, Maxillae and Hypopharynx
    1. 65. (0902) Left maxillary palpus and left mandible.
    2. 66. (2908) Galealacinia, palmate setae.
    3. 67. (2530) Galealacinia.
    4. 68. (2534) Galealacinia.
    5. 69. (2875) Maxillary palpus, distal segment.
    6. 70. (5422) Hypopharynx.
  23. Figures 71-76. Stenacron interpunctatum mouthparts, Hypopharynx to Labium
    1. 71. (2904) Lingua.
    2. 72. (2901) Superlingua.
    3. 73. (5427) Labium.
    4. 74. (2800) Paraglossa.
    5. 75. (2801) Paraglossa.
    6. 76. (2803) Paraglossa.
  24. Figures 77-80. Stenacron interpunctatum mouthparts, Labium
    1. 77. (2852) Labial palpus.
    2. 78. (5433) Labial palpus.
    3. 79. (3059) Labial palpus.
    4. 80. (2855) Labial palpus.
  25. Figures 81-86. Rhithrogena pellucida mouthparts, Labrum to Mandibles
    1. 81. (2695) Labrum.
    2. 82. (2433) Labrum.
    3. 83. (5518) Right mandible.
    4. 84. (5517) Left mandible.
    5. 85. (5514) Left mandible, apical plate.
    6. 86. (5477) Left mandible, mola.
  26. Figures 87-92. Rhithrogena pellucida mouthparts, Mandibles
    1. 87. (2925) Left mandible, mola.
    2. 88. (2926) Left mandible, mola.
    3. 89. (2429) Left mandible, mola.
    4. 90. (2430) Left mandible, mola.
    5. 91. (5519) Right mandible, apical plate.
    6. 92. (5474) Right mandible.
  27. Figures 93-98. Rhithrogena pellucida mouthparts, Mandibles to Hypopharynx
    1. 93. (5472) Right mandible, mola.
    2. 94. (5513) Left mandible.
    3. 95. (2239) Right maxilla.
    4. 96. (2240) Right maxilla.
    5. 97. (5523) Hypopharynx.
    6. 98. (5524) Hypopharynx.
  28. Figures 99-104. Rhithrogena pellucida mouthparts, Hypopharynx to Labium
    1. 99. (3289) Hypopharynx.
    2. 100. (5525) Superlingua.
    3. 101. (2696) Labium.
    4. 102. (2920) Paraglossa.
    5. 103. (2447) Labial palpus.
    6. 104. (2914) Labial palpus.
  29. Figures 105-107. Rhithrogena pellucida mouthparts, Labium
    1. 105. (2278) Labial palpus.
    2. 106. (2647) Paraglossa.
    3. 107. (3025) Paraglossa.
  30. Figures 108-109. Ephemerella needhami mouthparts
    1. 108. Lateral view.
    2. 109. Ventral view.
  31. Figure 110. Diagrammatic cross-section of the mouthparts of Ephemerella needhami
  32. Figures 111-114. Schematic diagram of the maxillae and mandibles of Ephemerella needhami during feeding
    1. 111. Resting position, all cycles.
    2. 112. End of Stage 2 of the Maxillary Brushing Cycle.
    3. 113. End of Stage 3 of the Maxillary Brushing Cycle.
    4. 114. Beginning of Stage 3 of the Mandibular Biting Cycle.
  33. Figures 115-118. My other Ephemerella needhami mouthparts
    1. 115. Distal end of galealacinia.
    2. 116. Bipectinate-hairlike filtering setae.
    3. 117. Right mandible, posterior view.
    4. 118. Right mandible, distal end, dorsal view.
  34. Figures 119-124. Ephemerella needhami and Cladophora
    1. 119. Ephemerella needhami larva clinging to Cladophora.
    2. 120. Dorsal abdominal spines of Ephemerella needhami.
    3. 121. Cladophora clumps in natural habitat.
    4. 122. Cladophora filaments with and without epiphytes.
    5. 123. Epiphyte community on Cladophora.
    6. 124. Cladophora filament and associated epiphytes taken from the gut of an Ephemerella needhami larva.
  35. Figure 125. Number of epiphytes found per mm2 of Cladophora filament
  36. Figure 126. Width of randomly selected Cladophora filaments
  37. Figure 127. Gut contents of Ephemerella needhami larvae

Preface to the HTML Edition

It didn't really take me long to convert my dissertation from Wordstar version 1.ancient_history to HTML.  However, as I look at the translation, I am bothered by a few things. Most important, there seem to be a number of errors in the literature citations, so please use these with care.  I have noticed some inconsistencies in either the date of publication or in the spelling of names that I will attempt to correct in later versions.  Also, it will take some time to get the figures scanned in. Finally, I noticed many other errors that have gotten past me and some good editors.  Students - proofread, proofread, proofread, then set it aside for a few years and proofread again.

I have made a conscious decision, for now at least, to leave the manuscript pretty much as it was.  Things cited as being in press that have since been published will be updated when I get a chance, but I will not revise my thoughts from the original. I may, however, add links to additional pages in the future where hindsight and other research has led me to reconsider.

ABSTRACT

McShaffrey, Dave. Ph.D., Purdue University, August 1988. Behavior, functional morphology, and ecology related to feeding in aquatic insects, with particular reference to Stenacron interpunctatum, Rhithrogena pellucida (Ephemeroptera: Heptageniidae), and Ephemerella needhami (Ephemeroptera: Ephemerellidae). Major Professor: W.P. McCafferty.

KEYWORDS: Videomacroscopy, scanning electron microscopy, gut content analysis, and other techniques were used to investigate the diet, feeding behavior, and functional morphology of three species of mayflies: Stenacron interpunctatum, Rhithrogena pellucida (Ephemeroptera: Heptageniidae), and Ephemerella needhami (Ephemeroptera: Ephemerellidae).

Stenacron interpunctatum was found to feed on detritus, which it collected using three distinct stereotypic feeding cycles; a brushing cycle was used to feed on loosely attached detritus, a gathering cycle was used to feed on unattached detritus, and a filtering cycle was used to feed on suspended material. Stenacron interpunctatum was classified as a collector-gatherer.

Rhithrogena pellucida used two feeding cycles to ingest a diet consisting mostly of periphyton. One cycle utilized the labial palps to brush up loosely attached material, the other cycle utilized the maxillary palps to scrape up tightly accreted material. Rhithrogena pellucida was classified as a scraper.

Ephemerella needhami used two feeding cycles to feed on filaments of Cladophora and associated material. One cycle used the maxillae to remove detritus from the filaments, the other used the mandibles to bite off filaments. Ephemerella needhami was classified as a collector-gatherer.

Comparative studies of the mouthpart morphology of all three species at the ultrastructural level revealed morphological differences closely associated with microhabitat and diet.

Brief accounts of feeding behavior are listed for gastropods, amphipods, other mayflies including: Callibaetis, Isonychia, Potamanthus, Heptagenia, Stenonema, Ephoron, and Caenidae; the caddisfly Macrostemmum, water pennies, larval blackflies, and the midge Cricotopus. The application of the techniques developed in this study to such diverse organisms proves the utility of these techniques for future studies.

Return to Table of Contents

CHAPTER I - INTRODUCTION

This dissertation concerns natural history, a scientific style at the heart of the scientific method. Here observational methods are emphasized and pushed to their limits by state-of-the-art technology.

The central question addressed here is simple: How do organisms feed? That topic in itself is rather broad, and here it is applied to three organisms: Stenacron interpunctatum, Rhithrogena pellucida, and Ephemerella needhami. These organisms play an important, but as yet poorly understood, role in stream ecology. In addition, understanding how these organisms feed may shed light on evolutionary relationships. The last two points deserve elaboration.

An important idea surfaced in 1980 in the form of the river continuum concept (RCC) (Vannote et al. 1980). Essentially, this was an attempt to describe the association between changing physical characteristics and differing biological communities along a river from headwaters to mouth. Rooted in earlier works, particularly that of Cummins (e.g. 1973, 1974), the RCC attempted to link a number of ecological concepts and observations.

A basic component of the RCC was the description of energy flow through the macroinvertebrate component of the ecosystem in terms of functional feeding groups (FFGs). These groupings attempted to reduce the manifold feeding adaptations of freshwater crustacea, mollusca, and insecta into a few basic groups. With these groupings in hand, ecologists could then formulate ecosystem-level theories. From an ecological perspective, two of the goals of this study are: 1) to expand our knowledge of the feeding ecology of a few organisms; 2) to provide techniques for a systematic approach to the feeding ecology of a wider range of macroinvertebrates. An overview of the techniques used in this study is presented in Chapter II, and accounts of the feeding ecology of the 3 organisms studied are given in Chapters III, IV, and VII.

Aside from the ecological ramifications, this study is important from an evolutionary and taxonomic standpoint. The feeding structures of aquatic macroinvertebrates are often used as identifying characters in taxonomy, or as a basis for comparison in systematics. An underlying knowledge of the function of these structures, why they take on a particular shape or configuration, will obviously enhance their use or disuse as taxonomic or phylogenetic characters. In particular, an ability to distinguish between homology and convergence is critical, this question is addressed in Chapter VI.

The three organisms studied were not chosen at random. Stenacron interpunctatum was chosen as the prototypic subject at a time when the study included 11 different species, all of which were selected for local availability. Because of its secretive ways and prognathous mouthparts, S. interpunctatum was viewed as the most challenging to film, hence the decision to study it first. Rhithrogena pellucida was selected next because it was related to S. interpunctatum, was found in similar habitat, but occupied a different microhabitat and had different mouthpart morphology. Together these species represent microhabitat extremes among the non-predaceous Heptageniinae, and provide a good basis for explaining some of the peculiarities of heptageniid mouthpart morphology. The comparative mouthpart ultra-morphology of these species is the subject of Chapter V.

Ephemerella needhami possesses more generalized, hypognathous mouthpart morphology, and was chosen to round out this study for comparative purposes. Ephemerella needhami turned out to be a fascinating subject because of its intimate association with the filamentous green alga Cladophora. The feeding behavior and morphology of E. needhami, and comparisons of E. needhami with other hypognathous mayflies and with heptageniids is covered in Chapter VI. The association with Cladophora is described in Chapter VII.

During the course of this study, a wide range of organisms were filmed in addition to the 3 species studied in detail. The casual observations of these other species may provide useful information to specialists in those groups, or act as a springboard for future research. A collection of anecdotal accounts appears in Chapter VIII to fill these needs.

Most of this dissertation has already been published or is currently in press. Chapter III was published as McShaffrey and McCafferty (1986), Chapter IV as McShaffrey and McCafferty (1988), and Chapters V, VI, and VII are in press as separate papers. Slight differences in style between the chapters reflect the different journal formats. All of the references and figures have been collected at the end of the paper. At this point, I would like to especially acknowledge the contributions of Pat McCafferty, who was truly a co-author on these chapters.

Since each of the main chapters was written to stand on its own, the traditional introduction, and methods sections are somewhat condensed, and pertinent literature reviews will be found in the chapters. Despite this, there is some redundancy in several areas, but overall it makes the dissertation much more readable by placing the appropriate background, methodology, and conclusions near the data concerned.

Because this dissertation has almost as much to do with techniques as with results, Chapter II has been written to guide the reader through the evolution of the observational protocol and associated equipment. It is historical in style; again, the rigorous description of the methods is contained in each chapter as appropriate. Some readers may wish to read the methods chapter last, after they have a feel for the methodology and equipment that were used.

Chapter IX is a brief discussion of the future applications of this study. It focuses on the techniques evolved during the course of the project, and how they might be used in future studies. It also looks at the types of information generated by these techniques and how they may be applied to different areas of science.

Return to Table of Contents

CHAPTER II - METHODS

A number of different techniques were used in this study; together they constitute a much more holistic approach to the study of feeding ecology and functional morphology than other previous studies. Figure 1 depicts the various methods used and the areas of investigation that the various methodologies treat. The overall design of this project was to deal with the central question - How do these organisms feed? - by investigating their microhabitat, diet, functional morphology, and feeding behavior. These areas of investigation were studied by means of field observations, gut content analysis, scanning electron microscopy (SEM) and videomacroscopy, as appropriate. Each area of investigation was thus treated by multiple techniques; this helped to eliminate the bias inherent in any one technique. The exact methods used in the study of each species is included with the chapters dealing with that species; this chapter is designed only to provide on overview of the techniques used, as well as some historical background on their evolution over the lifespan of this project. For purpose of discussion, the methods are divided into field observations, videomacroscopy, behavioral analysis, scanning electron microscopy, gut content analysis, and synthesis.

Field observations

Careful field collection and observation were a critical component of this project; much of the ecological data acquired was obtained in this way. Most of the organisms studied, including all of the S. interpunctatum, R. pellucida, and E. needhami, were collected at one site, the Tippecanoe River, at the point where it intersects state highway 18 at the Carroll County - White County border. This site was initially chosen for accessibility, but a number of advantages grew out of continued use. Repeated sampling and observation revealed ecological patterns, particularly seasonal patterns, which might have gone undetected otherwise. From May 1985 to November 1985 weekly presence/absence records of all species at the site were made, and the results entered into a computerized database. Additional sampling of this type, but on a more irregular basis, continued through May 1986. These records provided the baseline data from which patterns of seasonal activity were discerned. Such data led to the study of E. needhami, which had an interesting seasonal activity pattern closely linked to that of the alga Cladophora.

For all species, careful observations were made of microhabitat associations. Organisms were often collected individually, usually by picking up a piece of substrate. Location of the individual was noted, as well as its behavior; i.e. did the larva immediately move to the underside of the stone? Considerable time was spent closely examining the substrate with the aid of mask and snorkel to determine exactly where the larvae were located when undisturbed. All field observations were made in daylight.

Videomacroscopy

Videomacroscopy is the use of electronic image gathering, processing, and recording devices (video) to observe, analyze, and record moving images at magnifications greater than 1x. It has its origins in cinematography, where moving images are captured on film. Videomacroscopy has existed almost as long as video cameras, but the recent availability of high-quality, low-cost consumer equipment has greatly enhanced its appeal. Videomacroscopy has several advantages over cinematography: 1) Costs are much lower for video; whereas the initial investment for a video or cine set-up may be similar, videotape is less expensive than film, and there are no processing costs. 2) Videotape can easily be copied; its electronic format makes editing, image processing, titling, etc. much simpler. 3) Since the video signal is monitored in real time, the exposure can be corrected immediately with little loss of data; with film, exposure problems are only detected when the film is processed. 4) The video signal is routed electronically through wires, thus the camera operator may be some distance away; this enhances observational flexibility. 5) Video image brightness can be amplified electronically, making observations at low light levels possible. 6) Video image size is also amplified electronically, thus reducing the amount of magnification that must be accomplished by optics.

Video does have drawbacks; its main disadvantages are poor resolution and blurring of single-frame images. Both are related to the scan lines that make up a video image. The number of scan lines determines resolution to a great extent, the more scan lines there are, and the smaller they are, the better the resolution.

The equipment used in this study was limited by the resolution of the videocassette recorder (VCR). Resolution in video work is measured in terms of the number of horizontal lines that can be distinguished on a test pattern. The camera used had a resolution of 800 lines, the monitors had resolution up to 700 lines, and the VCR had resolution of about 240 lines.

In terms of obtaining clear single-frame images, the main limitation is the scan speed. Each scan of the image area takes 1/60 of a second, but two scans are needed to make a complete picture. Thus, the effective speed corresponds to a 1/30 second shutter speed in 35mm photography. This in turn means that almost any image will be blurred to some extent. Unlike 35mm photography, where shutter speeds can be increased, the speed of the standard video camera is fixed.

There are improvements in sight. High-speed shutters are already available in cameras. Although they require more light, they are able to freeze motion by allowing only a brief exposure of The video equipment used in this study was assembled by J. Keltner for his study of burrowing in mayflies (Keltner 1983, Keltner and McCafferty 1986). During the course of this study, the equipment was modified and augmented for observing feeding behavior. These modifications came in four areas: electronics, optics, observational arenas, and documentation.

Electronics

Few changes were made in this area. The WV-1850 video camera and its Extended Red Newvicon pickup tube (S4119) are extremely effective at low light levels (down to 0.1 foot-candles), and, as already noted, have relatively good resolution for video equipment. The basic configuration used by Keltner (1983), with the video signal routed through a Panasonic time-date generator (WV-810) to the VCR (Panasonic NV-8950) and monitors, was not changed. By 1987, a color monitor and a second VCR had been added to the basic configuration, but were not used in making observations. The time-date generator was modified so that its stopwatch functions were fully enabled and could be operated remotely.

Optics

The optical system used by Keltner consisted of a 50 mm Macro-Switar lens and 75 mm of extension tubes. The 35x magnification (on a 9" monitor) was not sufficient for observing mouthpart movement. Several different configurations were tested to increase magnification. A Fujinon 20-100mm remotely controlled zoom lens gave acceptable performance only when fitted with close-up lenses or extension tubes, both of which eliminated the remote zoom function. Ultimately, this lens, coupled with the low-light capability of the WV-1850 surveillance camera, found its best use in covert monitoring of such nocturnal laboratory events as students previewing examinations (Keltner, personal communication), or janitors surreptitiously using the telephone for personal calls (McShaffrey, unpublished data). Another lens, a modified Fujinon 50 mm lens, gave performance similar to the Macro-Switar, with the added convenience of remote control of the iris.

The main improvement to optics configuration in the system was the purchase of an adapter that permitted Nikon F mount equipment to be mated to the "c" mount of the videocamera. Magnifications up to 80x were achieved using the extension tubes, adapter, a bellows unit, and 50 mm Nikon lens. The bellows unit was adapted by adding a remotely controlled electric motor with elastic drive to the forward focusing knob, this made remote zooming possible.

The high magnification achieved by using this configuration reduced working distance and depth-of-field. At maximum extension, the lens was so close to the observation cell that it was very difficult to get light around the lens to the subject. At the same time, increased light was needed because lens apertures had to be reduced to increase depth-of-field. While adequate light could be obtained for visual-spectrum observations by a difficult arrangement of the fiber optics light pipes, infrared illumination was insufficient. A partial solution was achieved by attaching infrared light-emitting diodes (LED's) to the surface of the observation cell to provide spot illumination.

The Nikon to "C" adapter also made it possible to mount the videocamera on a trinocular-head dissecting microscope via a Nikon AFM photomicrography unit. This required that the shutter in the photomicrography unit be kept open by setting it on the "bulb" position. The stereomicroscope provided high-magnification views (up to 120x) with ample working distance, but focusing was difficult since the heavy camera had to be moved. In addition, depth-of-field and observational flexibility were severely limited. Still, it was used with great success in the study of E. needhami.

A final comment should be made concerning the effects various sources of light used in this study. Keltner (1983) used near infrared light almost exclusively. His organisms lived in low-light situations and he was concerned that high levels of human visual-spectrum light would affect their behavior. There is general agreement that most insects are not sensitive to infrared light, and in particular, aquatic insects would not be expected to sense infrared since it is absorbed quickly in water. Still, there have been few if any studies on the visual spectrum of aquatic insects; the assumptions about their visual spectra are based on extrapolations from other insects such as the honeybee Apis mellifera. The fiber optics lighting system used in this study provided infrared light by filtration, thus reducing the amount of light available. When the enhancements were made to the optical system to increase magnification, an increase in light was also necessary, and there was no longer sufficient infrared light available. Close examination of videotapes made with infrared and human visual spectrum light revealed little difference in the stereotypic feeding behavior of S. interpunctatum, although its overall behavior was certainly affected, since this species normally avoids light. Thus, while feeding activity was diminished (as the larvae sought to escape the light), the feeding that did occur was normal, and the resulting observations could be made at higher magnification and with greater clarity and depth-of-field. After the study of S. interpunctatum, the decision to use infrared or human visual spectrum lighting was made on a case-by-case basis; for instance, R. pellucida, which feeds on exposed rock surfaces in daylight, was filmed with human visual spectrum light, while Ephoron sp., a more nocturnal bottom dweller, was observed with infrared light.

Observational arenas

Keltner (1983) stated that the key to detailed behavioral observations was confinement of the organism to a small area, and he developed a series of small observation cells (aquaria) for this purpose. These observation cells were not suitable for observations of S. interpunctatum feeding; there were two problems that had to be solved: a requirement for flowing water (to make conditions more natural) and the need to view feeding underneath the head capsule. These problems were solved by the development of the flow cell described in detail in the next chapter. The flow cell essentially simulated the crevice microhabitat of S. interpunctatum, and the microscope slide with cultured periphyton which made up the front wall/viewing port of the observation cell simulated the rock surface with its biofilm food source.

Culture of periphyton and other material onto the glass slides was problematic; slides used often were essentially a monoculture of Cocconeis sp. diatoms. This problem was turned to an advantage by the observation that Cocconeis is among the periphyton most resistant to removal by macroinvertebrates. Cocconeis removal by an organism under laboratory conditions became the test criterion for scraping function.

The flow cell, as used in the study of S. interpunctatum proved useful, with modifications, in studying R. pellucida, E. needhami, and other organisms. The cell was easily adapted for tests of filtering and gathering functions by adding detritus in suspension and adjusting water flow. Flow cells were constructed in various sizes and enclosure depths. Current flow cells have been refined by increasing the width and bolt spacing so that the camera lens can approach the viewing port more closely in high magnification/short working distance situations. The front surface surrounding the viewing port was beveled in later models to allow more light to reach the viewing port.

Despite improvements to the flow cells, they are not suitable at maximum magnifications where the short working distance demands a flush front surface with no protruding bolt heads. The tank cell described in detail in Chapter IV was developed for that purpose. Since the front surface could not be removed, it was only suitable for tests of gathering function, where the food material could be placed directly into the enclosure. The tank cell also had little flow or aeration. The tank cell was employed to some extent in the study of R. pellucida, but its major use was in studying lentic organisms.

Although it was much easier to film organisms when they were confined to small observation cells, it was necessary to make observations of organisms in more natural surroundings in order to gauge the effects of the enclosures on behavior. For S. interpunctatum, such observations were made by placing the videocamera, bellows unit, and lens on a tripod next to the holding aquaria. Similar observations were made of R. pellucida and E. needhami in an artificial stream described in Chapter VI.

Documentation

This area was a constant problem. It is very difficult to get a publication-quality photograph from a video source, and presentation of moving video images in a published format is even more challenging. I have had to rely on written descriptions of feeding movements and line illustrations to document behavioral observations made using video. Other methods were attempted: plastic 3-D models, animated drawings, computer-generated drawings, and various techniques for reproducing video images on film. No method has approached the quality of well-drawn illustrations of each stage of feeding. A recent addition to the video equipment, a Polaroid Freeze-Frame unit, produces relatively good images from video, but images reproduced from videotape are not of sufficient quality for publication.

Behavioral analysis

Behavioral analysis was carried out in 3 stages. The first was a careful planning of observational conditions, the second a detailed analysis of recorded behavior, and the third an evaluation of results. Often the third stage prompted additional observations, sometimes under different conditions, and the process was repeated. Observational design meant insuring that each species was placed in appropriate circumstances so that all possible feeding modes might be observed. Normal practice was to initially pair the organism with a food source that the organism had been reported to feed on in other studies. After such initial tests, other food sources were introduced. The importance of testing organisms with food sources other than those reported in the literature cannot be overemphasized. Tests of filtering function in S. interpunctatum showed that this was a possible but totally unsuspected method of feeding. A similar situation occurred with the observation of a predatory function in E. needhami.

In this study, only mature larvae were studied. This minimized problems associated with accurate species identification and filming of the smaller, less mature larvae. Unfortunately, since organisms often change their feeding habits as they develop, these data are only applicable to a small segment of the total life span of the species studied.

Once appropriate observations had been made on as many organisms as practical, detailed analysis began. Feeding sequences were chosen, on a basis of clarity and completeness, for each feeding function. Feeding sequences were divided into cycles based on the movement of a key mouthpart (i.e. the labium of S. interpunctatum and R. pellucida, or the maxillae of E. needhami.

Each cycle was analyzed separately by freeze-frame techniques. In this process, the positions of all mouthparts at the beginning of each cycle were marked on a clear acetate sheet laid over the monitor screen. The tape was advanced one frame (typically 0.03 second) and the new mouthpart positions were noted. Time data were recorded from the on-screen display (accurate to 0.01 second).

Once composite behavioral cycle descriptions for each type of feeding had been assembled, these descriptions were checked against remaining videotape that was of lesser quality. Particular care was taken to determine if the behavioral descriptions, which were often based on observations from the more restricted observational arenas (hence higher quality), matched observations from more natural conditions (aquaria, artificial streams), which were usually of lower quality.

Scanning electron microscopy

With mayflies, it is vital to understand the form and function of the setae on the mouthparts if one is to understand the functional morphology of the mouthparts. Examination of the setae with a light microscope is not sufficient; SEM is necessary. Over 500 SEM micrographs were made on 3 different instruments during the course of this project. The following is a summary of the techniques in use at the end of the project.

Larvae were killed and stored in 70% ethanol. Periods of storage ranged up to 1 year; some specimens examined for comparative purposes were taken from the museum where they had been stored for up to 20 years. Larvae were dehydrated by placing them in 9ml vials with successive solutions of 80%, 90%, and 100% ethanol for 10 minutes at each step. They were then placed in 50% ethanol, 50% xylene overnight; the xylene increased hardness of the specimens. The ethanol/xylene solution was replaced by several successive 9 ml aliquots of ethyl acetate. If it was desirable to remove food material from the mouthparts, they were sonicated for 30 seconds in an ultrasonic cleaning bath after the first aliquot of ethyl acetate was added.

If dissection was required, this was performed with the mouthparts submerged in ethyl acetate. Mouthparts were transferred to SEM stubs coated with a thin layer of adhesive made by dissolving the adhesive from transparent tape in ethyl acetate, painting this material onto the stub, and allowing it to dry. Care was taken to insure that the mouthparts transferred to the stubs were dry before being set in place. This method of dehydrating specimens has the advantage of being much faster than critical-point drying, and it produces few artifacts.

Specimens were coated with gold-palladium for 5-8 minutes at 10 microvolts in a Hummer 1 evaporative coater; then the stubs were placed on their sides (approximately 70o) and coated for an additional 3-5 minutes.

Specimens were examined via secondary electron emission with a JSM 840 SEM, normally operating at an accelerating voltage of 15 kv. Micrographs were made using Polaroid Type 55 film. MIcrographs were made of whole larvae, head capsules, mouthparts, and all setal types. Dorsal, ventral, anterior, lateral, posterior, medial and other views were employed as needed. As a historical note, some of the initial SEM work in this project was done on a JSM 1 instrument at the University of Akron.

Gut content analysis

Gut content analysis, used alone, has been the chief method of determining both diet and FFG classification; the problems associated with this are discussed in Chapters III and IV. Still, gut content analysis is an important tool in determining diet, and, when coupled with behavioral observations, in determining FFG classification.

Because gut content analysis was a supporting method and not the primary means of investigation, qualitative methods were chosen initially. This in essence meant macerating the gut contents of individual larvae in euparol on a glass microscope slide, and examining the slide for content.

For the study of E. needhami, a more rigorous methodology was employed. Gut contents were analyzed quantitatively with the aid of an ocular grid and computerized data entry and summarization. In all cases, gut contents from individuals collected in the field were compared with those from organisms that had been videotaped to determine if laboratory conditions were affecting diet.

An adjunct to gut content analysis was the examination of substrates taken from the field to see what food items were available. Such examinations were conducted by light microscope and SEM. Similar examinations were made of substrates used in behavioral studies in the laboratory.

Synthesis

Once the data from the techniques described above had been gathered and analyzed, it had to be synthesized into a holistic overview of the feeding ecology and functional morphology of the organism in question. Much of the synthesis involved correlation of morphological data with behavioral observations to reach conclusions concerning functional morphology. This meant determining function based on what had been observed (rather than determining function based on pre-conceived notions; the reader should consult Keltner [1983] for an excellent treatment of functional morphology and associated sciences and their methodologies). Examples of situations where function did not follow preconceptions are given in Chapter V.

Other correlations included gut contents vs. material observed to be ingested, and microhabitat data vs. food availability vs. gut contents. Discrepancies were noted and the relevant data re-examined for possible explanations. At times, this stage of analysis suggested further observations, which were made to test the validity of the explanations. An example of this type of feedback is given in Chapter VII where additional gut content and field data was collected to investigate hypotheses of E. needhami's ecology that were generated by the previous year's data.

Keltner's (1983) review of the rich history of this type of study is unsurpassed. His ultimate assessment of the state of what he referred to as adaptive analysis and the methodologies employed in such analysis (functional morphology, biomechanics, etc.) was that no single methodology was sufficient. Whereas the present study does not claim to be a single, inclusive framework for adaptive analysis, it does present a holistic approach to the questions of feeding ecology and the function and form of mouthparts. Although specialists in the fields of ethology, morphology, and ecology may perceive a lack of rigor in the techniques used here, the methodologies borrowed from those disciplines form a synergistic approach that surpasses any of the methodologies alone.

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CHAPTER III - FEEDING BEHAVIOR OF STENACRON INTERPUNCTATUM

An important aspect of any animal's ecology is its role as a consumer. In freshwater ecology, particularly in studies of running water, the consideration of feeding relationships of benthic macroinvertebrates has been crucial to conceptualizing community dynamics and predicting ecological relationships (Cummins 1973, 1974, Vannote et al. 1980). Despite the fundamental importance of such information, feeding habits are often inferred from reports of similar taxa because most species have not received critical autecological study. Conclusions about feeding have usually been based on gut content analyses and casual observations. Unfortunately, gut contents can be misleading because of seasonality and errors introduced in sampling, dissection, and identification of partially digested material (Shapas and Hilsenhoff 1976). It may not be clear which material in the gut is nutritionally important. Casual observations of behavior can seriously underestimate the complexity of an organism's feeding activities.

Perhaps the most difficult task in organizing feeding ecology data is the grouping of organisms into categories descriptive of similar feeding habits. Classifications based on food types are the oldest. Cummins (1973), however, has argued for the need of categories based on functional characteristics. As an example, larvae of the mayfly genus Stenacron, which is the subject of the research reported here, have been placed in a "scraper" functional category by Cummins et al. (1984), defined as consisting of organisms feeding on "attached algae and associated material" (Cummins and Merritt 1984). "Gatherers", defined as organisms that collect detritus from sedimentary deposits, is another functional category in which Stenacron was placed (Cummins et al. 1984). Other authors have defined these groups differently; for example Lamberti and Moore (1984) broaden the definition of scrapers to include organisms that collect material other than algae from crevices in the substrate.

I have studied the functional feeding role of Stenacron interpunctatum by analyzing functional morphology and feeding behavior. This approach allows me to define more accurately the feeding role of the animal, and also to make informed estimates about the functional roles of other organisms with similar morphology. Other studies of mayflies have combined observation and morphological analyses (Brown 1960, 1961, Froehlich 1964, Rawlinson 1939, Schonmann 1981, Strenger 1953, 1975). My study, however, differs considerably by incorporating a recording device for detailed motion analyses, scanning electron microscopy (SEM) for analysis of fine structure and topology, and novel viewing angles. In addition, my primary observation system was designed to duplicate natural stream conditions (i.e. flow, light levels, orientation) more closely than the studies cited above.

I have chosen S. interpunctatum as my prototypic subject because it is common in the eastern half of North America and easily cultured. Furthermore, mature larvae are large enough (head capsule width >2.5mm) to observe with ease, and individuals continue to feed even under the high illumination often required for documentation. I view this study as ecologically important because Stenacron and its relatives in the family Heptageniidae are often important or even dominant in certain lotic habitats (Bednarik and McCafferty, 1979). My objectives in this study were to document the feeding behavior of S. interpunctatum and identify the structures utilized in feeding. Because this study is part of a larger, comparative investigation of the feeding behavior and associated morphology of larval mayflies, I have deferred critical study of the nutrition of this species, and I have not attempted to prepare a complete ethogram of its behavioral repertoire.

Methods

Organisms were collected from early spring to late fall from cobble substrate in the Tippecanoe River in north-central Indiana, and were maintained in the laboratory in large aquaria filled with substrate and water from the collection site. Circulation in the aquaria was provided by airstones placed on the bottom. Only large (head capsule width >2.5mm) larvae were used for observation and videotaping.

Individuals were observed by the following techniques. Initially, to minimize possible distractions, individuals were observed in a large light proof observational theater insulated to reduce sound vibrations. The observer was outside the theater, and by remote control operated the video camera (Panasonic WV-1850 with 1" Extended Red Newvicon S4119 pickup tube) mounted inside the theater for observation. The theater and monitoring system, which were designed for a study of burrowing behavior, were described in more detail by Keltner and McCafferty (1986). Light was provided either by an externally mounted fiber optic illuminator equipped with Kodak Wratten No. 87c filters or by infrared light-emitting diodes (LEDs). The filters transmit less than 0.1% of the light below 780nm, the LEDs transmit at 880nm; thus the light used was essentially non-visible to humans. High reflectiveness by Stenacron eyes and the lack of observed responses to changes in infrared light intensity indicated that the infrared light sources were non-visible to the study organisms. Other observations were carried out under visible light which allowed smaller lens diaphragm openings to be used, thus increasing resolution and depth of field. A variety of lenses and lens extensions were employed to allow magnification up to 80x on a 9" (23cm) high resolution black and white video monitor. A 1/2" (12.7mm) VHS format Panasonic NV-8950 video recorder with freeze-frame, slow motion, and reverse motion capabilities allowed images to be recorded and analyzed.

Organisms were placed for observation in a cell built of 1/2" (12.7mm) thick clear acrylic (Fig. 2), its center excavated to form a 1 x 3cm rectangular depression. Several cells were constructed with depths ranging from 1 to 5mm. At each end of the depression several small holes were drilled down into the material to join with larger holes leading out to the edge of the acrylic. Aquarium tubing was cemented into these larger holes. The depression was surrounded with self-adhesive automotive gasket material and covered with a 75 x 25mm glass microscope slide. The slide was held in place by a second piece of clear acrylic, 1/4" (6.35mm) thick, that had its center removed to correspond with the depression; this hole was also surrounded by gasket material. The apparatus was held together by bolts inserted through holes in both pieces and secured with wing nuts. The observation cell was placed in the theater with the long axis in a vertical position and the glass slide facing the camera. The stage on which the cell was mounted was moved in three axes by electric motors.

Water at room temperature (20oC) was circulated through the cell by pumping or siphoning it into the cell through the lower tube and out the upper. The flow was controlled by a valve; however, current velocities around the larvae (as measured by timing the movement of small particles) varied even within a given experiment. The larvae could occupy a significant portion of the cross-sectional space within the shallowest observation cells; this had a dynamic effect on local current velocities and produced a complicated pattern of streamlines of varying current velocities within the cell. For most experiments flow was adjusted until the larvae began to orient. This adjustment was necessary because of the difficulty in measuring current velocity (or deciding which velocity was significant to the organism). Orientation usually occurred at "low" current velocities (0-5cm/s).

Food was introduced into the observation cell by several means. Algae taken from the Tippecanoe River were cultured in the laboratory directly on the glass slide forming the viewing wall of the observation cell. The slides were covered primarily (95%) with Cocconeis; bacteria were common between these diatoms. The remaining 5% was covered by scattered fungal hyphae, Oscillatoria, Cladophora, Melosira, Fragilaria, Chlorella, rotifers, and protozoa. Slides were examined microscopically before and after the observation period. Feeding was considered effective if the slides contained patches where the algae had been removed, or if actual removal and ingestion were observed. Detritus taken from the holding tanks was placed in the cell and feeding was observed at low and high flow rates. Feeding on detritus was considered effective if ingestion was observed.

Motion analyses were carried out primarily by examination of the videotape recordings of the observation sessions of the organisms in the cells; however, this was supplemented by direct video observation. Over 20 individuals of S. interpunctatum were observed for a total period of over 50 hours in the course of this study. Ten of these individuals were videotaped in the observation cells; 236 feeding cycles from 8 of these individuals (4 larvae feeding on attached algae and 4 larvae feeding on detritus) were analyzed in detail using the single frame advance feature of the videotape recorder. This information was used to assemble a chronology of mouthpart movements which was checked against the remaining videotape. In all, several thousand feeding cycles were examined. Following the protocol of Keltner and McCafferty (1986), the observed feeding behavior was classified into stereotypic cycles describing different methods of obtaining and handling possible food material. Each cycle was divided into stages to describe the choreography of movement of the suite of mouthparts For our purposes the stages are delineated by changes in the movements of the labium and the labial palps.

Additional observations were made of organisms on more natural substrate in aquaria and in small artificial streams to assess the effect of the theater and observation cell on behavior, and to check the accuracy of the descriptions of the feeding cycles. These observations were made by the unaided eye, through a stereomicroscope, and with the video camera. These methods did not provide the detail and/or repeatability necessary for critical analysis of mouthpart movement, however, resolution is greater during the initial, direct observation than on videotape playback. I have found this also to be true for other species of mayflies and other aquatic invertebrates that have or are being studied using similar techniques.

Mouthpart structures were examined by light microscopy and SEM. This information was used to construct a movable plastic model of the mouthparts. Gut contents of living larvae were placed on glass microscope slides and examined immediately with a compound microscope. Gut contents of individuals freshly collected in the field were compared to those held in the laboratory. Gut contents were classified as being mineral, organic detritus, diatoms, filamentous algae, unicellular algae, animal remains, or bacteria. No attempt was made to quantify proportions other than to note what type predominated.

Results

Field observations

The open canopy at the collection site permitted algal growth to cover the substrate. The aufwuchs community was more diverse than that cultured on slides in the laboratory. Additional taxa of diatoms such as Gomphonema, Gyrosigma, Navicula, and Synedra were also present. Certain taxa such as Cladophora, Melosira, and Fragilaria exhibited seasonal blooms to a point where they visibly dominated the periphyton community. At times large areas of the substrate were covered by blackfly larvae and pupae, or by the silk retreats of Petrophila (Lepidoptera) or chironomid larvae.

The larvae collected in the field were, without exception, collected from the bottoms of stones. Although sampling was not quantitative, comparable numbers of S. interpunctatum larvae were found among the cobble substrate both in the open canopy areas and under an adjacent highway bridge where shading visibly reduced algal growth. Larvae were more commonly collected from areas of slower current.

Effects of experimental conditions on behavior

Mature S. interpunctatum was highly stereotypic in the movement of its mouthparts in all experimental enclosures (observation cells, artificial streams, holding aquaria with natural substrate). The few variations in feeding movements observed were apparently associated with food type and are described below. In every case, movements were independent of experimental enclosures.

External factors such as noise, vibration, changes in visible light intensity or water velocity did not affect the qualitative performance of feeding movements of S. interpunctatum. They did, however, affect the duration of movements and the release of stereotypic feeding movements. Feeding movements induced by external stimuli ceased after 1-2 cycles of the mouthparts; those that appeared to be induced by the organism typically continued for at least 5 cycles. Feeding movements observed under visible light were identical to those viewed with infrared light. Specimens observed in the artificial streams and holding aquaria were negatively phototactic, positively thigmotactic and positively rheotactic.

Stereotypic feeding behavior

Resting position: When the subjects are not feeding, their mouthparts (Figs. 3, 4, 5-9) are held in a characteristic resting position (Fig. 10). The distal segments of the labial palps are held folded inward so that they lie over the glossae and paraglossae. The labium as a whole is held adducted dorsally to close the preoral cavity ventrally. The galealaciniae of the maxillae are positioned with their median setae touching the lingua of the hypopharynx; the galealaciniae also are in contact ventrally with the labial palps and dorsally with the superlinguae of the hypopharynx. The distal segments of the maxillary palps are held folded over the anterior opening of the preoral cavity. The mandibles are held with the denticles separated, and the molae presumably closed. The labrum is held pressed against the head capsule dorsally and the mandibles ventrally.

Brushing cycle: Individuals placed in the observation cell with algae grown on the glass slide performed a behavioral cycle I term brushing. A brushing cycle is comprised of several stages delineated by the movements of the labium. A single brushing cycle, as described below, takes approximately 1 second at 20o C. The mouthpart movements described below are often preceded by vigorous movement of the forelegs ahead (upstream) of the individual. The legs are repeatedly extended and then retracted, drawing the claws over the substrate; this movement ceases during the first cycle. I did not find any evidence that such movement of the forelegs displaces tightly accreted material by the forelegs.

Stage 1: -- Abduction of the basal segments of the labial palps: As feeding commences, the labium begins to swing ventrally towards the substrate. The basal segments of the labial palps swing posteriorly about 10o (Fig. 11); this movement partially displaces the distal segments of the labial palps from contact with the glossae and paraglossae. At this point the basal segments of the maxillary palps swing posteriorly. The galealaciniae do not move in Stage 1 of the first brushing cycle, but in all subsequent brushing cycles they move medially at this point. The labrum begins to move down and backwards to shield the front of the other mouthparts.

Stage 2: -- Abduction of the distal segments of the labial palps: Once the basal segments of the labial palps reach their posterior position, the distal segments of the labial palps are abducted, moving through a range of about 30o. As the apical tips of the labial palps clear the glossae and paraglossae, the labium completes its ventral swing coming to rest against the substrate with the labial palps fully extended, yet remaining under the contour of the head capsule (Fig. 12). At this point the galealaciniae are abducted, moving laterally outward. The maxillary palps have been extended and reach beyond the lateral borders of the head capsule. The denticles of the mandibles begin to move laterally outward. The labrum completes its movement posteriorly and ventrally, effectively shielding the mouthparts under the head capsule from the current.

Stage 3: -- Retraction of the labial palps: The labial palps are retracted from their extended position by a combination of an anterior swing of the basal segments of the palps and adduction of the distal segments of the palps. This movement is continuous and coordinated so that the palps always remain under the head capsule. As the distal segments of the labial palps move they brush (sweep) up material from the substrate with the profuse setae on the ventral and anterior surfaces. When the distal segments of the labial palps reach the paraglossae they are raised dorsally by a rotation of the basal segments of the palps so that they pass dorsal to the paraglossae and glossae. The distal segments of the palps reach a position that is more median than in the resting position (Fig. 13). The entire labium is raised as soon as the distal segments of the labial palps are dorsal to the glossae and paraglossae. The galealaciniae follow the labial palps in moving medially; they also move somewhat ventrally. The maxillary palps sweep inward, brush over the substrate, and enter the preoral cavity dorsal to the superlinguae. The denticles of the mandibles complete their lateral outward movement and the maxillary palps are inserted among these denticles (Fig. 14). The labrum moves dorsally.

As feeding continues, this brushing cycle is repeated with the abduction of the basal segments of the labial palps as in Stage 1 (foreleg movement is not repeated). The brushing cycle is repeated unchanged except that in Stage 1 of subsequent cycles the galealaciniae are adducted medially, and the posterior movement of the basal segments of the maxillary palps draws the apical setae of the maxillary palps through the combs on the denticles of the mandibles.

Gathering cycle: When S. interpunctatum is presented with loose detritus, a gathering cycle is performed. The gathering cycle is similar to the brushing cycle except for the following. The mouthparts are not pressed tightly against the substrate in Stage 2, and the legs, particularly the forelegs, may bring detritus to the preoral cavity where it is swept up by the labial palps as they move inward in Stage 3. The lateral setae on the distal segments of the labial palps carry more material from the substrate than the ventral setae. In Stage 3 the apical setae of the maxillary palps bring a larger volume of detritus to the preoral cavity than in the brushing cycle; this material is combed out by the mandibular denticles when the maxillary palps are withdrawn in Stage 1.

When presented with filamentous algae such as Cladophora sp., the mouthparts are positioned in Stages 2 and 3 so that the labial palps brush over the filaments, removing epiphytes. The maxillary palps play a role in manipulating the filaments. It is possible for the organism to manipulate filaments into the preoral cavity; however, what could be interpreted as attempts to bite off and swallow pieces of such filaments were ineffective.

Filtering cycle: When a heavy load of detritus is moving in the current, S. interpunctatum alters its feeding behavior radically and becomes a passive filter feeder. The filtering cycle is less complex than the other cycles, and the labial palps are not used to gather food. The larva orients so that its head faces into the current and the longitudinal axis of the body is parallel to the flow. The mouthparts are held in the resting position except for the maxillary palps; these are extended until they are perpendicular to the flow. Particles of detritus adhere to the apical setae. Periodically the maxillary palps are retracted into the preoral cavity as in Stage 3 of the brushing cycle. The detritus brought to the preoral cavity is combed out from the apical setae of the maxillary palps by the denticles of the mandibles as in Stage 1 of the brushing and gathering cycles; the detritus is then further processed in the manner described below, except that the labial palps do not gather additional food.

Food processing

Once food material has been acquired by either the palps of the labium or maxillae in any of the cycles above, it must be positioned for ingestion. This process is carried on continuously during feeding and for several seconds after the last material is acquired from the substrate. The transfer is accomplished by successive fields of setae on the various mouthparts as described below (Fig. 3).

After completion of at least one brushing or gathering cycle, and when the labial palps are extended again (Stage 1), the material on their setae is removed in two ways. The outward lateral movement (Stage 1) of the distal segments of the labial palps causes material to be combed from the ventral setae of the labial palps by the setae on the dorsal surfaces of the glossae and paraglossae. Material on the lateral setae of the distal segments of the labial palps is removed by the comb setae of the galealaciniae as they move medially in opposition to the outward movement of the labial palps (Stage 1). The material in the comb setae is brushed out as these setae are drawn base-first through the setae on the glossae and paraglossae by the ensuing outward lateral movement of the galealaciniae (Stage 2).

In all cycles, material on the maxillary palps is removed by the combs on the mandibular denticles as the palps are withdrawn (Stage 2). In the filtering cycle all material is brought to the mouth in this way.

Material left on the glossae and paraglossae is moved further through a number of processes. As the labial palps return to the preoral cavity (Stage 3), this material is displaced posteriorly, medially, and dorsally by the palps and the new load of material they bear. As the galealaciniae move medially (Stage 1), their median setae push the material between the superlinguae and the lingua (Fig. 15). The lingua bears rows of setae on its dorsum which guides the material to a point where it can be pressed against the mandibular molae for straining and ingestion. Presumably, material left on the denticles is brushed directly onto the dorsum of the hypopharynx by movements of the mandibles; I have not been able to observe this since this area is obscured by the other mouthparts.

The maxillary palps are used in all cycles to displace larger particles from the preoral cavity and to brush material from the comb setae on the galealaciniae. Once food acquisition ceases, the mouthparts (except for the labial palps) remain in motion as described above for several cycles, moving material into position to be ingested.

Gut content analysis

Organisms collected in the field had little variety in their gut contents. All guts contained mineral material and organic detritus. Diatoms were observed in the guts of only a few individuals collected early in November, 1985, when a dense growth of diatoms, predominately species of Melosira and Fragilaria, covered the substrate in the Tippecanoe River. Whereas some of these diatoms were present in the gut, they were not predominant, and the diatom frustules were intact. None of the larvae collected in the field had filamentous algae, unicellular algae, or animal remains in their guts. Individuals held in the lab had gut contents similar to those newly collected in the field, although some of the individuals held in the laboratory did contain the diatom Cocconeis sp.

Discussion

In any behavioral study, it is important to assess the impact of experimental protocol on behavior. In this study the need to confine the organisms to a small area to allow for high magnification observation required an enclosure that did affect the overall behavior of the organism. Since my objective was only to document a small portion of the species' overall behavioral repertoire, this was not a significant constraint because the portion of the behavior in which I was interested in was not affected. The brushing cycle and the gathering cycle were performed in an identical fashion in the observation cell as well as in aquaria and in the artificial stream. My observations of other Ephemeroptera, Trichoptera, Diptera, Odonata, Coleoptera, Amphipoda, Gastropoda, and other freshwater macroinvertebrates (See Chapter VIII) suggest that the cyclic movements of the mouthparts are stereotypic and vary little if any between individuals of the same age class of any one species. This conclusion is consistent with other insect feeding studies (Brown 1960, Chance 1970, Devitt and Smith 1985, Froehlich 1964, Pucat 1965).

This study shows that S. interpunctatum is an opportunistic feeder with a behavioral repertoire sufficient to allow it to feed in a number of different ways. The importance of detritus as a food source for this species has been documented by Lamp and Britt (1981); their quantitative gut content data agree with my qualitative observations. My laboratory observations combined with ecological observations from the field suggest that this species is better classified as a collector (gatherer) than as a scraper according to the categories of Cummins and Merritt (1984). In laboratory tests S. interpunctatum was not able to remove tightly adhering diatoms such as Cocconeis sp. from a glass slide; however, individuals on more natural substrates are apparently able to remove this diatom to some extent. The only alga that S. interpunctatum was observed to ingest was an extremely flocculent mass of diatoms with physical characteristics similar to loose detritus. Although the organisms are capable of manipulating filamentous algae and removing epiphytes from surfaces of these plants, they have difficulty biting off sections of the filaments. Filamentous algae, however, have been reported in the gut of Stenonema modestum, a closely related and morphologically similar species (Kondratieff and Voshell 1980).

Microhabitat data also suggest that this species is a collector. I consistently found these larvae on the undersides of stones in crevices where detritus was deposited. Other studies (Wiley and Kohler 1980, Wodsedalek 1912) also stressed the occurrence of S. interpunctatum larvae on the undersides of stones.

Although direct observation and analysis of feeding behavior of earlier instars of S. interpunctatum were not conducted in this study, earlier instars of many species may be detritivores even if they later specialize on other food sources (Cummins 1973, Cummins and Merritt 1984). Since mature S. interpunctatum larvae primarily ingest detritus (Lamp and Britt 1981), the importance of this food source in the diet of this species is significant.

The ability of S. interpunctatum to vary its feeding behavior to either filter or gather depending on the circumstances demonstrates its independence from a single resource such as periphyton. A more tenable classification of S. interpunctatum as primarily a collector (gatherer) has important consequences for ecological investigations incorporating or extrapolating from the river continuum concept.

On the basis of their mouthpart structure, heptageniids, in general, have sometimes been assumed to scrape their food. For example, Brown (1960) stated that the crown setae of the maxillae are used to rake up diatoms; however, this actually was a misinterpretation of Strenger (1953) (in German) who described the function of these setae ("Kammborsten") as combs for the labial palps. I found that the orientation of these setae aligns them precisely with the labial palps, and that they cannot reach the substrate because of the position of the large labium between the maxillae and the substrate. The external chitinized "scraping bars" reported by Morgan (1911) on the distal segments of the labial palps in the heptageniid Epeorus fragilis are not present in S. interpunctatum. Examination of the distal segments of the labial palps of S. interpunctatum by light microscopy revealed structures which could be interpreted as scraping bars, but examination with SEM revealed that they were internal structures (probably apodemes). Further investigations will be needed to see if this is not the case for other heptageniids.

The only mouthparts of S. interpunctatum that reach the substrate are the labial palps and the tips of the maxillary palps. Both of these structures are heavily setose and not fit for scraping in the manner I have observed for radulae of gastropods and the mandibles of water penny beetle larvae (Psephenus) (Chapter VII). I have, however, seen the heptageniid Heptagenia flavescens scrape bacterial film from the substrate (Chapter VII). The terminal ends of the maxillary palps of that species are reinforced and modified as a scraping tool, quite unlike the brush-like maxillary palps of S. interpunctatum. It is possible that the forelegs of S. interpunctatum may be capable of some scraping. They are active before the brushing cycle and are important in bringing food to the mouth in the collecting cycle. The fact that I did not observe them to be effective in removing material from the substrate may be an experimental artifact.

From a functional-morphological standpoint, it is desirable to erect a new category of feeding -- 'brushing', distinguished from scraping by the morphological structures utilized in acquiring material from the substrate. I propose that brushers include organisms that remove material from the substrate using setae, and that scrapers include organisms that use hardened structures such as mandibles or radulae to remove accreted material. Whereas such a distinction is valuable from a functional standpoint, its ecological significance may be less apparent and awaits data about what precise components of the aufwuchs can be removed by each of these functions and what components are nutritionally significant.

Analyses of the feeding behavior and functional morphology of a species are important tools in describing the ecological relationships of that species. Such studies are necessary before ecological assumptions based on morphology are made, and provide additional insight into the relevance of gut content data. As more of these studies are completed within taxonomic groupings and among taxa with similar morphology their usefulness will increase as an aid in understanding community dynamics and evolutionary trends.

Acknowledgments

I thank John Keltner for his technical assistance and advice; Arwin Provonsha, whose illustrations translate complex three-dimensional relationships to the two-dimensional world of the journal page; and Dan Bloodgood for assisting with field work, construction of the observation cells, and preparation of Figure 2. Purdue University Agricultural Experiment Station Journal No. 10,178.

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CHAPTER IV - FEEDING BEHAVIOR OF RHITHROGENA PELLUCIDA

The feeding behavior and functional morphology of the heptageniid mayfly Stenacron interpunctatum and new methodology for studying such was reported by McShaffrey and McCafferty (1986). In order that these data can begin to be viewed within a larger comparative context I subsequently studied another midwestern mayfly, Rhithrogena pellucida. This mayfly provides an interesting basis of comparison because it is also a stream-dwelling member of the family Heptageniidae but lives in different habitats and possesses somewhat different mouthpart morphology. As with S. interpunctatum, my objectives with R. pellucida were to document both the feeding behavior and feeding structures. I did not attempt to document the entire range of behavior of the species, or to determine the nutritional significance of the material ingested.

My functional morphology/behavioral approach to studying feeding incorporates field observations, gut content analysis, scanning electron microscopy, and videomacroscopy into a comprehensive research protocol. This allows critical examination of questions concerning diet and microhabitat and the related adaptations of feeding behavior and mouthpart morphology. An important feature of this system is its redundancy; all questions are investigated using multiple techniques. Besides the comparative data that may have relevance to the study of adaptation and evolution, there are also obvious ecological applications. One that I have pursued involves the common practice of assigning functional feeding groups (FFG) (Cummins 1973) to the various members of aquatic communities.

Strenger (1953) studied the functional morphology of European species of Rhithrogena and included observations of behavior and interpretation of morphology. Strenger's work, although important, was limited by her inability to film behavior for detailed analysis and the fact that mouthpart morphology was not studied at the ultrastructural level. I was fortunate to have been able to extend such research by having SEM and videomacroscopic techniques available to me. Rhithrogena mayflies have previously been regarded as both collector-gatherers and scrapers (Cummins et al. 1984), with the primary designation being collectors.

Methods

Organisms were collected in April, 1986, from cobble substrate in the Tippecanoe River in north-central Indiana. They were maintained in the laboratory at room temperature (20-25oC) in 75x30x30 cm aquaria filled with substrate and water from the collection site; circulation was provided by airstones. Only mature larvae, as judged by relative wingpad development in Ephemeroptera larvae (McCafferty and Huff 1978), were used for observation and videotaping.

Observations were carried out using the techniques outlined in McShaffrey and McCafferty (1986), with some modifications. When studying R. pellucida, all observations were made using visible light supplied by a fiber optic illuminator to maximize resolution. My experience showed that S. interpunctatum feeding movements were the same in visible and infrared light (McShaffrey and McCafferty 1986).

Most observations were made in an observation flow cell as described and illustrated in Figure 2. With R. pellucida, only a shallow (1mm deep) flow cell was used. Water at room temperature (20-25oC) was fed into the cell by gravity from an aquarium mounted on the roof of the observational theater. Water leaving the cell collected in another aquarium; a pump controlled automatically by a depth-sensing switch periodically returned water to the upper tank. Water in both aquaria was aerated. The flow of water was regulated by a valve; current speed was adjusted as described in the study of S. interpunctatum, with the same inherent difficulties in determining precise current speed (McShaffrey and McCafferty 1986). In most cases the flow was increased until the individuals began to orient to the current, this usually occurred at low current velocities (1-5 cm/s).

In addition to the shallow flow cell, a tank cell was also employed. The tank cell (Fig. 16) was constructed of 2 mm thick acrylic, 10.5 cm long, 4 cm wide, and 4 cm deep, and held a volume of 150 ml of water when in use. The front face had a 1 X 3 cm rectangular section removed from the center; a portion of a standard glass microscope slide was attached over the outside of this hole, and a 1 mm2 mesh screen was attached over the inside to produce an enclosure into which the individuals were placed. This enclosure measured 1 cm long, 2mm wide, with a water depth up to 3 cm. A hemicylinder of black plastic (radius 1.5 cm), with 30 2.5 mm diameter holes, was placed around the enclosure on the inside of the tank. This plastic served to isolate the organism visually and reduce unwanted reflections during filming.

Water flow was provided as in the shallow flow cell; the water entered the tank cell through a tube attached with its outlet below the waterline inside the plastic hemicylinder directly behind the enclosure, and exited through another tube attached to one side of the tank cell. Water level in the cell was controlled by regulating inflow and outflow with valves. There was no noticeable water current in the enclosure. The tank cell was mounted in place of the shallow flow cell on the stage of the observational theater. The final position of the organism, vertical with the mouthparts facing the glass slide and the videocamera, was the same as in the flow cell.

Food for the experiments was provided as described in McShaffrey and McCafferty (1986). Diatoms and other periphyton collected in the Tippecanoe River were cultured on glass slides in the laboratory. The composition of the periphyton community on the slides was the same as the previous study - a 95% covering of the diatoms Cocconeis and Achnanthes, numerous bacteria between the diatoms, and the remaining 5% comprised of scattered fungal hyphae, Oscillatoria, Cladophora, Melosira, Fragilaria, Chlorella, rotifers and protozoa. Scanning electron micrographs of typical growth on a slide surface are shown in Figures 17 and 18. Detritus taken from the holding tanks was introduced into the water flow in the shallow flow cell at both high (>5 cm/s) and low (<5 cm/s) flow rates; detritus was placed directly into the enclosure of the tank cell. Detritus settled to the bottom of the tank cell and the shallow flow cell at low flow rates; at high flow rates detritus moved through the shallow flow cell in suspension. I considered feeding effective if ingestion of the material was observed.

Motion analyses were carried out by examination of the videotape recordings of the observation sessions as in McShaffrey and McCafferty (1986). Owing to sparseness of specimens in the field, the need to sacrifice some individuals upon return to the laboratory for gut content analysis, and natural mortality, only 10 individuals were videotaped. Of the several hundred feeding cycles videotaped, 50 were analyzed in detail using single frame advance. The 50 feeding cycles chosen for detailed analysis were selected for visual clarity and presence of repeated feeding activity. Once a chronology of mouthpart movement was determined within these cycles, the chronology was tested against the remaining cycles. The feeding cycles are classified into stereotypic cycles as outlined in McShaffrey and McCafferty (1986) and adapted from the methods used by Trueman (1968) and Keltner and McCafferty (1986). Each stage is delineated by specific movements of the labial palps.

Mouthpart structures were examined using light microscopy and SEM. Specimens were prepared for SEM by dehydration in increasing concentrations of ethanol from 75% to 100%, then transferred to 100% ethyl acetate. Some specimens were sonicated in 100% ethyl acetate for one minute in an ultrasonic cleaner to remove food material from the mouthparts, others were not sonicated so that the position of material on the mouthparts might be determined. The larvae were then either dissected or transferred whole to the SEM stubs after air drying. This method produces very little distortion of mouthparts and is much faster than critical point drying.

Gut contents of living larvae were placed on glass microscope slides and examined immediately with a compound microscope. Gut contents were classified as being mineral, organic detritus, diatoms, filamentous algae, unicellular green algae, animal remains, or bacteria. No attempt was made to quantify proportions other than to determine what type predominated. Periphyton species present in the field were determined by returning substrate to the laboratory, carefully removing the periphyton, and examining it under a compound microscope.

Results

Field observations

My knowledge of the life cycle of R. pellucida in the Tippecanoe River is based on extensive collections from the same site from 1983 to 1987. Diligent searching of all microhabitats during weekly intervals from April 1985 to July 1986 produced R. pellucida samples only during April and May. Most of these larvae were relatively mature and were always collected off the upper surfaces of stones or collected in kick screen samples. The larvae were also always found in mid-stream where the substrate receives direct sunlight and the periphyton community is well developed.

When R. pellucida larvae were found, the cobble substrate was covered by a diverse aufwuchs community, including diatoms such as Gomphonema, Navicula, Fragilaria, Melosira, Cocconeis, Achnanthes, and Synedra. Cladophora, which was also present throughout the period when R. pellucida larvae were found, reached a growth peak in early May at the same time R. pellucida adults emerge. It appears that R. pellucida in the Tippecanoe River is univoltine, with emergence and egg-laying in early May. Although I do not have data on the egg stage or the early instars, it is probable that the eggs hatch in winter, with mature larvae appearing on the upper surfaces of rocks in April.

Effects of experimental conditions on behavior

Rhithrogena pellucida larvae were highly stereotypic in their mouthpart movements. External factors such as noise, vibration or change in light intensity often appeared to initiate feeding movements, however, these movements were qualitatively similar to those observed under other conditions. Feeding cycles induced by external stimuli usually ceased after 1-2 cycles, whereas non-induced feeding cycles were usually repeated at least 5 times. Specimens in the holding aquaria were positively rheotactic. During the course of this study, larvae never attempted to feed on detritus or to filter feed.

Stereotypic feeding behavior

Resting position: When a larva is not feeding, the mouthparts (Figs. 19, 20, 21-25) are held in a characteristic resting position (Fig. 26). The distal segments of the labial palps are held folded inward so that they lie over (dorsal to) the paraglossae but not the glossae. The labium as a whole is held adducted dorsally to close the preoral cavity ventrally. The galealaciniae of the maxillae are positioned with their median setae touching the lingua of the hypopharynx; the galealaciniae also are in contact ventrally with the labial palps and dorsally with both the superlinguae of the hypopharynx and the mandibles. The maxillary palps are held with the basal segments perpendicular to the long axis of the body; the distal segments are folded forward at a 45o angle to the basal segments. The basal segments of the maxillary palps do not extend beyond the contour of the head capsule; the distal segments of the maxillary palps parallel the contour, extending just slightly beyond it. The apices of the distal segments of the maxillary palps do not meet but extend only to the lateral edges of the labrum. The labrum and the maxillary palps together cover the anterior and lateral aspects of the preoral cavity. The labrum is in contact with the denticles of the mandibles ventrally. The denticles of the mandibles are held separated, and presumably the molae are closed.

Feeding cycles: Larvae placed in the observation cell with diatoms grown on the glass slide performed two different behavioral cycles; I term these brushing and scraping cycles. The two cycles differ in the degree to which the maxillary palps are employed. In the Labial Brushing Cycle, the labial palps are used as brushing tools; in the Maxillary Scraping Cycle, the maxillary palps are used as scraping tools. Each cycle consists of several stages that can be delineated by specific movements of the labium. A single cycle of either type takes 1.2 seconds at 20oC. Mouthpart movements described below are often preceded by and concurrent with movement of the forelegs ahead (upstream) of the individual. Legs are alternately and repeatedly extended and then retracted, drawing the claws over the substratum. These leg movements were capable of removing tightly adhering material from the substrate and bringing it to the mouthparts.

Labial brushing cycle:

Stage 1: -- Abduction of the basal segments of the labial palps: As feeding commences, the labium begins to swing ventrally towards the substrate. The basal segments of the labial palps swing posteriorly about 20o (Fig. 27); this movement draws the distal segments of the labial palps away from contact with the paraglossae. Once the apices of the labial palps clear the paraglossae, the labium completes its ventral movement and comes to rest against the substrate. At this point the basal segments of the maxillary palps swing posteriorly about 20o. The galealaciniae do not move in Stage 1 of the initial brushing cycle in the series, but in all subsequent cycles of the series they move medially at this point in Stage 1. The labrum begins to move down slightly to shield the front of the other mouthparts.

Stage 2: -- Abduction of the distal segments of the labial palps: Once the basal segments of the labial palps reach their posterior position, the distal segments of the labial palps are abducted, moving through a range of about 50o until they are at an angle of 125o to the basal segments (Fig. 28). When the labial palps are fully extended they extend just beyond the contour of the head capsule. At this point the galealaciniae complete their medial movement and are now abducted, moving laterally outward. The distal segments of the maxillary palps are extended at the same time as the labial palps, moving through an angle of about 60o to make an angle of 105o with the basal segments. The maxillary palps reach well beyond the lateral borders of the head capsule. The labrum completes its movements ventrally and helps shield the mouthparts under the head capsule from the current.

Stage 3: -- Retraction of the labial palps: The labial palps are retracted from their extended position by a combination of an anterior swing of the basal segments of the labial palps and adduction of the distal segments of the palps. This movement is continuous and coordinated so that the labial palps always remain under the head capsule. The maxillary palps follow this movement closely; the labial palps lead the movement and remain positioned just anterior to the articulations between the segments of the maxillary palps (Fig. 29). During this movement the distal segments of the labial palps brush (sweep) up material from the substratum with the profuse setae on their ventral and anterior surfaces. The trailing maxillary palps are not in contact with the substratum but are positioned to capture any particles dislodged by the labial palps. If foreleg movement is concurrent with feeding, the claw of one foreleg is brought to the corresponding labial palp as it begins to be adducted during this stage.

When the distal segments of the labial palps reach the paraglossae they are raised dorsally by a rotation of the basal segments of the labial palps so that they pass dorsal to the glossae and paraglossae. The distal segments of the labial palps reach a position that is more median than in the resting position (Fig. 30). The entire labium is raised as soon as the distal segments of the labial palps are dorsal to the glossae and paraglossae. The galealaciniae follow the labial palps in moving medially; they also move somewhat ventrally. The denticles of the mandibles complete their lateral outward movement and the distal segments of the maxillary palps rotate and are inserted among these denticles (Fig. 31). The labrum moves dorsally.

As feeding continues, this brushing cycle is repeated in a series of these cycles. Subsequent cycles in the series are initiated by a slightly further adduction of the distal segments of the labial palps (Fig. 32), followed by the abduction of the basal segments of the labial palps as in Stage 1. The brushing cycle is repeated unchanged except that in Stage 1 of subsequent cycles the galealaciniae are moving medially and brush across the labial palps as they move outward, and the posterior movement of the basal segments of the maxillary palps draws the setae on the distal segments of the maxillary palps through the combs on the denticles of the mandibles. If leg movements accompany the brushing cycle, then the left and right forelegs alternate in moving to the mouthparts.

Maxillary scraping cycle: This cycle differs from a Labial Brushing Cycle in that the labial palps do not initiate all the movements, and the maxillary palps are brought to the substrate to scrape. Stage 1 is similar to Stage 1 of a Labial Brushing Cycle. In Stage 2, the maxillary palps are extended and lowered to the substrate; the labial palps are positioned in the articulation between the maxillary palp segments. The ventral surfaces of the distal segments of the maxillary palps are covered with unique pectinate setae (Fig. 33) which effectively remove tightly adhering material from the substrate. In Stage 3, the maxillary palps initiate the closing movements, and both sets of palps move over the substrate. The forelegs may also be active in the Maxillary Scraping Cycle.

Food processing

Once food has been brushed or scraped up by either the labial palps or the maxillary palps, respectively, it must be positioned for ingestion. This process is carried on continuously during feeding and for several seconds after the last material is procured from the substrate. Transfer is accomplished by successive fields of setae on the various mouthparts (Fig. 19) as described below. Food transport is similar for both the Labial and Maxillary Feeding Cycles.

After completion of the first cycle, and when the labial palps are extended again (Stage 1), material on the setae of the distal segments of the labial palps is removed in two ways. Outward lateral movement (Stage 1) of the distal segments of the labial palps causes material to be combed from the ventral setae of the labial palps by setae on the dorsal surfaces of the glossae and paraglossae. Most of the material on the distal segments of the labial palps is carried by the lateral and dorsal setae. This material is removed by the comb setae on the crown of the galealaciniae as the distal segments of the labial palps are adducted further and then moved laterally outward by the posterior movement of the basal segments of the labial palps (Stage 1). The comb setae of the galealaciniae are brought into contact with the distal segments of the labial palps by the slight adduction of the distal segments of the labial palps at the beginning of the outward movement and with a ventral movement of the galealaciniae. Material in the comb setae is removed by stout setae on the dorsal sides of the distal segments of the labial palps when the comb setae are drawn base-first through them as the distal segments of the labial palps are adducted medially (Stage 3). Material on the comb setae may also be removed when the galealaciniae move laterally outward (Stage 2); this draws the comb setae base-first through the setae on the dorsal sides of the glossae and paraglossae. In both types of cycles, any material on the maxillary palps is removed by combs on the mandibular denticles as the palps are withdrawn (Stage 2).

Material left on the glossae and paraglossae and on the dorsal surfaces of the distal segments of the labial palps is moved further through a number of processes. As the labial palps return to the preoral cavity (Stage 3), this material is displaced posteriorly, medially, and dorsally by the labial palps and the new load of material they bear. As the galealaciniae move medially (Stage 1 and Stage 2), their median setae push the material between the superlinguae and the lingua of the hypopharynx (Fig. 31). The lingua bears dorsal rows of setae which guide the material to a point where it can be pressed against the mandibular molae for straining and ingestion (Fig 34). Presumably, material left on the denticles of the mandibles is brushed directly onto the dorsum of the hypopharynx or the venter of the epipharynx by the movements of the mandibles; I have not been able to observe this directly since this area is obscured by the other mouthparts.

In both types of cycles the maxillary palps are used to displace larger particles from the preoral cavity and to brush material from the comb setae on the galealaciniae. Once food acquisition ceases, the mouthparts remain in motion as described above for several cycles, moving material into position to be ingested. During this phase, the labial palps make only small movements, primarily slight adductions of the distal segments, and the maxillary palps only brush against the mandibular denticles and the galealaciniae (they do not sweep the substratum).

Gut content analysis

Gut contents of four larvae collected in the field were analyzed along with those of one larva that had been videotaped. All five guts were similar in content; they contained diatoms, organic detritus and sediment. The predominate material was intact diatom frustules; there was also considerable organic detritus apparently of algal origin. Diatoms found in the gut included: Navicula, Gomphonema, Melosira, Cocconeis, Achnanthes, Fragilaria, and Rhoicosphenia curvata; Navicula species were the most common. None of the guts contained intact filamentous algae or recognizable animal remains.

Discussion

My observational procedures required the organisms to be confined inside a small cell made of synthetic materials, and it exposed them to unnatural light and water flow regimes. In my study of S. interpunctatum (McShaffrey and McCafferty 1986), I found that the experimental conditions did affect the overall behavior of that species, but did not affect the actual feeding movements reported. With S. interpunctatum, I was able to study individuals not confined to an observation cell, and I made observations using infrared light to simulate the low-light nature of the crevices in which I collected the larvae in the field (McShaffrey and McCafferty 1986). I was not able to collect enough R. pellucida larvae to permit observations outside the observation cell, but experience with S. interpunctatum, Ephemerella needhami, and various other Ephemeroptera, Trichoptera, Diptera, Odonata, Coleoptera, Amphipoda, and other freshwater macroinvertebrates (unpublished data) suggests that the cyclic movements of the mouthparts are stereotypic and vary little between conspecifics of the same age class. This conclusion is consistent with data from other insect feeding studies (Brown 1960, Chance 1970, Devitt and Smith 1985, Froehlich 1964, Pucat 1965).

My observations are similar to those made by Strenger (1953) for a European species of Rhithrogena. I was not able to confirm her suggestion that the distal segments of the labial palps deform their surfaces; this reportedly would enable their setal patches to open and close and thus increase feeding efficiency (Strenger 1953). I also was not able to document the function of the brush on the mola of the right mandible; Strenger (1953) stated that it is used to carry food from the hypopharynx to the left mola. In both cases, it is possible that Strenger (1953) did not actually observe these movements but inferred them based on her extensive morphological studies. I was able to observe some opening and closing of the setae of the distal segments of the labial palps, but can not eliminate the possibility that such movements are passive responses to drag as the palps are moved against the substrate, the water, or the other mouthparts. The molar region of the mandibles is completely obscured by the other mouthparts, and I was not able to observe this area directly. I had to estimate molar action based on the movement of the denticles, which were visible.

Because R. pellucida and S. interpunctatum are both flatheaded mayflies of the subfamily Heptageniinae, and can be found coexisting in the same general area of streams, it is interesting to compare their morphology, microhabitat, and feeding movements. Stenacron interpunctatum is found in slow-current areas, on the bottom of stones, away from the current (McShaffrey and McCafferty 1986, Flowers and Hilsenhoff 1978, Wiley and Kohler 1980, Wodsedalek 1912). I observed R. pellucida living on the tops of stones in high-current areas; Flowers and Hilsenhoff (1978) also found this species in high-current microhabitats. Strenger (1953) reported that the European species she studied were also collected on the tops of stones. The difference in microhabitat is further reflected when comparing the gut contents of these species. Whereas S. interpunctatum is primarily a detritivore (Lamp and Britt 1981, McShaffrey and McCafferty 1986), the R. pellucida studied here are primarily periphyton feeders.

The short developmental period for R. pellucida in the spring (see also Flowers and Hilsenhoff 1978, Clifford 1982) is associated with its feeding habits. Rhithrogena pellucida in the Tippecanoe River is only found in early spring before Cladophora growth is significant, while S. interpunctatum occurs year-round at the same site. There is a narrow temporal window in which there is good periphyton development on the rock substrate. In the Tippecanoe river, this period is during the later part of April to early May. The end of this period is marked by Cladophora overgrowth of the substrate; presumably the filaments of the Cladophora interfere with the feeding of R. pellucida, which requires a relatively flat substrate free of obstructions to the movements of the maxillary palps. Stenacron interpunctatum feeds on detritus, which is always available, and exhibits a complex life history with larvae present year-round (McCafferty and Huff 1978).

Morphological differences in R. pellucida correspond to its different microhabitat and are apparently adaptations allowing it to remove the tightly-bound material on the upper surfaces of rocks. The primary difference is the increased development of the maxillary palps [compare Fig. 22 with Figs. 3 and 6]. Corresponding to increased development of the maxillary palps is a flattening and broadening of the mandibular denticles [compare Fig. 24 with Figs. 3, 4, and 8] to accommodate the larger maxillary palps involved in removal of food particles. Other secondary morphological differences between R. pellucida and S. interpunctatum reflect a decrease in importance of the labial palps as food-gathering organs. There are fewer comb setae on the galealaciniae. Also, the hypopharynx, which is important in transferring material from the labium to the molae, is greatly reduced [compare Fig. 23 with Fig. 7]. Strenger (1953) noted similar differences when comparing Rhithrogena and Ecdyonurus in Europe.

The brushing cycle of S. interpunctatum is very similar to the Labial Brushing Cycle of R. pellucida, the major difference being an improved coordination of the movements of the maxillary and labial palps in R. pellucida. This improved coordination places the maxillary palps in a position closely following the labial palps to capture any material swept up by the labial palps but not captured by them. Such improved efficiency may be vital in a higher-current microenvironment. The Maxillary Scraping Cycle of R. pellucida is much more effective than either the Labial Brushing Cycle of R. pellucida or the Brushing Cycle of S. interpunctatum in removing tightly adhering material from the substrate due to the use of the large maxillary palps with their unique setae.

I did not observe R. pellucida performing cycles similar to the filtering or gathering cycles I found in S. interpunctatum (McShaffrey and McCafferty 1986). Although none of the R. pellucida in this study would feed on deposits of detritus, such feeding probably would be effective when the Labial Brushing Cycle is employed. Similarly, whereas none of the R. pellucida were observed filter-feeding, the large, well-muscled maxillary palps, with their fringe of bipectinate setae (similar to those present on the small maxillary palps of S. interpunctatum) appear adequate for filtering. At this time I cannot discount the possibility of R. pellucida filter-feeding or deposit-feeding (gathering). I had been unable to confirm a scraping function for the forelegs of S. interpunctatum (McShaffrey and McCafferty 1986); however, I was able to document such a capability for R. pellucida in the present study.

In regards to FFG classification, R. pellucida is best defined as a scraper based on the ability of the maxillary palps and legs to remove tightly adhering material from the substrate, and the presence of significant amounts of periphyton in the gut contents. It also, however, brushes, particularly when using the Labial Brushing Cycle.

The ability of an organism to use more than one mode of feeding, whether it be R. pellucida scraping and brushing, or S. interpunctatum using the same mouthparts to brush, collect-gather, or filter; or different developmental stages of a single species using different feeding methods (Cummins 1973, Cummins and Merrit 1984), complicates the problem of pigeonholing species into FFGs. This complexity is aggravated by attempting to assign FFGs based on gut contents that are often unidentifiable. My study of R. pellucida and other aquatic insects suggests that a more mechanical approach to delineating FFGs for benthic insects that feed on relatively small materials (microvores, sensu McCafferty 1981) may be appropriate. In this scheme, potential food material, regardless of origin, is viewed in terms of a continuum ranging from material suspended in the water, to material settled on the substrate, to material bound or growing attached to the substrate. Various feeding strategies are adapted to deal with various sections of this continuum.

In my scheme, benthic microvores can be divided into two basic groups, Filterers and Collectors. Filterers derive food material from the water, and the filter may consist of either parts of the body or manufactured devices such as silk nets. Passive filterers rely on seston already moving to their filtering apparatus, whereas active filterers generally resuspend deposits to filter. Thus, depending on the immediate source of the food material, I classify benthic filterers as either seston filterers, or deposit filterers. Collectors remove deposits or attached material from a substrate by direct contact of the feeding structures with the food material. Collectors can be divided into three groups: brushers, gatherers, and scrapers. Brushers use setae to obtain loose or lightly attached material and are often morphologically, functionally and behaviorally close to deposit filterers. Gatherers feed on similar materials but primarily use structures other than setae for food-gathering. Scrapers have adaptations that allow them to feed on tightly accreted material.

To summarize, benthic filterers and collectors exhibit a range of feeding strategies for feeding on a spectrum of small particles ranging from suspended materials (seston filterers) to deposits of various integrity (deposit filterers, brushers, gatherers) to firmly attached materials (scrapers). As is the case for R. pellucida and S. interpunctatum, species may not be limited to one strategy. This scheme is attractive because habitats can be characterized hydraulically, and the FFG composition of the microvore community can be estimated based on the physical state in which the hydraulic forces will place the food material. In this context, concepts of community development can be based on the relative amounts of microhabitat available to provide food material in different positions in the environment and with different propensities for attachment. One drawback of such a classification system is that it provides little information about the origin of the material (i.e. primary production, detritus, etc.); however, under natural conditions many benthic macroinvertebrate microvores feed on a variety of materials and only a relative few species are dependent on a single trophic level for their food resources. As indicated above, the system excludes macrovores such as shredders, miners, engulfers, and many predators.

It remains premature to erect a new FFG classification system, however, such a system may become necessary as the behavior of more species of aquatic insects is studied in detail. For instance, Dahl et al. (1988) recently presented a classification scheme of FFGs for the Culicidae that also further divided the FFGs of Cummins and Klug (1979) into more discreet groupings based on the mechanism of food acquisition. It is notable that that Dahl et al. (1988) chose to use the term brushers to describe a feeding system found in certain Culicidae. Those authors were evidently unaware of the brusher concept of McShaffrey and McCafferty (1986); from their discussion it is not clear whether their mosquito "brushers" feed by direct contact of the mouthparts with deposits (as is the case with S. interpunctatum), or if the mouthparts create a current which carries the material to the mouthparts (deposit filtering, herein), or both. This study of R. pellucida shows the utility of my approach in investigating questions about the functional morphology of feeding and adaptations to differing environments. Comparisons between this study and the study of S. interpunctatum illustrate how these techniques, when applied to coexisting species, increase understanding of problems related to resource partitioning.

Acknowledgments

Arwin Provonsha provided Figures 19 and 20; Annn Delleur provided Figure 16. Dan Bloodgood assisted with the construction of the observation flow cell. The scanning electron microscope was made available by the Electron Microscopy Center in Agriculture at Purdue University with support from NSF grant PCM-8400133.

Purdue University Agricultural Experiment Station Journal No. 11,359.

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CHAPTER V - COMPARATIVE FUNCTIONAL MORPHOLOGY OF STENACRON INTERPUNCTATUM AND RHITHROGENA PELLUCIDA, WITH COMPARISONS TO OTHER HEPTAGENIIDAE

Morphological examinations are often used to make estimations about an organism's ecological role. In most cases, these estimations are merely extrapolations from a species whose morphology and behavior are well-known, to a morphologically similar species whose natural history is lesser known. If valid, these extrapolations are valuable tools in many areas such as systematics, biomechanics, ethology, etc. However, if the behavior differs, or if the morphology is not well known, misunderstood, or not studied at the correct level of detail, serious errors may be made.

The study of aquatic insects has included numerous such errors, especially in hydrodynamics and feeding ecology. These two areas are interrelated; flow around organisms influences both the distribution of food and the mode of feeding. Hydrodynamics are often misunderstood because many flow characteristics are counter-intuitive; Nielsen (1950) lists several examples. More recently, Smith and Dartnall (1980) and McShaffrey and McCafferty (1987) suggested that the shape of water-penny beetle larvae is not related to attachment by suction as has often been proposed, and Statzner and Holm (1982) pointed out that flattening in heptageniid mayflies cannot be explained by a simple model of current avoidance.

When considering feeding morphology, the problems often arise from improper comparisons. Strenger (1973, 1975a) gave examples of this, stating that the long mandibular tusks of some mayflies are not indicators of carnivory but are related to burrowing. Even this revised assessment lacked strong supporting data until the detailed analysis by Keltner and McCafferty (1986) allowed for a precise evaluation of the functional morphology of burrowing in two species. Other problems may arise in determining functional morphology when a study does not examine all the relevant structures, for example, a study of burrowing in mayflies would be incomplete if the mouthparts were not included. Further errors may occur if the study is not done in enough detail, since even the smallest structures can affect overall function.

Behavior and functional morphology of feeding in aquatic invertebrates are largely unexplored at the ultrastructural level, although there has been significant work in a few groups such as Crustacea (Miller 1961, Farmer 1974, Kunze and Anderson 1979, Crittenden 1981, Kropp 1981,1986, Vogel 1984) and Diptera (Surtees 1959, Pucat 1965, Chance 1970, Craig and Chance 1982, Nubel 1984, Chance and Craig 1986, Hart 1986, Dahl et al. 1988). Although a few studies of other aquatic insect groups are scattered throughout the literature, the majority of aquatic insect species have not been examined closely in terms of functional morphology or food acquisition behavior. Many of the species of aquatic insects that have been studied in detail feed by filtering; these include Trichoptera (Wallace 1975, Wallace and Sherberger 1975, Wallace et al. 1977), Diptera (Craig and Chance 1982, Chance and Craig 1986, Dahl et al. 1988) and Ephemeroptera (Braimah 1987a, 1987b Wallace and O'Hop 1979). The study of these insects has contributed to a growing body of knowledge about suspension feeding in aquatic organisms (LaBarbera 1984, Nilsson 1984, Rubenstein and Koehl 1977). Feeding functional morphology in mayflies has been studied in detail for only a few species: Baetis rhodani and Cloeon dipterum (Brown 1960, 1961), Arthroplea cogener (Froehlich 1964, Soldan 1979), Siphlonurus aestivalis (Schonmann 1975, 1981), Lepeorus goyi (Schonmann 1981), Ecdyonurus sp. and Rhithrogena sp. (Strenger 1953), Palingenia longicauda (Strenger 1970), Ephemera danica (Strenger 1975b), Proboscidiplocia skoria (Strenger 1977) Isonychia campestris (Braimah 1987a, 1987b), Isonychia sp. (Wallace and O'Hop 1979) Ametropus neavei (Soluk and Craig 1988) Stenacron interpunctatum (McShaffrey and McCafferty 1986) and Rhithrogena pellucida (McShaffrey and McCafferty 1988). With the exception of the last four species, all of these studies relied on light microscopy for both morphological examinations and behavioral observations. The need for close examination of ultrastructural features was demonstrated by Wallace and O'Hop (1979); McShaffrey and McCafferty (1986, 1988), Dahl et al. (1988) and Soluk and Craig (1988) showed the importance of using videomacroscopic techniques for observation of feeding behavior.

Detailed analysis of functional morphology was confined to the mouthparts, but some general statements can be made regarding the form of the body in heptageniids. The body is strongly dorsoventrally flattened, a general characteristic of heptageniids. The flattening allows the larvae to access crevices in the substrate, a common microhabitat of some heptageniids (Lamp and Britt 1981, McShaffrey and McCafferty 1986). A flattened shape will also have a different interaction with the surrounding water (Smith and Dartnall 1980, Statzner and Holm 1982, McShaffrey and McCafferty 1987), but the hydrodynamic significance of flattening to organisms like Stenacron interpunctatum, which is usually found under rocks away from the current (Wodsedalek 1912, McShaffrey and McCafferty 1986), is not clear.

The shape of the head capsule has direct impact on the morphology and function of the mouthparts in heptageniids. Like the rest of the body, it is strongly dorsoventrally flattened (Fig. 35), and the mouthparts are carried prognathously, lying at only a slight inclination to the substrate. The head is convex dorsally; the two compound eyes, themselves convex, project upward from the curve of the head capsule. The eyes are thus oriented dorsally. The mouthparts are located on the underside of the head capsule in a space formed by the concave inner wall of the head capsule. The small antennae are located dorsally between the compound eyes and the anterior border of the head capsule.

The orientation and shape of the head capsule in heptageniids has several important functional implications. Because of the flattening and prognathous orientation of the mouthparts (Figs. 4, 20, 35), only the labium is in direct contact with the substrate; thus, food gathering is largely limited to the labium. The head because of its shape and position, shields the mouthparts so that water flow does not reach the area of active feeding. Since the eyes face upward, they do not play a direct role in food acquisition, but may be important in locating patches of food and/or avoiding predators.

The Heptageniidae is believed to have evolved from stream-dwelling, seston filtering forms that were minnow-like and held their bodies above the substrate to utilize filtering forelegs for food acquisition (McCafferty 1988). It is likely that the flat shape of the head is the result of selective forces acting on the overall body shape of heptageniids, resulting in dorsal-ventral flattening associated with exploiting new food sources deposited or growing on the substrate.

Stenacron interpunctatum and Rhithrogena pellucida use their flattened body shapes to utilize quite different food sources. A flattened body gives S. interpunctatum access to crevices where it feeds on detritus (Wodsedalek 1912, Lamp and Britt 1981, McShaffrey and McCafferty 1986). Stenacron interpunctatum feeds on detritus using its labial and maxillary palps in three different feeding cycles (McShaffrey and McCafferty 1986); in two of these cycles it feeds directly on detrital deposits or detritus loosely attached to the substrate, and in the third it filters detritus suspended in the water. Rhithrogena pellucida feeds on periphyton on the upper surfaces of stones exposed to current (McShaffrey and McCafferty 1988). The flat heptageniid head capsule deflects the current away from the area of feeding, thus prevented dislodged food from being swept away (Strenger 1953). Rhithrogena pellucida shows little behavioral flexibility in its feeding; it is only able to feed on deposited material (McShaffrey and McCafferty 1988); on these deposits the highly modified mouthparts of R. pellucida are capable of removing material that is tightly bound to the substrate McShaffrey and McCafferty 1988).

The earlier studies of S. interpunctatum and R. pellucida revealed many morphological differences apparently related to the different microhabitat and feeding ecology of these two species. Although some functional morphology was introduced in those studies, a complete assessment of the functional morphology of the mouthparts of these two species has not been published. The following account is a comprehensive description of the morphology of the mouthparts of S. interpunctatum and R. pellucida, with assignment of function based on behavioral observations presented wherever possible. These complete descriptions also allow some hypotheses concerning trends in heptageniid feeding ecology and associated mouthpart morphology to be formulated and presented.

Methods

Observational methods have already been detailed in McShaffrey and McCafferty (1986, 1988). Briefly, larvae were observed using videomacroscopy; this allows images up to 80x to be displayed on a 23 cm video monitor. The larvae were observed in a variety of observation cells, aquaria, and artificial streams designed to mimic natural conditions with respect to light, current, and orientation. Morphological examinations were conducted with both light and scanning electron microscopy (SEM). For light microscopy, mouthparts were dissected from the head capsule and mounted on glass microscope slides in euparol mounting medium.

Specimens for SEM were preserved first in 70% ethanol, then dehydrated by increasing ethanol concentration to 100% in 10% steps. In the basic treatment, specimens are transferred to 100% ethyl acetate, air dried, and mounted on stubs with double-side tape. Near the end of the study, a modified treatment was utilized, and specimens were transferred from the 100% ethanol to a 50% ethanol, 50% xylene mixture and left overnight; these specimens were then transferred to 100% ethyl acetate. While in the ethyl acetate, some specimens from both treatments were cleaned by sonication for 30 seconds, other specimens were left uncleaned so that food deposition on the various mouthparts could be observed. Specimens were coated with gold-palladium and examined with a JSM-840 scanning electron microscope. These methods have the advantages of being more rapid than critical point drying, and produce few artifacts. Only the dorsal side of the labial palps collapses when these methods are used. The treatment with xylene allows for easier dissection and better preparation of soft or delicate structures such as the hypopharynx or mandibles.

A total of 28 mature S. interpunctatum larvae and 11 mature R. pellucida larvae were observed by SEM; these observations were then checked against slide-mounted material obtained from 12 S. interpunctatum and 5 R. pellucida larvae. Maturity was based on wingpad development (McCafferty and Huff 1979), and only penultimate and ultimate larvae were used for morphological analysis. The descriptions below are based on 165 SEM micrographs of S. interpunctatum and 129 SEM micrographs of R. pellucida. The sizes given for the various structures are not absolute because of the great expense involved in preparing enough material from enough different larvae. The purpose of providing measurements here is simply to facilitate comparisons between mouthparts. In cases where the structures are uniform in size, a single measurement is given as representative. If the structures vary greatly in size, a range is given between the lowest and highest representative numbers.

Results and Discussion

Glossary of setae

The functional morphology of the mouthparts of mayflies is determined to a large extent by the functional morphology of the setae found on the mouthparts. It is therefore necessary to describe the setae themselves before discussing the mouthparts on which they are found.

There are many schemes for classifying setae. The proposal of Massoud and Ellis (1977) is complete in that it describes most of the setal types found in heptageniids, but it is written in French and thus not descriptive for English-speaking workers. The classification system used here is loosely based on that of Farmer (1974). In the scheme presented here, there are 7 basic setal types: simple, pilose, pinnate, pectinate, bipectinate, furcate, and palmate; each basic type may have variations.

Simple setae: surface of seta smooth, without setules; end whole, no divisions.

Simple-stout: blunt apically; small length-to-width ratios.

Simple-hairlike: (Fig. 36) pointed apically; large length- to-width ratio.

Simple-triangular: (Fig. 37) relatively short, triangular.

Pilose setae: (Fig. 38) hairlike seta bearing numerous small setules arising randomly from the surface.

Pinnate setae: seta bearing two rows of setules arising on opposite sides of the setal shaft.

Pinnate-hairlike: (Figs. 39, 40, 41) hairlike seta bearing two rows of short, laterally directed, pointed setules.

Pinnate-plumose: (Figs. 42, 43) hairlike seta bearing two rows of long, slender setules.

Pinnate-lanceolate: (Fig. 44) flattened, blade-like seta bearing two rows of short, pointed setules laterally.

Pectinate setae: seta bearing one row of setules.

Pectinate-stout: (Fig. 33) stout seta bearing a row of stout, pointed, laterally-directed setules laterally.

Pectinate-hairlike: (Fig. 45) hairlike seta bearing a row of long, slender setules.

Bipectinate setae: seta bearing two parallel rows of setules arising on one surface of the seta.

Bipectinate-stout: (Fig. 46) stout seta with two rows of pointed setules. Bipectinate-hairlike Type I: (Fig. 47) short, hairlike seta with apical hook; two rows of small tubercles on inside of hook. Bipectinate-hairlike Type II: (Fig. 47) hairlike seta with apical hook; two rows of tubercles and/or small peglike setules on inside of hook.

Bipectinate-hairlike Type III: (Fig. 48) hairlike seta with apical hook composed of fused setules, inside surface of hook and contiguous setal surface with two rows of peg-like setules at least as long as width of seta. Bipectinate hairlike setae of Types I,II, and III often grade into one another (Fig. 47).

Furcate setae: seta divided apically one or more times.

Furcate-stout: (Fig. 49) stout, peglike seta with one or more short clefts apically.

Furcate-hairlike: hairlike seta with one (bifurcate, Fig. 50) or more (polyfurcate, not pictured) apical divisions.

Palmate setae: (Fig. 52). Stout, flattened seta with apex divided into long, stout, pointed setules. These setae are similar to pectinate-stout setae; more work is needed on their development.

Morphological and functional descriptions: Stenacron interpunctatum

Labrum:

Description: -- The labrum's width is 1/2 the width of the head capsule, and it is 1/3 as long as wide. The shape is shown in Figs. 9 and 53; it is slightly emarginate anteriorly, tapers to sharp points laterally, and basally grades into clypeus dorsally and epipharynx ventrally. The anterior margin protrudes slightly from under the anterior margin of the head capsule. The dorsal surface is covered with scattered simple-hairlike setae, and resembles the dorsal surface of head capsule in texture. The anterior margin consists of an elliptical area (Fig. 53), with short simple-hairlike setae and simple-triangular setae, fringed by very long (>100 um) simple-hairlike setae; the lateral areas bear simple-hairlike setae which continue to lateral labrum edge. The ventral surface is covered with mat of simple-hairlike setae directed medially and posteriorly; it grades basally into epipharynx, which is similarly covered.

Function: -- The functional role of the labrum was difficult to determine. In orthognathous mayflies the labrum functions as the anterior boundary of the preoral cavity this role could be served by the head capsule in heptageniids. The labrum is extended during feeding and may play a role in deflecting current, but these movements may also be a passive result of the movements of the epipharynx during swallowing. The function of the dorsal setae is not clear either. Keltner and McCafferty (1986) state that simple hairlike setae in burrowing mayflies may play important roles in mechanoreception and protection of delicate surfaces. It is likely that the simple-hairlike setae on the labrum, including those on the anterior margin, are used primarily as mechanoreceptors. The simple-triangular setae bear a resemblance to proposed chemoreceptors in other aquatic species (Zacharuk 1980) and may serve a similar function here. The simple-hairlike setae on the anterior edge, as well as those on the ventral surface, probably also serve to retain food material in the mouth cavity. The simple-hairlike setae on the anterior edge are directed medially; this would enhance a food-retaining capability. Although these setae bear no obvious structures for food particle capture, there is no doubt that they can serve in this capacity (Fig. 54). Food particle retention may be increased by some sort of mucous secretion (Ross and Craig 1980), but this was not observed in S. interpunctatum. Still, as shown by Rubenstein and Koehl (1977), there are a number of ways in which small particles may be filtered out, even by single strands.

Mandibles:

Description: -- The mandibles in S. interpunctatum are asymmetrical (Figs. 8, 55, 56). Their dorsal surfaces lie along the curving head capsule wall, thus the mandibles themselves are strongly curved. The mandibles are roughly triangular, with the lateral articulations at one corner, the molar region at another, and the apical projections at the third. The lateral articulations and the molar regions are co-planar; because of the curvature of the mandibles the apical projections lie up to 100 um ventral to that plane.

The musculature, articulations, and movements of the mandibles are complex. The movements are difficult to observe because they are obscured by the other mouthparts. The musculature and articulations of heptageniid mandibles are described in detail by Strenger (1953). The most important consideration in analyzing the movements of the mandibles of mayflies is their unique tricondylic articulation; one of these articulations is on the apex of the lateral extension, and one is on the medial surface at the midpoint of the lateral extension. The third articulation is a strengthened area in the middle of the mandible on the dorsal surface. This area fits into an area on the head capsule and serves to limit the lateral movements of the mandibles.

The left mandible (Fig. 56) is about 500 um long at the median edge and extends about 700 um from the median margin at the mola to the lateral apex. The anterior apex consists of three apical projections (Fig. 57). The most apical is about 120 um long, 30 um wide, and terminates in three blunt teeth; its ventral surface bears a row of 13 sharp spines up to 20 um long (Fig. 57) and 1-5 um apart. The middle projection is slightly shorter than the apical one; it is bowed ventrally and ends in three blunt teeth. The ventral surface of the median projection bears a row of 15 sharp spines up to 25 um long and 1-5 um apart (Fig. 57). The basal projection is a single long spine 85 um long. It is also bowed ventrally and bears several small spurs.

Two rows of simple-hairlike setae 40 um long run along the medial edge to the mola, a distance of about 150 um. The molar region (Fig. 58) consists of several areas. Most of the surface is part of the ventral surface of the mandible, additional surface is provided by ridges produced from the medial margin. Several crescent-shaped rows of long furcate setae delimit the interior boundaries of the molar region. These setae are up to 30 um long and are irregularly furcate (Fig. 59). Towards the base a number of stout simple setae up to 10 um long replace the furcate setae. Inside the crescent much of the surface is smooth with a few stout simple setae. Near the base of the ridges there is a dense patch of short furcate setae oriented toward the ridges (Figs. 58, 59). There are 13 ridges ranging from 10 to 90 um in length. The ridges are fringed with small, blunt lobes up to 5 um long (Fig. 58). On the basal portion of the ridges these lobes overlap and are directed ventrally; towards the distal ends they separate and are directed laterally; on the apices they are directed apically. Outside the molar region the ventral surface bears numerous small (1 um) spines directed toward the molar area (Fig. 60). The lateral margin of the left mandible bears a row of simple-hairlike setae up to 100 um long.

The right mandible (Fig. 55) bears only two apical projections (Fig. 61). The distal apical projection is about 120 um long with three blunt apical teeth, 4 lateral (dorsal) blunt teeth, and a row of 15 sharp spines up to 20 um long on the ventral surface. The basal projection is the same length, with a sharp apical tooth, a smooth dorsal surface, and a row of 13 sharp spines up to 25 um long on the ventral surface.

A row of long pectinate setae 40 um long runs 100 um along the medial margin to the molar region. The molar region on the right mandible is on the medial surface, which widens to provide a working surface. At the distal end of the molar region there is a dense row of relatively stout simple-hairlike setae up to 45 um long, curving basally over the molar surface. This row continues for 100 um basally along the dorsal margin of the molar region until it reaches the ridges making up much of the molar surface. On the ventral margin this row continues as a row of more slender simple-hairlike setae up to 30 um long, interspersed with shorter (10-15 um) long furcate setae similar to those of the left mandible. The molar surface proper is composed of two distinct surfaces. The distal portion is covered by dense irregularly furcate, short (3 um) setae with bulbous bases; these are interspersed with scattered stout furcate setae up to 7 um long and 1 um wide (Fig. 62). The basal portion is about 100 um long and is composed of 10 transverse ridges. The ridges are fused ventrally, but are free dorsally where they form a series of sharp edges (Fig. 63). The longest ridge stretches across 70 um (the width of the molar surface); the ridges are about 6 um wide (Fig. 64). The ridges bear blunt lobes about 5 um long and 3 um wide along one margin, the other margin is smooth. As in the left mandible, the lobes are crowded ventrally and separated dorsally. Several rows of setae up to 30 um long line the ventral surface of the basal portion of the mola (Fig. 64); these setae are irregularly furcate distally. A row of about 7 simple-stout setae 20 um long lies ventral to the furcate setae (Fig. 64). Much of the ventral surface of the mandible in the region surrounding the mola is covered with small spines similar to those on the left mandible. There are 4-6 pinnate-plumose setae up to 100 um long at the base of the mola, and a row of simple-hairlike setae each 100 um long along the entire lateral margin.

Function: -- I use the term "apical projections" as a name for the medial toothed projections of mayfly mandibles. "Apical projections" is functionally neutral and thus preferable to the term "incisors" or "canines", which imply a cutting function. In many mayflies, including the heptageniids and many of the burrowing species, these structures do not reach the substrate and are used as combs rather than cutting tools. In other mayflies, including such hypognathous species as Ephemerella needhami, these projections are used to scrape or bite. In still others, such as the carnivorous heptageniidae, the projections are modified to impale or hold prey. Thus, there is no single function attributable to these structures throughout the mayflies, and it is preferable to use a functionally neutral term. "Apical projections" is also preferable to "denticles," another term which has been used for these structures, since denticles literally means "small teeth," which is not descriptive of the range of forms taken on by these structures in the various mayfly species.

The mandibles have two major tasks: removal of material from the maxillary palps and packing food into the mouth. The apical projections are responsible for the former, the molae for the latter.

The apical projections act as combs for the bipectinate-hairlike type III setae on the maxillary palps (Fig. 65). The primary comb structures are the sharp spines on the ventral surfaces. The material left on the apical projections is held by setae on the hypopharynx and labrum and is pressed toward the mouth by the material brought in the next load.

The molar region compacts food for swallowing. Material at the base of the hypopharynx and epipharynx is packed between the molar surfaces when they are separated. As the molae close, the material is packed together and water is removed. The asymmetry of the mandibles, combined with the rolling motion they make as they close, forces material through them in a ventral to dorsal direction. The compressed bolus is packed into the pharynx (which lies dorsal to the molae) by the loose material packed in during the next cycle. The elaborate setation of the mandibles is used to retain food material and insure that it moves toward the molae. In particular, the long simple-hairlike setae and long furcate setae on the ventral margins of the molae probably rake material from the hypopharynx as the molae close.

Maxillae:

Description: -- The maxillae in S. interpunctatum are about 1/2 the length of the head capsule (Fig. 6). The distal ends of the maxillae reach to the labrum and the basal ends are inserted 2/3 of the length of the head capsule from the anterior margin of the head capsule. The galea and the lacinia are fused into a galealacinia which is flattened dorsoventrally and somewhat rectangular when viewed from a dorsal or ventral aspect. The medial-apical corner of the galealacinia is produced into 3 spines. The cardo and the galealacinia are equal in length. The maxillary palps are long and slender, elliptical in cross-section, again reflecting the overall dorsoventral flattening. The basal segments are inserted onto small stipes and are equal in length to the galealacinia. The distal segments are about 1.3x the length of the basal segments and reach their greatest width at a point 3/4 of the distance from the base to the tip. The distal segments are pointed apically.

The movements of the maxillae are complex. The basic movement is an inward and outward lateral displacement of the galealaciniae. At their inward limit, the galealaciniae meet along the midline; at their outward limit, the distal-medial corners of the galealaciniae approach the lateral margins of the labrum. This lateral swinging movement has the basal attachment point of the cardo as its fulcrum. The galealaciniae can also be moved either dorsally towards the mandibles or ventrally towards the labium. To some extent, the maxillary palps follow the movements of the galealaciniae. Their displacement, however, is small compared to the movement of the tips of the galealaciniae; this is due to their insertion close to the fulcrum. Independent movement of the maxillary palps is both possible and complex in its own right. The basal segment can be adducted anteriorly and abducted posteriorly and can rotate dorsoventrally. The distal segment can be abducted and adducted with respect to the basal segment, and rotated to a small extent.

The maxilla has several different types of setae. The most conspicuous of the setae are the 9-12 palmate setae on the crown of the galealacinia (Fig. 66). The palmate setae are about 55 um long with 10-15 setules up to 25 um long. The setules are spaced 5 um or less at the tips. On the ventral surface of the galealaciniae there is a row of 20-30 pinnate-plumose setae (Fig. 67). This row parallels the median margin of the galealacinia except at the ends where it curves laterally. On the median margin of the galealacinia are two rows of medially pointing setae (Fig. 67). The ventral row consists of 60-90 pinnate-hairlike setae approximately 75 um in length, 2 um wide, with 2 rows of pointed setules 1-1.5 um long spaced 0.5 um apart at the bases. These setules arise dorsolaterally and make a 45o angle with the shaft (Fig. 68). Dorsal to the pinnate-hairlike setae there is a row of about 150 pectinate-hairlike setae (Fig. 45) approximately 120 um long and 2 um wide. The basal 30 or so pectinate-hairlike setae in this row are longer than the others, with the approximate length equal to 200-240 um. The pectinate-hairlike setae bear long pointed setules 6-7 um in length (Fig. 45), spaced approximately 0.3 um apart. The setae are directed medially with the setules pointing posteriorly.

There are several different types of setae on the maxillary palp. On the medial and lateral margins of the basal segment there are single rows of simple-hairlike setae approximately 150 um long. A row of 18-20 pinnate-lanceolate setae 40-80 um long runs along the midline of the ventral surface of the distal segment (Fig. 44). The distal segment also bears a sparse row of simple-hairlike setae on its medial and lateral margins; these setae are approximately 120 um long. An apodeme runs along the lateral margin of the distal segment (Fig. 69), this apodeme widens apically making the apex of the distal segment somewhat bilobed. The apical 1/3 of the distal segment bears a dense patch of bipectinate-hairlike type III setae. These setae may be over 200 um long, 1.5 um wide, and bear two rows of blunt setules on their inner surfaces (Fig. 48). The setules are approximately 1 um long, spaced 0.5 um apart, and the two rows are about 1 um apart.

Function: -- The functional aspects of the maxillae are fairly well understood. The palmate setae on the crown of the galealacinia, together with the terminal spines, remove food material from the bipectinate-hairlike III setae on the dorsolateral margins of the labial palp (Strenger 1953, McShaffrey and McCafferty 1986). The setae along the medial margin of the galealacinia are used to trap small particles and push them between the lingua and the superlinguae of the hypopharynx. Of the two types of setae on the medial margin, the ventral pinnate-hairlike setae are likely to play a primary role in pushing food particles, and the dorsal pectinate-hairlike setae are probably more important in trapping and retaining particles. The role of the submedian row of pinnate-plumose setae is not clear; they may act to filter small particles, or as mechanoreceptors.

The simple-hairlike setae on the medial and lateral margins of the basal and distal segments of the maxillary palp are most likely mechanoreceptors. The function of the row of pinnate-lanceolate setae probably is filtering. Isonychia campestris uses similar setae on the forelegs for filtering (Braimah 1987a), therefore, it is possible that they may act as primary filter elements in the filtering behavior reported by McShaffrey and McCafferty (1986). The fact that they are only on the ventral side suggests that they may interact with the labial palp or even the substrate. They may be capable of trapping small particles or removing them from other setae for further processing.

The bipectinate-hairlike type III setae on the apex of the maxillary palp are primary filtering structures. They act to brush up and trap small particles from the substrate, or as primary filter elements in flowing water (McShaffrey and McCafferty 1986). Food particles adhering to these setae are removed by the mandibular apical projections (Fig. 65).

Hypopharynx:

Description: -- The hypopharynx (Fig. 7) is about 1 mm wide measured across the greatest width of the superlinguae, and is about 0.5 mm long measured from the base to the most anterior point on the superlinguae. The ventral side of the lingua is mostly fused to the labium leaving only the tip exposed. This region tapers to a blunt apex roughly quadrate in shape with sides approximately 200 um long. An apparent apodeme approximately 250 um long runs longitudinally down the midline of the dorsal surface of the lingua, but I have not been able to observe this structure with a light microscope.

The paired superlinguae attach to the lingua basally and arch out laterally, with an average width of about 300-500 um. The apices of the superlinguae are broadly rounded. The basal 2/5 of the superlinguae overlap the dorsal side of the lingua along the medial margins of the superlinguae.

The dorsal surface of the lingua is covered with a dense patch of simple-hairlike setae (Fig. 70). These setae are 10-20 um long and 0.2 um wide (Fig. 71). A row of longer, stouter (1 um wide) simple-hairlike setae runs around the lateral borders of the lingua. Three converging rows of simple-stout setae lie on the dorsal surface of the lingua at the base of the hypopharynx, on the dorsal surface of the lingua.

There is a single row of longer simple-hairlike setae running along the medial margins of the superlinguae. Patches of simple hairlike setae similar to those on the dorsal side of the lingua stand on the dorsal surfaces of the superlinguae along the bases on the medial margins where they overlap with the lingua. The remainder of the dorsal surfaces of the superlinguae are smooth except for a band of approximately 300 apparent sensillae 2.5 um wide set in 4.5 um wide pits (Fig. 72). This band runs along the midline of the superlinguae, following the arch. The ventral surfaces of the superlinguae are smooth.

Function: -- While I have not been able to observe the actions of the hypopharynx directly, it is possible to determine the functions of the various structures based on the actions of other structures whose movements were observed. The smooth ventral surfaces of the superlinguae provide a surface across which the medial setae of the maxillae push food. The food material is pushed between the superlinguae and the lingua. The stout setae bordering these structures comb food material from the pinnate-hairlike and pinnate-plumose setae of the maxillae. The simple-hairlike setae on the dorsal surfaces of the hypopharynx point toward the converging rows of simple-stout setae at the base of the hypopharynx, and are thus oriented to help trap material and insure that its movement is towards the base of the hypopharynx. Material collecting there is pressed against the mandibular molae. The sensillae on the dorsal surface of the superlinguae may function to taste material before it is swallowed. I have observed the maxillary palps removing material from this area, possibly this was a rejection of poor-tasting or potentially toxic material.

Labium:

Description: -- The labium (Figs. 5, 73) is large and covers the other mouthparts ventrally. The labium consists of a somewhat quadrate mentum; the mentum in turn bears a pair of glossae and a pair of paraglossae apically, and a pair of labial palps laterally (Fig. 73). The mentum measures about 900 um at its greatest width and is about 400 um long at the midline. There are a few scattered simple-hairlike setae on the ventral surface of the mentum. The dorsal surface of the mentum is largely fused to the lingua of the hypopharynx.

Movement of the labium is restricted to a dorsoventral swinging of the mentum and the independent movements of the labial palps. The swinging of the mentum occurs at the beginning of feeding as the mentum is lowered to the substrate. The fulcrum for this movement is located at the base of the mentum, thus the most anterior parts of the labium - the glossae, paraglossae, and the distal segments of the labial palps - have the greatest vertical displacement. The hypopharynx is also displaced by the swinging of the mentum.

The basal segments of the labial palps are able to swing through an angle of about 10o. The muscles for this movement are located in the mentum. The swinging of the basal segments is accompanied by the expansion and contraction of the flexible cuticle at the junction of the mentum and the basal segments (Fig. 73). The fulcrum for this movement thus lies at the midpoint of the basal segments. The basal segments contain large muscles used to abduct and adduct the distal segments. These muscles are large and fan-like, with tendons running to insertion points on the distal segments. The distal segments are joined to the basal segments at two articulation points, one dorsal and the other ventral. The dicondylic joint is the fulcrum for the movement of the distal segment, which can move through a range of about 30o with respect to the basal segment.

The glossae are somewhat triangular, about 350-500 um long, with the bases about 175 um wide. The sides of the glossae curve outward laterally. The glossae are separated by a distance of 100 um at the tips; this widens to 200 um at the midpoint, and tapers to 75 um at the bases. The dorsal surfaces are covered with scattered simple-hairlike setae 10-150 um long. The ventral surfaces are smooth. Several rows of stouter simple-hairlike setae approximately 100 um long are inserted on the medioventral surfaces, and an irregular row of fine serrate setae 50-100 um long and 1-2 um wide runs across the basoventral margins of the glossae.

The paraglossae lie laterally to the glossae with their insertion on the lateral/apical corners of the mentum. An apodeme divides the paraglossa into two parts, one lateral and the other medial. The ventral surface of the paraglossa is covered by scattered simple-hairlike setae resembling those on the glossae. Anteriorly these simple-hairlike setae are replaced by several rows of bipectinate-stout setae. The outer lateral margin of the paraglossa bears several rows of pinnate-lanceolate which range in length up to 100 um.

The setation on the dorsal surface of the paraglossa is complex. There are three main patches of setae: a basal patch of pinnate-plumose setae, several rows of pinnate-hairlike setae, and an apical band of pinnate-plumose setae. The basal patch is elliptical, running from the mediobasal margin of the paraglossa to the middle of the paraglossa, and consists of pinnate-plumose setae about 70 um long with setules up to 10 um long (Fig. 74). The pinnate-plumose setae are bounded apically by 10-15 rows of pinnate-hairlike setae (Fig. 75). These setae are about 100 um long and are directed apically with the setules facing the surface of the paraglossa. Several rows of very short (10-20 um) bipectinate-hairlike type III setae occur in the basal rows of this band. A second group of pinnate-plumose setae occurs apical of the pinnate-hairlike setae. These pinnate-plumose setae form a band that grades into the bipectinate-hairlike type III setae on the lateral margin of the paraglossa. The band of pinnate-plumose setae follows the general curve of the outside margin of the paraglossa anteriorly. The band spreads out from a point at the lateral margin into 10-15 rows covering the anterior 100 um of the dorsal surface of the paraglossa, leaving a smooth patch of exoskeleton bounded anteriorly by the pinnate-plumose setae and posteriorly by the bipectinate-hairlike type III setae. The pinnate-plumose setae in the anterior band may exceed 100 um in length and are directed anteriorly; their setules are about 10 um long (Fig. 76).

The labial palp consists of two somewhat oval segments. The basal segment is about 1 mm long and 0.5 mm wide. It is inserted with a flexible linkage along the lateral margin of the mentum. The distal segment is about the same size as the basal segment.

Most of the ventral surface of the labial palp is bare, although a few scattered simple-hairlike setae may be present. The lateral/apical portion of the distal segment bears a dense patch of setae. This patch begins about 2/5 of the length of the outer margin and runs to the apex. The patch consists of a series grading from bipectinate-hairlike type I setae ventrally to bipectinate-hairlike type II setae laterally and bipectinate-hairlike type III setae dorsolaterally (Fig. 47). The apical view (Fig. 77) shows how the rows of type I and type II setae are overlain by the much longer (200 um) type III setae. The type III setae have setules 2 um long spaced 0.5 um apart.

There are several types of setae on the dorsal surface of the labial palp (Fig. 78). These setae are located on the apical 1/3 of the distal segment, and occur as sparse rows running the width of the segment, with all setae directed apically. The basal rows are pinnate-hairlike setae up to 100 um long (Fig. 79). Apically, the pinnate-hairlike setae grade into several rows of much shorter setae. Some of the short setae are smooth, others are furcate. Along the medial margin of the distal segment some of the pinnate-hairlike setae bear much longer setules and are thus better classified as pinnate-plumose setae.

Function: -- Of all the mouthparts, the labium is the most visible during feeding, thus, I have better observational data to correlate with morphological data when determining function. The simple setae on the ventral surfaces of the mentum, glossae, and paraglossae probably serve as mechanoreceptors; they have not been observed to perform any specific function associated with feeding. The bipectinate-stout setae may also be mechanoreceptors, or they may filter material loosened from the substrate as they are often heavily coated with such material. The bipectinate-hairlike type I setae on the ventral/lateral surface of the distal segment of the labial palp brush material up from the substrate. If the material is thick enough, the distal ends of the bipectinate-hairlike type II setae come into play to brush up material. Material loosened by the type I or type II setae is filtered by the bipectinate-hairlike type III setae on the lateral margin of the distal segment of the labial palp (Fig. 80).

The setae on the dorsal surfaces of the labial palps, paraglossae and glossae perform 2 roles. Some setae, such as the short smooth and furcate setae and the pinnate-hairlike setae on the dorsal surface of the labial palp, act as rakes, removing particles from other structures such as the palmate pectinate setae on the galealaciniae. The bipectinate-hairlike type III setae and the pinnate-plumose setae on the paraglossa act to filter and retain food particles until they can be moved further, in this case by the medial setae on the galealaciniae.

Morphological and functional descriptions: Rhithrogena pellucida

Labrum:

Description: -- The labrum (Figs. 21, 81) is small; it occupies the middle 1/3 of the head capsule with a width of 600 um. The dorsal surface free of the head capsule is about 200 um long. The labrum is broadly rounded anteriorly and rounded laterally. The labrum tapers ventrally into the epipharynx, which is about 250 um wide.

The dorsal surface of the labrum is covered with scattered simple-hairlike setae up to 100 um long. Towards the anterior margin there are a number of simple-triangular setae about 1.2 um long and 1 um wide at the base (Fig. 37). These setae are set in circular pits 4 um in diameter and 0.5 um deep.

The apical margin of the labrum bears three types of setae. The middle 1/3 of the anterior margin is expanded to a width of about 30 um and bears scattered simple-triangular setae like those on the dorsal surface. Lateral to this area the anterior margin narrows to a width of 10-20 um (Fig. 82). The lateral areas are covered by several rows of medially directed setae; the dorsal rows consist of polyfurcate setae up to 50 um long and 2.5 um wide, and the ventral rows consist of simple-hairlike setae of the same size (Fig. 82). The medial 2/3 of the ventral surface of the labrum, and the entire epipharynx, are densely covered with simple-hairlike setae up to 50 um long.

Function: -- The functional role of the labrum in R. pellucida is difficult to determine. It is likely that the simple-hairlike setae act as mechanoreceptors. The presence of polyfurcate setae, with their greater surface area, reinforces the supposition that at least one role of the long setae on the labrum is to retain food particles. Any role of the labrum as a current shield becomes problematic with R. pellucida, since a smaller labral size is here correlated with a higher-current microhabitat.

Mandibles:

Description: -- The mandibles are somewhat asymmetrical (Figs. 24, 83, 84), with the apical projections of the two mandibles similar, but large differences in the molar areas. The apical projections are not in the same plane as the molae and lateral articulations; the apical projections are up to 100 um ventral of the molae.

The left mandible (Fig. 84) is about 400 um long at the median margin and extends 700 um from the median margin at the mola to the most lateral point. The apical projections consist of two laterally flattened triangular plates. The distal plate (Fig. 85) is larger, Starting at the apical projections, there is a row of 12-15 pectinate-hairlike setae (Fig. 85) running towards the mola for a distance of 30 um along the medial margin. These setae are up to 70 um long, 1 um wide, with setules 10 um long spaced 1 um apart at the bases. The setae are directed medially, the setules posteriorly. The remaining 100 um between the end of the setal row and the mola is bare. There is no crescent of setae delimiting the molar region, however, the ventral margin of the mola does bear one or two irregular rows of sharp, stout, simple setae about 10 um long pointing toward the mola (Fig. 86). The mola proper is composed of 17 ridges, ranging from 30 to 90 um long and 10 um wide (Fig. 86). The basal attachment of these ridges is raised into a row of low bumps (Fig. 87). The ridges bear a row of small projections along their posterior margins, the anterior margins are smooth. The projections are up to 3 um long, and are composed of blunt finger-like structures (Fig. 88); the projections are separated by up to 1 um. The ridges overlap when viewed from a ventral perspective, but a gap up to 5 um wide is visible between the ridges when viewed from a more posterior view. The ends of the ridges are palmate (Fig. 89), with the terminal projections up to 10 um long (Fig. 90). The posterior tip of the mola is ringed by 10 um long bluntly pointed spines. There are 4-6 pinnate-plumose setae up to 100 um long with 5 um long setules curving out from under the posterior tip of the mola (Fig. 86). There are no setae along the lateral margin of the mandible.

The right mandible (Fig. 83) is about 400 um long at the medial margin and extends 600 um from the medial margin at the mola to the most lateral point. The apical projections (Fig. 91) are similar to those of the left mandible.

The row of pinnate-plumose setae is similar to that on the left mandible, the distance between the end of that row and the mola is about 70 um. The anterior end of the mola is marked by a patch of about 15 pointed setae up to 30 um long (Fig. 92). The ventral surface of the mola is bounded by several parallel rows of tubercles (Fig. 93). There may be a few pointed simple-stout setae outside of the rows of tubercles. The tubercles are low and rounded anteriorly, becoming up to 10 um long and bluntly pointed posteriorly. The inner rows of tubercles are smaller than the outer ones. The molar surface is composed of 19 ridges which are up to 50 um long when viewed ventrally. The ventral margins of the ridges fuse into the ventral surfaces; the medial margins are free and bluntly rounded. The ridges bear small, blunt finger-like projections grouped in clusters about 3 um long on their anterior margins; the posterior margins are smooth. A gap up to 5 um wide separates the ridges; this gap narrows ventrally. There are 4-6 fine serrate setae up to 100 um long at the posterior end of the mandible. Both of the mandibles of R. pellucida bear numerous apparent sensillae on their ventral surfaces (Fig. 94). There are no similar structures on the hypopharynx of R. pellucida.

Function: -- The mandibles of R. pellucida function in the same way as those of S. interpunctatum. The apical projections remove food material from the maxillary palps, and the molae strain and grind the food.

Maxillae:

Description: -- The galealacinia is about 400 um long and 300 um wide (Figs. 22, 95). The maxilla is not as quadrate as in S. interpunctatum, the distal end sloping laterally. The galealacinia bears 3 spines at the medial/apical corner. The cardo is the same length as the galealacinia, but is only 1/2 as wide. The maxillary palp is highly modified compared to that of S. interpunctatum. The basal segment is somewhat triangular, measuring about 700 um long and 500 um wide (Fig. 95). The medial margin of the basal segment is grooved to receive the distal segment is fully adducted. The distal segment is somewhat elliptical, about 1200 um long and 300 um wide at its widest point (Fig. 95). The distal segment comes to a point apically.

The movements of the maxilla in R. pellucida are similar to those in S. interpunctatum. The main difference is in the increased size of the maxillary palp. The distal segment is increased in size to augment its food-gathering capability, and the basal segment is enlarged in order to support and move the larger distal segment.

For the most part, the setation of the maxilla in R. pellucida is similar to that of S. interpunctatum. There are 7-9 palmate pectinate setae on the crown of the galealacinia. The palmate pectinate setae are about 60 um long and bear 7-10 setules ranging from 10 to 50 um in length. The separation between the setules varies from 0 um at the bases to 10 um at the tips. The ventral submedian row of pinnate-plumose setae contains about 30 setae, each about 50 um long with setules up to 10 um long. The ventral median row of pinnate-hairlike setae consists of about 60 setae each 40-50 um long and about 2 um wide with the pointed setules reaching lengths up to 2 um, and spaced about 0.3 um apart. There are about 80 dorsal pectinate-hairlike setae; each is about 100 um long and 1.5 um wide. The setules on the pectinate-hairlike setae are up to 4 um long and are about 0.3 um apart at their bases. There are 4-6 longer setae basal to the other median setae on the galealacinia. These are fine serrate setae about 150 um long with setules up to 5 um long.

The setation on the maxillary palp of R. pellucida is much different than on S. interpunctatum. There are no pinnate-lanceolate setae on the ventral surface of the distal segment, but there is a row of 18 or more simple-hairlike setae present. Some of these simple-hairlike setae have tips with incipient setules; presumably these setae are homologous with those of S. interpunctatum. Much of the ventral surface of the distal maxillary palp segment is covered by an elliptical patch about 750 um long and 200 um wide. This patch is composed of 10 rows of apically directed pectinate-stout setae (Fig. 96). These pectinate-stout setae are about 30-40 um long, 5 um wide at the base, and bear up to 12 (typically 7) setules (Fig. 33). The setules arise in a straight row perpendicular to the setal shaft and are up to 25 um long, 2.5 um wide at the base, and about 0.3 um apart at the bases. The medial 7 rows have fully developed setules, the lateral rows have many setae with the setules absent or poorly developed. There is some evidence that the pectinate-stout setae may be hollow. The lateral margin of the elliptical patch is fringed by 6-8 rows of bipectinate-hairlike type III setae. These setae are up to 60 um long, about 1.5 um wide, with two parallel rows of setules. The setules are about 2 um long; those on the basal portion of the seta are spaced 1 um apart, but they are more dense apically with spacing about 0.3 um apart near the apex.

Function: -- The setae on the galealacinia of R. pellucida indubitably perform the same tasks as their counterparts in S. interpunctatum. The pectinate-stout setae on the maxillary palp are used to brush material up from the substrate. The bipectinate-hairlike type III setae filter out material dislodged by the pectinate-stout setae.

Hypopharynx:

Description: -- The hypopharynx (Figs. 23, 97, 98), is about 500 um across the superlinguae and about 500 um long, with about 200 um of the ventral side exposed between the glossae of the labium. The apex of the lingua is quadrate with a width of 200 um and a thickness of about 100 um. The dorsal apodeme is about 100 um long. The superlinguae are about 150 um wide and 400 um long. The superlinguae curve medially; they are roughly quadrate in shape and rounded apically.

Simple-hairlike setae cover the terminal 30 um of the ventral surface of the lingua as well as the apical surface and the entire dorsal surface. These setae are up to 40 um long and 0.2 um wide. There are shorter (5-15 um), stouter (1 um), furcate setae on the lateral surfaces of the lingua (Fig. 49). There are 3 converging rows of simple-stout setae at the base of the lingua (Fig. 97).

The superlinguae bear patches of simple-hairlike setae on their medial and dorsal borders. There are also simple-hairlike setae on the ventral/apical surfaces of the superlinguae (Fig. 99). The superlinguae bear rows of simple-hairlike setae up to 5 um long (Fig. 100) on the dorsal surfaces of the superlinguae. There are no apparent sensillae similar to those of S. interpunctatum on the hypopharynx of R. pellucida.

Function: -- The functional aspects of the hypopharynx in R. pellucida are similar to those of S. interpunctatum.

Labium:

Description: -- The labium of R. pellucida (Figs. 21, 101) is smaller than that of S. interpunctatum. The mentum is quadrate, about 600 um wide and 250 um long; there are a very few widely scattered simple-hairlike setae on the ventral surface. The dorsal surface of the mentum is again the point of attachment for the hypopharynx.

The movement of the labium is similar to that of S. interpunctatum, but the palps are more flexible. The basal segments can move through an angle of 20o with respect to the mentum, and the distal segments through an angle of 50o with respect to the basal segments.

The glossae are elongated, about 200 um long and 100 um wide at the widest point (Fig. 101). They taper to a blunt point apically, with the greatest width about 50 um from the base. The glossae are about 200 um apart at the tips and 100 um apart at the bases. The ventral surface bears widely scattered simple-hairlike setae, usually about 4 um long and 0.6 um wide. The medial margin bears several rows of pinnate-plumose setae up to 75 um long, 1.5 um wide, with setules up to 10 um in length. The ventral surface is bare.

The paraglossae are quadrate with rounded margins (Fig. 101), about 200 um long and wide. There is no evidence of an apodeme corresponding to that found in S. interpunctatum. The basal, medial, and lateral margins of the ventral surface of the paraglossa are covered by widely scattered simple-hairlike setae similar to those on the glossa. There is a triangular patch of bipectinate-stout setae on the ventral surface of the paraglossa; the base of this patch is the anterior margin and the apex is in the middle of the paraglossa. The bipectinate-stout setae are up to 40 um long; 25 um is typical. They are about 7 um wide and bear 2 rows of setules 5-10 um long on one surface (Fig. 46). There are no pinnate-lanceolate setae on the outer lateral margin of the paraglossa.

There are several types of setae on the dorsal surface of the paraglossa. Basally, there is a row of 8-12 pilose setae (Fig. 38) 45 um long, 3 um wide, with setules up to 3 um long. The lateral/basal surface is covered by scattered simple-hairlike setae and fine serrate setae up to 70 um long and 1-2 um wide. The medial margin and the apical 1/3 of the ventral surface is covered by a dense patch of pinnate-hairlike setae (Fig. 102). These setae are about 100 um long, 1 um wide, with setules 1 um long. The setae are directed apically and often curve medially. The apical margin of the paraglossa bears several very dense rows of pinnate-plumose setae 75-100 um long and 2 um wide. These setae bear 2 rows of setules up to 10 um long; the rows of setules are located 180o from each other (Fig. 43).

The labial palp in R. pellucida is similar to that of S. interpunctatum. The basal segment is about 500 um long and 250 um wide; the distal segment is about 550 um long and 200 um wide. The ventral surfaces of the basal segment and much of the distal segment are bare, with a very few scattered simple-hairlike setae to break the monotony.

The distal segment has an irregular row of 3-5 simple-stout-hairlike setae on the ventral surface. More prominent are the apical/lateral setae. These are arranged as in S. interpunctatum, with bipectinate-hairlike type I setae ventrally grading into type II and type III dorsally. The type I setae are about 30 um long; the type III are up to 150 um long, with 2 um long setules about 0.5-1 um apart (Fig. 103).

The dorsal surface of the distal segment of the labial palp bears several different types of setae in the apical 1/3 (Fig. 104). There is a clump of about 15 fine serrate setae about 70 um long, 3 um wide, with 1 um long setules, located on the dorsal/medial margin of the distal segment. There is a large patch of setae arranged in regular rows covering the remainder of the distal 1/3 of the segment. This patch consists of 6-10 rows of pinnate-plumose setae basally and 1-2 rows of long bifurcate setae apically (Fig. 105). The pinnate-plumose setae are 20-60 um long, 2 um wide, with the setules often as long as the seta (Fig. 51). The long bifurcate setae are 20-60 um long, with the bifurcation splitting the distal 1/3 of the seta into 2 apical setules separated by a space up to 3 um wide (Fig. 50).

Function: -- The setae on the labium of R. pellucida perform the same roles as their counterparts in S. interpunctatum. With R. pellucida, I have some evidence that the bipectinate-stout setae on the ventral surface of the paraglossae may gouge the substrate as they are often worn (Fig. 106). These setae also retain food material (Fig. 107), but the significance of either of these roles is unclear since no mechanism for transferring this material to the inner mouthparts has been observed.

Comparative functional morphology of Stenacron interpunctatum and Rhithrogena Pellucida

The body of R. pellucida is similar in many respects to S. interpunctatum. Both are strongly dorsoventrally flattened, but R. pellucida shows some unique structures which are apparently adaptations to a higher-current microhabitat. Most striking are the gills of the abdomen which form a ventral structure that appears to help the larvae remain attached to the substrate, perhaps by increasing friction.

The head capsule in R. pellucida is similar to S. interpunctatum. It is about the same width (2 mm), but is much shorter (1.2 mm) and more elliptical in shape. The labium is about 1/2 the size of the labium in S. interpunctatum and thus does not cover the other mouthparts or the posterior/ventral regions of the head capsule to the same degree. The smaller labium also does not prevent the maxillary palps from reaching the substrate (only the tips of the maxillary palps in S. interpunctatum can reach the substrate), and the mouthparts are more exposed to the current anteriorly when compared to S. interpunctatum. This does not necessarily mean that the current reaches the inner mouthparts, however, as it is likely that an area of stagnation would exist directly upstream from the head capsule. The stagnation and the resulting vortices may enhance feeding; Soluk and Craig (1988) have shown how one mayfly utilizes the horseshoe vortex upstream of the head capsule for feeding; but the results of Statzner and Holm (1972) do not suggest the formation of a horseshoe vortex upstream of heptageniids. Experimental data are needed before definitive statements can be made about the flow around the head capsules of heptageniids.

The mandibles of R. pellucida function in the same fashion as those of S. interpunctatum, but there are two main morphological differences: the modification of the apical projections into large, flat plates, and the reduction of setae in the molar region. The apical projections of R. pellucida are enlarged and flattened to receive the larger maxillary palps and remove material from them. The spines on the ventral sides of the plates are positioned to comb through the bipectinate-hairlike type III setae on the maxillary palps. The reduction of setae around the mola in R. pellucida may be related to a reduction of material taken up by the labium and/or an increase in particle size of acquired material.

Presumably the sensillae on the mandibles of R. pellucida perform the same function as those on the hypopharynx of S. interpunctatum. In both species these structures are oriented towards the same area. Another possibility is that these structures are chloride cells; however, the chloride cells on the gills of S. interpunctatum do not resemble the structures on the mouthparts. There would be significant advantages to having ion uptake structures in the vicinity of the mouthparts since the local ionic concentration might be higher due to the rupture of the cells of the food material. Resolution of the function of these structures will require electron microscopy of the underlying structures.

The setae on the galealacinia of R. pellucida indubitably perform the same tasks as their counterparts in S. interpunctatum. There is a great degree of similarity in numbers and size of setae on the galealaciniae of the two species, with a slight trend toward smaller numbers and reduced sizes in R. pellucida. The reductions correspond to the reduced food gathering role of the labium in R. pellucida.

The superlinguae are reduced in R. pellucida; this reduction corresponds to the reduced role of the labium in food gathering and thus the reduced need for food transport via the hypopharynx. A similar reduction is seen in the labium of R. pellucida; because of the increased role of the maxillary palps, the labium in R. pellucida has a reduced role overall.

General trends

The study of S. interpunctatum and R. pellucida suggests some general trends in mouthpart morphology among heptageniids in particular, and mayflies and aquatic invertebrates in general.

There is a suite of morphological differences between S. interpunctatum and R. pellucida that correlates to the different microhabitats in which they live. Stenacron interpunctatum occurs in slow, marginal areas of streams and is usually found in crevices (Wodsedalek 1912, McShaffrey and McCafferty 1986, B. Heising, personal communication). Rhithrogena pellucida is taken in faster currents and may be found on the upper surfaces of stones (McShaffrey and McCafferty 1988). The increased size and setation of the maxillary palpus of R. pellucida not only allows more efficient uptake of tightly accreted material (McShaffrey and McCafferty 1988), but they also allow for more effective feeding in currents (Strenger 1953). Correlated with the increase in size of the maxillary palpus is the modification of the mandible apex, and a reduction in importance of the labium as a food-gathering organ. The reduction in importance of the labium also means a lesser food load for the palmate pectinate setae on the crown of the galealacinia, and thus for the hypopharynx also. This in turn is correlated with a reduction of these structures.

Similar modifications of mouthparts in correlation with current velocity of microhabitats are found throughout the Heptageniidae. Unfortunately, accurate data on microhabitat and current velocity in such areas are lacking for most heptageniids. If sufficient data can be gathered in support of this correlation, it will link taxonomically important characters to their ecological function.

There are a number of modifications found in the mouthparts of carnivorous heptageniids. For example, the comb structures on the mandibles and maxillae are lacking, the molae are greatly reduced, and the superlinguae are small (McCafferty and Provonsha 1986). Since these structures are used for transport of microscopic particles in other heptageniids, their disappearance in the carnivorous heptageniids, which feed on macroscopic particles, is predictable.

Carnivorous heptageniids are also the only members of the taxon to ingest macroscopic food. The restrictive nature of the tricondylic mandibles may explain why feeding on macroscopic food rare among mayflies. In the carnivorous heptageniids, the reduction of the molae increases the size of the opening to the pharynx. The carnivorous heptageniidae, as well as most carnivorous mayflies, are midge predators, and midges are macroscopic only in one direction, length. The critical dimension of a midge larva, insofar as passage through the mandibles of a mayfly is concerned, is its diameter, which is small compared to its length.

Another interesting datum is the absence of any shredders among the Heptageniidae. This is not surprising, since the prognathous orientation and structure of the mouthparts is not suited to shredding. In particular, there are no hardened, pointed mouthparts in position to bite into vascular plant material. Shredding also seems to be rare among mayflies in general, although many species do have mouthparts which could bite into plant material. The paucity of mayfly shredders is perhaps again due in part to the nature of the mandibles, although other constraints such as evolutionary history and ecological factors are no doubt also very important.

Finally, there is a trend shown in S. interpunctatum, R. pellucida, and many other aquatic invertebrates, namely the use of pectinate and bipectinate setae as filtering structures. The convergence is striking and widespread; probably reflecting basic biophysical principles of filter design (Rubenstein and Kohl 1977, LaBarbera 1984, Nilsson 1984). Such setae have been demonstrated to act as filter structures in Heptageniidae (McShaffrey and McCafferty 1986, 1988), Isonychiidae (Braimah 1987a, Wallace and O'Hop 1979), Culicidae (Dahl et al. 1988), and Crustacea (Farmer 1974). I have additional observations of similar structure and function in the mayflies Ephoron (Polymitarcyidae), Callibaetis (Baetidae), Ephemerella needhami (Ephemerellidae), and Potamanthus (Potamanthidae), the caddisfly Macrostemmum (Hydropsychidae), ostracods, amphipods, and polychaetes.

The discovery of a common filtering structure is only one of the results of this detailed study of the mouthparts of S. interpunctatum and R. pellucida. Close examination of the morphology, feeding behavior, and ecology of these species has also shown that morphology is closely tied to function in these organisms, and that trends in mouthpart morphology observed in these two species are also apparent in other heptageniids. Such results illustrate the synergistic effect of the methodology developed in this study.

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CHAPTER VI - FEEDING BEHAVIOR AND RELATED FUNCTIONAL MORPHOLOGY OF EPHEMERELLA NEEDHAMI

Studies of behavior and functional morphology associated with feeding in aquatic insects are important tools in understanding ecological and evolutionary relationships. Historically, most of such work has been either anecdotal or speculative, with casual observations of feeding behavior in one species often applied to any organisms with similar morphology. More recent work has emphasized rigorous observational methodology such as videomacroscopy, combined with the detailed morphological descriptions made possible by scanning electron microscopy (SEM).

My previous studies (McShaffrey and McCafferty 1986, 1988) of functional morphology in mayflies have centered on Heptageniidae, a group with modified, prognathous mouthparts. For the present study, I examined the functional morphology and behavior associated with feeding in Ephemerella needhami, a member of the family Ephemerellidae. These mayflies have more generalized, hypognathous mouthparts that resemble those of such primitive mayfly lineages as the Siphlonuridae. Since several other mayfly families, including the primitive Siphlonuridae, have many hypognathous species, an understanding of the functionality of this type of mouthpart is critical for ecological interpretations. For similar reasons, comparative studies of mouthpart morphology and feeding behavior among these groups is important in understanding evolutionary relationships.

Unfortunately, little research has been completed on this subject. The work on the Heptageniidae includes Strenger (1953), Froehlich (1964), Soldan (1979), McShaffrey and McCafferty (1986, 1988). Studies on feeding and functional morphology of mouthparts among hypognathous mayflies were done by Brown (1961) on Cloeon dipterum L. and Baetis rhodani Pictet (Baetidae), and by Schonmann (1975, 1981) on Siphlonurus aestivalis Eaton (Siphlonuridae).

Little ecological information on E. needhami has been published. Ecological studies concerning the diet of organisms' may give clues to the function of their mouthparts. Whereas some work on the diet of some ephemerellids has been conducted, nothing has been published regarding the diet of E. needhami. According to Cummins et al. (1984) the Ephemerella belong to the collector-gatherer and scraper functional feeding groups (FFGs). Hawkins (1985) described a western species, E. infrequens as a diatom scraper, detritus shredder, and collector-gatherer. Sweeney and Vannote (1981) studied 6 species of ephemerellids, 2 of which belong to the genus Ephemerella; all of the ephemerellids in their study fed on diatoms and detritus.

The present study is the first to combine modern techniques such as scanning electron microscopy (SEM) and videomacroscopy to investigate the feeding habits and ecology of the Ephemerellidae. During the course of the study, additional data regarding the diet of E. needhami and its ecological association with the filamentous alga Cladophora were gathered; this information is presented in Chapter VII.

Methods

Field work was conducted over four successive years: 1985, 1986, 1987, and 1988. In 1985 weekly presence/absence surveys were conducted and similar sampling on a less regular basis was conducted in 1986. A total of 35 organisms collected on 1 May 1987 and 8 May 1987 were used for detailed videomacroscopic behavioral analysis and subsequent gut content analysis. Additional larvae were collected for less detailed videomacroscopic behavioral observation, gut content analysis, and morphological examinations by light microscope and SEM. Throughout the study, only mature larvae, as judged by relative wingpad development (McCafferty and Huff 1978) and head capsule width (1.1-1.6 mm), were used. To insure correct species identification, only larvae with the typical dorsal stripe of E. needhami were used in this study.

Larvae used in videomacroscopic behavioral analyses were maintained in the laboratory in an artificial stream consisting of two reservoirs constructed of clear acrylic measuring 30 x 30 x 30 cm with a mean depth in operation of 20 cm. The reservoirs were connected by a channel, also made of clear acrylic, measuring 15 cm deep by 11.5 cm wide by 1.2 m long. Water was pumped from the downstream reservoir to the upstream reservoir through a 1.25 cm diameter nalgene hose by a submerged pump provided with a silicon-controlled rectifier (SCR). The SCR allowed current speed in the channel to be regulated between 0-1.5 m s-1, depending on the configuration of substrate in the channel and depth of water in the reservoirs. Water in the channel varied between 0 and 7 cm over the substrate.

The channel was filled with substrate from the collection site, including rocks with attached Cladophora. Aeration was provided by airstones in the downstream reservoir; light was provided by a 45 cm, 15 watt fluorescent light placed 15 cm over the channel bottom and operated 24 hours/day. Water temperature was equal to room temperature, 20-25o C.

The videomacroscopic techniques used have been described in detail elsewhere (Keltner and McCafferty 1986, McShaffrey and McCafferty 1986, 1987, 1988). For this study, the observational methodology developed for the study of heptageniid mayflies was adapted to ephemerellids. Because of E. needhami's strong affiliation with Cladophora as both substrate and food source, the alga was always included in the observational arenas. Three configurations of observational arenas and appropriate videomacroscopic equipment were used. These were: a) a shallow observation flow cell (McShaffrey and McCafferty 1986, 1988) filled with Cladophora and operating at "high" (5-10 cm s-1) current speeds; b) the artificial stream described above, observed by means of the videocamera coupled to a 50 mm lens by a bellows unit; c) a 9 x 7.5 x 2 cm enclosure with 1.5 cm deep water into which the Cladophora and E. needhami were placed, observed by means of the videocamera attached to a stereoscope. No flow or aeration was used in this last configuration. Since E. needhami larvae in the field were observed to actively feed during daylight hours, visible light supplied by a fiber optic illuminator was used in all videomacroscopic observations.

In all cases, food for the experiments was Cladophora and associated detritus, periphyton, and animals. After videotaping, the organisms were sacrificed to obtain gut contents, and slide mounts were made of the mouthparts.

Several hundred complete feeding sessions were observed. Of these, 11 feeding sessions, each composed of multiple (> 10) feeding cycles were analyzed in detail. The 11 sessions analyzed in detail were performed by 9 different individuals and included 7 sessions of detritus feeding, 3 sessions of Cladophora feeding, and 1 predatory encounter. All of the sessions chosen for detailed study were observed using configurations a or c, the observations made in the artificial stream were not at a great enough magnification for detailed study. The descriptions generated by these analyses were then checked against the remaining sessions, including those made in the artificial stream, as recorded on videotape. Feeding behavior was described as stereotypic feeding cycles composed of definite stages, using the same methodology as employed in McShaffrey and McCafferty (1986, 1988); this methodology is adapted from that used by Trueman (1968) and Keltner and McCafferty (1986). Each stage is delineated by specific movements of either the maxillae or the mandibles.

Mouthpart structures were examined using light microscopy and SEM. Specimens were prepared for SEM by dehydration in increasing concentrations of ethanol from 75% to 100%., then transferred to 50% ethanol, 50% xylene and allowed to stand for 12 hours. The specimens were then transferred to 100% ethyl acetate. Some of the specimens in 100% ethyl acetate were sonicated for 1 minute in an ultrasonic cleaner to remove food material from the mouthparts, others were not sonicated so that the position of food on the mouthparts might be determined. The larvae were then either dissected or transferred whole to the SEM stubs after air drying.

Results

Effects of experimental conditions on behavior

Observations of feeding behavior of E. needhami larvae in the field and in the artificial stream were similar to those made under the more restricted conditions of the observation cells. In the field and in the laboratory, larvae were consistently rheotactic and tended to climb on Cladophora filaments as opposed to other substrates such as rock.

One aspect of the experimental conditions that did affect behavior was the visibly larger load of detritus borne by Cladophora in the laboratory. Whereas Cladophora used in behavioral observations was freshly collected and thus bore a similar number of epiphytes compared to those in the field, the Cladophora used in the observation cells and artificial stream quickly picked up additional detritus.

Stereotypic feeding behavior

Resting Position: Larvae not actively feeding hold their mouthparts in a characteristic resting position (Figs. 108, 109, 110, 111) which is nearly hypognathous. The labial palps are normally somewhat extended and free of the other mouthparts. In a foraging larva they will constantly be moving, touching the Cladophora filament and associated material. The remainder of the labium is drawn anteriorly and dorsally to form the ventro-posterior boundary of the pre-oral cavity. The glossae and paraglossae are in contact anteriorly with the posterior apices of the galealaciniae, which in turn are held with their anterior surfaces in contact with the superlinguae of the hypopharynx. The superlinguae contact the distal ends of the mandibles anteriorly; the lingua is held pressed against the mandibles at the molae. The mandibles are positioned with the distal apices separated by the width of the glossae. The labrum covers the mandibles anteriorly.

Feeding cycles: Two distinct behavioral cycles were observed. The one that occurs when the larvae are removing detritus and other material from the Cladophora filaments is designated the Maxillary Brushing Cycle. The second, which occurs when larvae bite off portions of Cladophora filaments, is designated the Mandibular Biting Cycle. In the Maxillary Brushing Cycle the maxillae are the primary food gathering tools, supplemented by the mandibles; the reverse is true for the Mandibular Biting Cycle. The cycles each consist of several stages that are here delineated by movements of the galealaciniae, although movements of the mandibles could also be used to delineate the cycles. A single cycle takes 0.3 - 0.5 seconds at room temperature.

Both cycles are preceded by specific orientation movements. First, the larva uses the labial palps to determine the position of a Cladophora filament relative to the body. The larva then positions itself so that the filament is aligned with its length parallel to the long axis of the body. Next, the foreclaws are hooked on either side of the filament aligned with the larva, or onto other nearby filaments, and are adducted, thus bringing the filament and the mouthparts together. The labial palps may assist in bringing the filament and mouthparts together. As the feeding cycles proper are about to commence, the larva is positioned with the algal filament traversing the pre-oral cavity, contacting the midline of the labrum anteriorly and the glossae posteriorly.

Maxillary brushing cycle:

Stage 1: -- Initial abduction of the galealaciniae: As feeding commences, the galealaciniae move laterally outward while the mandibles move slightly inward towards the midline.

Stage 2: -- Final orientation: The distal segments of the labial palps and/or the tibiae of the forelegs are adducted, bringing the head and filament into contact. This movement may be accompanied by a forward motion of the whole body induced by a rotation of the forelegs at the coxae. At this point the galealaciniae are fully open and the mandibles fully closed (Fig. 112).

Stage 3: -- Adduction of the galealaciniae: The galealaciniae are adducted, moving medially with the terminal spines and setae (Fig. 115) being drawn over the filament surface. The spinous processes dislodge material from the filament; this material is filtered out by the lanceolate bipectinate setae on the crown of the galealaciniae (Fig. 116). The final position of the galealaciniae is with the distal ends meeting at the midline and inserted between the superlinguae and the lingua of the hypopharynx. While the galealaciniae are moving medially, the mandibles are being abducted laterally (Fig 113).

Stage 4: -- Food deposition and subsequent abduction of the galealaciniae: As the galealaciniae are abducted from the midline to begin a new cycle, food material is deposited on the superlinguae and lingua, and, to a lesser degree, on the anterior surface of the paraglossae. The mandibles at this point are being adducted medially and may pick up material from the substrate. As the galealaciniae clear the hypopharynx, and the mandibles return to the midline, the mouthpart configuration is the same as it was in Stage 1. Feeding continues with a repetition of Stages 2, 3 and 4.

Mandibular biting cycle: The Mandibular Biting Cycle is similar to the Maxillary Brushing Cycle in many respects, with the main difference a replacement of the maxillae by the mandibles as the primary food-gathering tools. In my observations this cycle was performed only as a modification of an ongoing Maxillary Brushing Cycle.

Stage 1: -- Adduction of the galealaciniae: When feeding shifts from a Maxillary Brushing Cycle to a Mandibular Biting Cycle the Stage 3 adduction of the galealaciniae becomes the first stage of the Mandibular Biting Cycle.

Stage 2: -- Final Orientation: final orientation takes place with the galealaciniae closed and the mandibles fully open. The head and filament are brought into close contact in a fashion similar to that of the Maxillary Brushing Cycle.

Stage 3: -- Abduction of the galealaciniae and biting: As the galealaciniae are abducted laterally, the mandibles are adducted medially and the distal ends of the mandibles bite into the filament (Fig 114). Often the pressure of the mandibles will cause the filament to bend abruptly, usually 90o in a direction ventral to the larva. When the filament has already bent, the closing of the mandibles acts to pull the filament slightly further into the pre-oral cavity.

Stage 4: -- Adduction of the galealaciniae: As the mandibles reach the midline, the galealaciniae are adducted medially and may pass over the filament as they move inward. As they pass over the filament they may gather loose material or material partially dislodged by the mandibles. The mandibles are abducted laterally as the galealaciniae reach the pre-oral cavity. The closing of the galealaciniae at this point serves to hold the filament in place while the mandibles are positioned for the next bite and may also force the filament further into the mouth. Feeding continues with a repetition of stages 2, 3 and 4.

Predatory feeding: One individual was observed to attack and ingest a midge (Chironomidae: Orthocladiinae: Cricotopus sp.) that had been feeding on Cladophora and associated material. Because the mayfly was moving quickly, the initial stages of the attack and penetration of the midge cuticle were not observed. The mayfly fed using a Maxillary Brushing Cycle, with both the maxillae and the mandibles packing midge tissue into the preoral cavity. The cuticle of the midge was not eaten. The mayfly consumed the posterior half of the midge over a period of 319 seconds with individual cycles lasting about 0.3 seconds each.

The midge lived throughout the attack and even fed actively during its early stages; the midge was still alive when removed from the observation cell. The gut contents of both organisms were similar, containing Cladophora, detritus, and diatoms. The only recognizable midge body part in the gut of the mayfly was a small section of trachea.

Food processing

Food brought to the pre-oral cavity by the maxillae in either cycle is deposited on the hypopharynx and paraglossae. Medially directed setae on the superlinguae and paraglossae strip food material from the apical setae of the galealaciniae during Stage 4 of the Maxillary Brushing Cycle or stage 3 of the Mandibular Biting Cycle, and hold it in place. When the galealaciniae return in Stage 3 of the Maxillary Brushing Cycle or Stage 4 of the Mandibular Biting Cycle, they push the material stripped on the previous cycle further medially towards the base of the hypopharynx. The hypopharynx in turn pushes the material at its base between the molae, which are moving apart. As the molae close (Stage 4 of the Maxillary Brushing Cycle or Stage 3 of the Mandibular Biting Cycle) they strain excess water from the material and force the material into the mouth.

Small pieces of food brought to the mouth by the mandibles may be retained by dorso-medially projecting setae on the inside of the labrum, or by the medially projecting setae on the superlinguae. The material on the superlinguae is treated as already described; material on the labrum is forced dorsally by fresh material and is thus placed on the epipharynx to be pressed into the area between the molae. Filaments are directed into the oral cavity by the labial palps, galealaciniae, mandibles, and the tip of the hypopharynx Eventually the mandibles and/or the maxillae are able to cut the filament and it is swallowed. Filaments in the gut are reasonably intact.

Diet

Gut content analyses of larvae collected in the field and those observed in the laboratory revealed a diet consisting of detritus, Cladophora filaments, and epiphytic diatoms. Detritus was the most common component, and the larvae ingested relatively more detritus when detritus availability was high. Epiphytic diatoms were taken in similar proportions in all cases, but Cladophora intake declined when detritus intake increased.

Discussion

My observations of feeding behavior show that E. needhami is opportunistic, but relies on two simple, closely related feeding cycles to obtain its food. The observations themselves, while requiring the organisms to be confined to several different artificial situations, were nonetheless consistent with each other. They also approximated feeding in the field as shown by gut content analyses, with larvae in laboratory and field conditions all feeding on the same food items. The stereotypic nature of feeding movements in larval mayflies despite artificial conditions has been well-documented (Brown 1961, Froehlich 1964, McShaffrey and McCafferty 1986, 1988).

The two feeding cycles complement each other in the type of food that is acquired. The Maxillary Brushing Cycle allows loose material such as detritus that has been filtered out of the water column by Cladophora to be ingested; the Mandibular Biting Cycle allows Cladophora filaments and more tightly bound material to be eaten. Together the two cycles account for all of the 3 main components of the diet - detritus, Cladophora, and diatoms. The direct intake of epiphytic diatoms was not observed, many of these diatoms are taken into the gut when the Cladophora filament to which they are attached is ingested. It is probable that some diatoms are removed from the algal filaments by the mandibles (Fig. 117) or the spinous processes on the ends of the galealaciniae (Fig. 115), particularly during the Maxillary Brushing Cycle. In addition, measurements of the widths of Cladophora filaments found in the gut contents suggests that a filament width of about 0.1 mm may be the upper size limit for ingestion by E. needhami. Cladophora in the field was measured at widths up to 0.15 mm; presumably this is a function of some mechanical factor such as molar opening width or mandible strength.

Brown (1961) reported a feeding sequence for Cloeon dipterum that was essentially the same as the Mandibular Biting Cycle described for E. needhami. The main difference was that C. dipterum used its labial palps to obtain food. Brown's (1961) account of C. dipterum feeding on filamentous algae was also similar to my observations of E. needhami. The tips of the mandibles were the primary food-gathering organs of Baetis rhodani (Brown 1961), but otherwise it was similar also.

Although his descriptions did not give a precise accounting of sequential movements of the mouthparts, it is clear from Schonmann (1975, 1981) that the overall feeding and food transport in Siphlonurus aestivalis was similar to that of E. needhami, the main difference being the use of the labial and maxillary palps in S. aestivalis to acquire food. Schonmann (1975) was also able to document the use of the labial palps in manipulating algal filaments.

A comparison of the functional morphology of the mouthparts of E. needhami with C. dipterum, B. rhodani, S. aestivalis, and other mayflies is useful because it illustrates how behavioral and morphological differences reflect differing habitat and function. In my observations to date (McShaffrey and McCafferty 1986, 1988, unpublished), I have observed a consistent pattern among mayflies in the primary food-gathering appendages used to obtain detritus or other fine particulate food. This pattern involved the presence of specialized filtering setae that are either pectinate or bipectinate, bearing one or two rows of setules perpendicular to the main setal shaft. Such setae are also found on internal mouthparts where it is important that food material be retained for further processing.

Ephemerella needhami has greatly reduced maxillary palps and the labial palps do not bear filtering setae (Fig. 108); neither of these mouthparts plays an active role in feeding on small particles. The labial palps of C. dipterum (Brown 1961) bear numerous setae, although the type was not specified, and the palps are active in feeding. In contrast, the maxillary palps and labial palps of B. rhodani are relatively bare and are not as active in feeding on fine detritus (Brown 1961). In S. aestivalis, both the labial and maxillary palps are provided with setae and are active in feeding. Heptageniid mayflies also commonly have extensive fields of filtering setae on the labial and maxillary palps (Strenger 1953, McShaffrey and McCafferty 1986, 1988).

The differences in the distribution of these setae are probably related to habitat. The heptageniid mayflies and S. aestivalis feed on flat surfaces where detritus collects on surfaces that are wide relative to the width of the head capsule (Strenger 1953, Schonmann 1975, McShaffrey and McCafferty 1986, 1988). On such surfaces it would be advantageous to cover a larger area with each sweep of the mouthparts, and thus adaptive to use more of the mouthparts in food gathering. Ephemerella needhami, in contrast, feeds in an area where the surface on which the food lies is narrow, and extra coverage by additional mouthparts is not necessary. In any event, the labial palps of E. needhami are active in positioning the head in relation to the Cladophora filament and are thus not available for a food-gathering role. Cloeon dipterum is somewhat intermediate, feeding on both filaments and on flat surfaces, and the labial palps can be used for food acquisition (Brown 1961). Baetis rhodani faces a different problem, it feeds on particles that are firmly attached to the substrate and that are relatively large in comparison to the mouthparts (Brown 1961). Filtering setae would presumably be of little use in such a situation.

Another pattern related to food-gathering appendages is the modification of parts of the mandibles into combs used for removing particles from the maxillary palps. Among Heptageniidae, in which the distal ends of the mandibles do not reach the substrate, the comb structure of the mandibles is particularly noticeable (Strenger 1953, McShaffrey and McCafferty 1986); the trend itself is evident within the Heptageniidae, with well-developed maxillary palps correlated with highly comblike mandibles (McShaffrey and McCafferty 1988). Comblike structures are not well developed in E. needhami, and are represented only by a relatively few setae on the posterior surface of the mandible (Figs. 117, 118) where they may act to strip food material from the galealaciniae. Comblike structures are present in S. aestivalis (Schonmann 1981, Fig. 13d), which does utilize the maxillary palps to obtain food. Cloeon dipterum represents a condition similar to E. needhami with only slight development of comblike structures on the mandibles (Brown 1961, Figs. 6e-g). Baetis rhodani, with its mandibles used as the primary food-gathering organs (Brown 1961), apparently does not possess comblike structures on the mandibles (Brown 1961, Fig. 5d).

In conclusion, it appears that strong similarities in feeding behavior and functional morphology exists in these mayflies (from several different families) that retain the generalized, plesiomorphic, hypognathous type of mouthparts. Variations are primarily related to the development of setal fields and combs and the degree to which mouthparts, including palps, are involved in food gathering. These variations, in turn, appear to be generally related to the feeding microhabitat and substrate.

Obviously, use of feeding structures to decipher evolutionary relationships must be done with extreme caution since modifications are prima facie candidates for convergent evolution. They are in essence ecological characteristics that may best be used for studying relationships of small groups of related species.

Acknowledgments

The Scanning Electron Microscope was made available by the Electron Microscope Center in Agriculture at Purdue University with support from NSF grant PCM-8400133. Arwin Provonsha provided Figures 111-114. Dan Bloodgood and Yeon J. Bae assisted with fieldwork.

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CHAPTER VII - ECOLOGICAL ASSOCIATION OF EPHEMERELLA NEEDHAMI AND THE GREEN ALGA CLADOPHORA

Larvae of the mayfly Ephemerella needhami McDunnough (family Ephemerellidae, or spiny crawlers as they are sometimes known) are extremely common each spring in the Tippecanoe River in north central Indiana, U.S.A. Several aspects of this species' life history and ecology make it an interesting and important subject for a thorough study of its feeding regimen. The larvae (Fig. 119, 120) are intimately associated with the filamentous green alga Cladophora (Figs. 119), and the life cycle of the mayfly is timed so that most larval development corresponds to maximum Cladophora growth. The alga itself serves as a substrate, refuge, food, and food-gathering system for the mayfly; it is also possible that the mayfly provides symbiotic benefits to the alga.

Whereas minimal information about the feeding ecology of Ephemerella species in general is available, almost nothing has been reported for E. needhami. Shapas and Hilsenhoff (1976) reported that E. needhami fed on detritus, diatoms, and filamentous algae, and that other Ephemerella species had similar diets. Cummins et al. (1984) list the genus as belonging to the collector-gatherer and scraper functional feeding groups (FFGs). Hawkins (1985) described a western North American cogener, E. infrequens, as a diatom scraper, detritus shredder, and collector-gatherer. Other species studied by Hawkins (1985) fed on a variety of materials including diatoms, detritus, animals, and moss; Gilpin and Brusven (1970) reported similar diets for western North American ephemerellids. Hawkins (1984) found ephemerellids in a variety of habitats, including moss. Sweeney and Vannote (1981) studied six species of ephemerellids, two of which belonged to Ephemerella and fed on diatoms and detritus. Gray and Ward (1978) found E. inermis feeding primarily on detritus even when filamentous algae were present; a similar diet was reported by Hamilton and Clifford (1983) for this species. Hamilton and Tarter (1977) reported that E. funeralis was primarily a leaf shredder.

Association of Ephemerella with vegetation, including filamentous algae, appears to be common, particularly in the European species. Percival and Whitehead (1929) and Hynes (1961) found Ephemerella ignita in England associated with vegetation including Cladophora    and moss; according to Percival and Whitehead (1929), the two plants were also common in the diet of E. ignita. Jones (1950) found E. notata in the River Rheidol associated with filamentous algae and reported that its diet consisted of filamentous green algae, diatoms, and detritus. Jones also found that E. notata prefers Ulothrix, but would eat moss (Fontinalis antipyretica) when Ulothrix was scarce (Jones 1949).

The present study describes the feeding ecology of E. needhami and its association with the filamentous alga Cladophora. It is the first study to combine conventional gut content analysis with modern techniques such as scanning electron microscopy (SEM) and videomacroscopy to investigate the feeding ecology of the Ephemerellidae.

Methods

Ephemerella needhami were collected at the Tippecanoe River in north central Indiana. At the collection point, the river is 6th order, about 50 m wide, with an open canopy and cobble-gravel substrate. The greatest current velocity was 1 m s-1 at a water depth of 0.4 m. The river Field work was conducted over four successive years: 1985-1988. In 1985 weekly presence/absence surveys were conducted and similar sampling on a less regular basis was conducted in 1986.

A total of 35 organisms collected on 1 May 1987 and 8 May 1987 were used for detailed videomacroscopic behavioral analysis and subsequent gut content analysis and were designated as the Laboratory subgroup. An additional 50 larvae collected in groups of 25 on 8 May 1987 and 24 May 1988 were used only for gut content analysis; these were designated the Field 1987 and Field 1988 subgroups, respectively. Additional specimens collected on various dates were used for morphological examinations by light microscope and SEM, as well as less detailed videomacroscopic behavioral observation. Throughout the study, only mature larvae, as judged by relative wingpad development (McCafferty and Huff 1978) and head capsule width (1.1-1.6 mm), were used. To insure correct species identification, only larvae bearing the typical prominent dorsal stripe were used.

Cladophora was collected on 24 May 1988 and 31 May 1988. Five clumps of Cladophora were collected, placed in 90% ethanol, and returned to the laboratory. The number of E. needhami per clump was determined, as well as damp weight (excess ethanol removed) and volume.

Cladophora filament widths and epiphyte cover were evaluated by cutting the filaments into 0.5 cm segments and mounting a random subsample from each clump in euparol on a glass slide. The widths of 50 randomly chosen filaments and 50 randomly chosen terminal filaments were measured at 200x using a Whipple disk ocular grid. The number of epiphytes per 0.0147 mm2 of surface area was determined at 200x using the same grid. Similar measurements were made for Cladophora filaments among the gut contents of E. needhami, however, only 40 filaments and 40 terminal filaments were examined from the gut contents of specimens collected on 24, May 1988.

Gut contents of E. needhami larvae were analyzed by removing the gut and macerating the contents in euparol on a glass microscope slide. Gut contents were classified as being animal remains (other than midges), midge remains, plant remains, Cladophora, diatoms, unicellular algae, filamentous algae (other than Cladophora), or detritus. Slides were examined at 400x with proportions of each type of food determined by measuring the area covered by each type. Area was measured by using a 10 x 10 ocular grid measuring 0.175 mm on a side. Ten randomly selected fields were evaluated for each slide.

Larvae used for videomacroscopic behavioral analyses and some gut content analyses were maintained in the laboratory in an artificial stream with current speeds varying between 0-1.5 m s-1, depending on the configuration of substrate in the channel. Water in the channel varied between 0 and 7 cm over the substrate, which was taken from the collection site and included rocks with attached Cladophora. Aeration was provided by airstones; light was provided by a 45 cm, 15 watt fluorescent light placed 15 cm over the channel bottom and operated 24 hours/day. Water temperature was equal to room temperature, 20-25o C.

Technological aspects and methodology for my videomacroscopic observations have been described in detail by Keltner and McCafferty (1986) and McShaffrey and McCafferty (1986, 1987, 1988). After videotaping, the organisms were sacrificed to obtain gut contents, and slide mounts were made of the mouthparts. Videotaped feeding behavior from nine larvae was analyzed in detail, the resulting descriptions were then checked against the remaining videotape. Stereotypic feeding behavior and mouthpart morphology were determined as per McShaffrey and McCafferty (1986, 1988), and detailed descriptions are found in Chapter VI.

Results

Field observations

Ephemerella needhami larvae were collected from mid-April to late May in 1985-1988. All of the larvae collected had head capsule widths greater than 1mm; smaller larvae were not taken and their habits are unknown. Cladophora first became noticeable in late April and formed extensive mats that began to break up by late May when the water temperature passed 20o C, although some smaller Cladophora clumps did persist throughout each summer. Most E. needhami larvae were collected in association with Cladophora, only a very few larvae were taken on or under stones away from Cladophora. Emergence of E. needhami took place in late May. Quantitative samples of E. needhami taken on 24 May 1988 showed an average of 5.8 E. needhami per cubic centimeter of Cladophora and 8.2 E. needhami per gram of Cladophora.

Food availability and diet

Cladophora in the Tippecanoe River provided E. needhami with several potential food items. Besides the filaments themselves, an extensive epiphyte community was often present on the Cladophora, and the filaments also collected detritus (Figs. 122, 123, 124). Cocconeis sp. was by far the most abundant epiphyte, with Achnanthes sp., Navicula sp., Gomphonema sp., Fragilaria sp., and Melosira sp. present in smaller percentages. The epiphyte community increased as the season progressed (Fig. 125). Visual observations revealed that detritus also accumulated as the season progressed, but because of the difficulty in removing the Cladophora without losing the unsecured material, quantitative measurements of detritus were not made. A similar increased loading of detritus on Cladophora was noted in the artificial stream and other observational arenas; this meant that E. needhami larvae observed in the laboratory in early May were exposed to a higher level of detritus than was present in the field at the time. Cladophora growth, as measured by filament width (Fig. 126) showed an increase between 28 April 1988 and 24 May 1988, but remained the same between 24 May 1988 and 31 May 1988.

In addition to the Cladophora, diatoms, and detritus, a number of potential prey animals were found among the Cladophora filaments including individuals of Chironomidae, Simuliidae, Hydropsychidae, and other mayflies from families such as Baetidae and Caenidae. Two species of Plecoptera were also present; their gut contents indicated that one was a detritivore and the other a predator feeding mainly on midges and baetid mayflies.

Ephemerella needhami in the field and in the laboratory primarily consumed Cladophora, diatoms and detritus (Fig. 127). All three subgroups (Laboratory, Field 1987, Field 1988) contained the same percentage of diatoms, but differed significantly (Student's T-test, 95% confidence level) in the relative amounts of Cladophora and detritus consumed. Animal parts, midge remains, filamentous algae other than Cladophora, and unicellular algae accounted for less than 1% of the gut contents in all cases. The average width of Cladophora filaments in the gut was less than that available in the field, only slightly larger than the average width of terminal filaments (Fig. 126).

Stereotypic feeding behavior

Larvae used 2 distinct methods of feeding on Cladophora, one method occurred when the larvae were removing detritus and other material from the Cladophora filaments primarily by using their maxillae; the second occurred when larvae bit off portions of Cladophora filaments primarily by using their mandibles. The cycles differed in both the type of material ingested and the mouthpart used as the primary food gathering tool. Filaments and diatoms in the gut were often relatively intact (Fig. 124).

One individual was fortuitously observed to attack and feed on a midge (Chironomidae: Orthocladiinae: Cricotopus) that had been feeding on Cladophora and associated material. Soft tissues and internal materials, including the gut and its contents, were ingested beginning at the posterior end, but the cuticle of the midge was not eaten. The midge lived throughout the attack and even fed actively during its early stages; the midge was still alive when removed from the observation cell. The gut contents of both organisms were similar, containing Cladophora, detritus, and diatoms. The only recognizable midge body part in the gut of the mayfly was a small section of trachea.

Discussion

The life history of E. needhami is well-adapted to its reliance on Cladophora. Data collected over 4 years show that the presence of late instar larvae was always correlated with presence of Cladophora. Leonard and Leonard (1962) also found the larvae associated with vegetation, and their emergence data is also consistent with a univoltine life cycle with emergence occurring when Cladophora mats begin to break up (Leonard and Leonard 1962). Although I have not observed young larvae, it is probable that they are detritivores, given the habits of the later instars and the presence of detritivorous habits in other young mayfly larvae, even those which later specialize on other foods (Edmunds 1984).

Other aspects of E. needhami that suit them to life among Cladophora include the dorsal spines (Fig. 120) on the abdomen, and the coloration of the larvae. The dorsal spines curve posteriorly and may help anchor or brace the larvae against the Cladophora and thus aid in maintaining position against the current. Similar morphology and function has been observed for other aquatic insects (Hynes 1970) and for Ephemerella ignita, a European species found among vegetation (Hynes 1961).

Color patterns among the larvae at the Tippecanoe River range from a dark brown to dull green, with a large proportion of the larvae black with a green dorsal stripe. The dark brown color is typical of old, epiphyte and detritus encrusted Cladophora, while the green is typical of newer growth. These colors may help the larvae avoid visual predators. The combination of a dark larva with a green dorsal stripe may be an adaptation to disguise body outlines (Fig. 119), or a compromise between coloration matching old or new Cladophora growth. Much work on the population genetics and ecological significance of these color morphs is needed.

The diet of E. needhami also showed its close ties to Cladophora. Based on gut content data and feeding observations, E. needhami is best described as a collector-gatherer according to the FFG categories of Cummins and Merritt (1984). Such a classification would also be consistent with the diet of this species as reported by Shapas and Hilsenhoff (1976), their results are very similar to those reported here. This species is opportunistic, able to function to some extent as an herbivore feeding on Cladophora (shredder FFG) or as a predator (predator - engulfer FFG). Because it does not use setae to gather its food, I do not consider E. needhami to be a brusher (McShaffrey and McCafferty 1986, 1988). I also find no evidence to support the classification of E. needhami as a scraper.

The observation of a predatory encounter was important because it illustrated the opportunistic nature of E. needhami. The degree of importance of carnivory in the life cycle of E. needhami is not clear, and I was not able to document any additional instances of predation. The fact that the mayfly consumed the midge without ingesting any recognizable hard body parts of the midge is disturbing because it exemplifies the danger of assessing feeding habits based only on gut contents; in this case the gut contents of the predaceous mayfly were similar to both the midge and other E. needhami which had fed on detritus, Cladophora, and diatoms. Ephemerella needhami larvae do appear to be selective in ingesting food. The proportion of diatoms ingested was the same in the 3 subgroups tested (Fig. 127), but the number of epiphytes available was significantly different for the 3 groups (Fig. 125). In particular, the data for 24 May 1988 show that the larvae consumed filaments with significantly fewer epiphytes than were present on filaments in the field. In addition, individuals in situations where more detritus was available, namely the late season (24 May 1988) field subgroup and the laboratory subgroup consumed relatively more detritus and less Cladophora.

The apparent preference of detritus over other food items has several possible explanations. The observed response could be a simple response to increased detritus availability with no selection involved. Also, the detritus was the easiest food to ingest. Observations of feeding revealed the relative difficulty of biting off Cladophora filaments, with resulting decrease in feeding efficiency (as measured by handling time and energy expenditure). There was an upper size limit of filament width that could be ingested; the largest filaments ingested were only about 0.1mm wide (Fig. 126), with average filament sizes smaller than those available in the field. The average filament width of Cladophora in the gut samples was comparable to the width of terminal filaments in the field, suggesting some preference for the terminal filaments, which were smaller (Fig. 126). Epiphytes, particularly Cocconeis sp., were also difficult to remove and ingest. I did not observe any direct removal of Cocconeis, although some frustules not associated with filaments were found in the gut. The smaller number of epiphytes on Cladophora in the gut samples compared to the field samples was probably due to the ingestion of a higher proportion of terminal filaments which, being younger, bore fewer epiphytes.

Another possible explanation of the preference of detritus could be related to the nutritive values of the food materials. However, whereas all 3 types of food have similar caloric values, the epiphytes and Cladophora normally have higher Carbon to Nitrogen ratios (Cummins and Klug 1979) and are thus more nutritionally complete. Anderson and Cummins (1979) rated detritus as the least nutritious and living algae, particularly diatoms, as being more nutritious. Shapas and Hilsenhoff (1976) documented Ephemerella spp. that shifted their diets seasonally; some species increased relative amounts of detritus in their diet while others reduced detritus in their diet from season to season. Obviously, much work needs to be done in this area.

Among other aquatic insects, feeding on Cladophora is apparently rare (Hutchinson 1981), perhaps because Cladophora contains poisonous fatty acids (Lalonde et al. 1979). Brown (1960) did find that the mayfly Cloeon dipterum Linnaeus would feed on Cladophora, but Cladophora was not ingested as frequently as other filamentous algae unless it was chopped up. Mechanically cutting the filaments would decrease the mechanical feeding problems discussed above, and it might release potential toxins before they are ingested by the larvae.

Overall, E. needhami's diet appeared to balance the relative advantages and disadvantages well. Early in the season, when detritus was rare, relatively more Cladophora was consumed. Later in the season, when detritus is abundant, detritus was taken in greater proportions than Cladophora, and any potential problems with toxins were thus reduced. The relatively constant ingestion of epiphytes may reflect a need for some portion of high-quality food. Whether this shift in feeding was an active decision by the individuals or a passive response to changing environmental conditions (detritus availability, filament width) was not apparent and requires further study.

It is not clear what effect the feeding of E. needhami larvae has on the Cladophora. Generally, removal of detritus and epiphytes, which block out sunlight and nutrients, may be beneficial to the Cladophora (Whitton 1970). Since it appears that E. needhami feeds primarily on terminal filaments, the removal of these filaments may stimulate growth and branching of the Cladophora, but this has not been demonstrated. If effect of E. needhami larvae feeding on the Cladophora is negative, then perhaps the epiphytes and detritus are beneficial to the Cladophora by either shielding it mechanically from ingestion, or serving as an alternate food source. The reduction of Cladophora in the Tippecanoe River in late May is probably not the result of E. needhami feeding. For example, E. needhami may feed only on the terminal filaments; it may not be able to ingest the wider basal filaments. In any case, Cladophora shows seasonal growth patterns (Brown 1908, Whitton 1970), whose origins are unclear (Whitton 1970). Other explanations for the decline of Cladophora include temperature increases, accumulation of epiphytes and detritus reducing light and nutrient availability, and changing nutrient levels (Whitton 1970). Recently, Creed (1988) suggested that crayfish might be responsible for reduction of Cladophora mats. Ephemerella needhami is an opportunistic collector-gatherer which obtains food from Cladophora filaments and associated material. Detritus is apparently the preferred food when available, due to its abundance and ease of ingestion. In addition to its role in the diet of E. needhami, Cladophora serves as a support, food collector, refuge, hunting ground and camouflage. Ephemerella needhami is well adapted to life in the Cladophora, with a life cycle synchronized to Cladophora development, and morphological and color adaptations that allow it to move about in the filaments and remain hidden from visual predators.

Acknowledgments

The Scanning Electron Microscope was made available by the Electron Microscope Center in Agriculture at Purdue University with support from NSF grant PCM-8400133. Dan Bloodgood and Yeon Jae Bae assisted with field work.

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CHAPTER VIII - MISCELLANEOUS FEEDING OBSERVATIONS

In addition to the 3 species studied in detail, many other organisms were observed during the course of this study. The purpose of this chapter is to relate, in an anecdotal form, some of these observations.

Gastropoda

Snails on the walls of aquaria and artificial streams in the laboratory were observed to scrape material from the walls with their radulae. Some species were able to remove Cocconeis.

Amphipoda

An unidentified amphipod, possibly Gammarus sp., was observed to scrape algae (including Cocconeis) from the walls of an aquarium with its mouthparts. These amphipods are one of four taxa in which I have been able to document scraping function.

Ephemeroptera

Callibaetis sp.:

These mayflies fed on detritus in a tank cell in a fashion very similar to that of E. needhami. They were also able to create a current by rapid movements of their maxillary and labial palps. This current stirred up small particles of detritus that were subsequently filtered out by the bipectinate setae on the palps. When perched near the air-water interface, this current moved neuston to the mouthparts, which then filtered the neuston. This is an example of interfacial feeding as noted by Dahl et al. (1988) for Culicidae.

Isonychia sp.:

Isonychia larvae were observed to filter-feed in aquaria, artificial streams, and in flow cells. Feeding was similar to published accounts (e.g. Wallace and O'Hop 1979). Larvae faced into the current and material was filtered out by the pectinate setae on the forelegs. Periodically the legs would alternately be brought to the mouth to be cleaned by the mouthparts, particularly the labial palps.

Potamanthus sp.:

These larvae were observed to collect detritus with the labial palps, and to filter material with the pectinate setae on their forelegs. There are also pectinate setae on the mandibular tusks; presumably these are used for feeding. Food transport and processing seems to be similar to that in the Heptageniidae.

Heptagenia flavescens:

Although few observational data are available, they and gut content data suggest that these larvae use the hardened tips of their maxillary palps as scraping tools to loosen biofilm on the bottom surfaces of rocks and logs. Other feeding behavior is similar to S. interpunctatum.

Stenonema sp.:

The small amount of videotape available for this species revealed feeding behavior similar to S. interpunctatum. The gut contents were more varied, with some periphyton present. This species occupied a wider range of habitats than S. interpunctatum, and was sometimes taken on the upper surfaces of stones. In the laboratory, it was not as negatively phototactic as S. interpunctatum, and was often observed on the upper surfaces of stones.

Caenis sp.:

Small caenid larvae found in Cladophora with E. needhami were observed to feed on detritus associated with the filament in a fashion similar to that of E. needhami. No filaments were ingested while the larvae were under observation.

Ephoron sp.:

On one rare occasion when several mature larvae were returned to the laboratory alive, it was possible to make observations of their feeding behavior. The larvae were placed in the artificial stream, where they made unlined burrows in the coarse gravel substrate. The larvae fed by extending their heads and forelegs from the burrow. Particles retained by the pectinate-hairlike setae on the forelegs was combed out by the labial palps as the legs were periodically drawn through the mouthparts. The labial palps were also equipped with pectinate-hairlike setae.

Trichoptera

Macrostemmum zebratum:

These caddisflies filter material through a very fine mesh net built in an ingenious retreat which takes advantage of hydrodynamics to provide current without pumping activity by the larvae. The larvae remove material from the net with the pectinate-hairlike setae on their forelegs. This observation confirms the speculation by Wallace and Sherberger (1975) that Macrostemmum larvae clean the catchnets with the setae on their forelegs.

Coleoptera

Psephenus herricki:

These larvae were observed to feed by scraping material from the sides of aquaria with their mandibles. They are able to remove Cocconeis from the glass. Further observations of the hydrodynamics and behavior of these organisms is found in McShaffrey and McCafferty (1987).

Diptera

Eusimulium spp.:

Observations of this genus support published accounts of feeding behavior, for example, Craig and Chance (1982). In one session, simuliid larvae in a cell without flow attached themselves to E. needhami larvae.

Cricotopus sp.:

These midge larvae, tentatively identified as Cricotopus, were commonly found in Cladophora with E. needhami. They used the mandibles and hypostoma to dislodge detritus and other material from Cladophora. The mandibles are able to break off Cladophora filaments. The gut contents closely resembled those of E. needhami. Other unidentified Orthocladiinae midge larvae were observed to comb detrital material from their anterior prolegs using the hypostoma. This material was then packed into the gut by the maxillae and mandibles.

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CHAPTER IX - CONCLUSIONS AND HORIZONS

Many conclusions flow from this study. Most have been dealt with in the preceding chapters; what remains is an assessment of the impact of this study and the methodology developed for it.

From the standpoint of pure results, the feeding ecology, functional morphology of the mouthparts, and feeding behavior of 3 species of mayflies has been studied in detail. The usefulness of those data is directly proportional to the abundance of these organisms in the ecosystems being studied. Of greater significance is the development of a comprehensive observational protocol for obtaining this type of information for a wide variety of organisms. The data gained through the application of this protocol will directly affect 4 areas of biological science: ethology, functional morphology, systematics, and ecology. The applications for ethology are obvious; there is now an inexpensive, reliable way to obtain behavioral data in a form amenable to detailed analysis and long-term storage. Although video techniques themselves are not new, the variations of them pioneered by Keltner (1983) and in the present study are. Importantly, these techniques extend video observational capability to a new class of organisms, namely those which are small, feed in the darkness, in flowing water, in silty situations, in cracks and crevices, and with a host of other complications which make cinematography difficult. Still, it is necessary to include these 'complications' in the observational protocol if 'normal' behavior is to be observed.

For the functional morphologist, the applications are also apparent. The techniques used here bring together the study of morphology and behavior and allow the researcher to determine the function of a structure the way it should be determined - by watching the structure in use. No longer are appeals to common sense, comparisons with human-engineered structures or similar structures in other organisms needed to assign function. Despite their lack of scientific rigor and their subtle but unmistakable teleological or anthropomorphic underpinnings, such comparisons are still fairly common, particularly in the more popular media.

Another area of functional morphology, which is yet to be explored using these methods, involves a still more rigorous treatment of functional morphological theories. As Keltner (1986) stated, to truly test theories derived from observations it is possible to perform experiments based on modifications of the structures involved. There is certainly potential for that type of experimentation here, and hopefully future studies of aquatic organisms will incorporate such methodology.

The systematist also has much to gain by the application of the methods used here to the taxa under study. Many functionally significant structures are also useful taxonomic characters; a case in point are the palmate setae on the crown of the galealaciniae in heptageniids. These setae function to move food material from one set of filtering setae to another, and the number of such setae is used to separate species in such groups as Stenacron and Stenonema. Understanding their function adds a level of predictability and testability to their use as taxonomic characters. Variability in such structures now becomes less of a mere statistical hurdle and more of a tool for separating populations and taxa, particularly those which inhabit discrete microhabitats. In the case cited, understanding that the number of setae is relative to the role of the labial palps as food-gathering organs cues the systematist to expect variability in this structure to be correlated with current speed; individuals and/or taxa associated with higher current speeds (where the labial palps are less effective) will tend to have fewer palmate setae on the galealaciniae. The character is now predictable, not some whim of nature or arbitrary pronouncement.

At higher levels of systematics, an understanding of function will help the systematist discern patterns of evolution and aid in the determination of homologous vs. convergent characters. For example, the bipectinate filtering setae found on the forelegs of such mayflies as Ephoron and Isonychia might at first seem to be a character linking these groups, but the knowledge that such setae are widespread among not only mayflies but also most other aquatic organisms feeding on microscopic food illustrates that the foreleg setae are likely a convergence, since for such a structure to be both widespread and discontinuous strongly implies separate evolution. The knowledge of function in this case supports the case for convergence since it reveals the selective advantage necessary to explain multiple evolution.

Another possibility for the application of these methods to taxonomy lies in the use of the behavior itself as a taxonomic character. This is commonly done with bird and insect sound production, and there are many other species-specific behaviors, particularly in association with mating 'rituals.' Although there is potential to use feeding behavior as a taxonomic character, observations to date suggest that this will be difficult. First, despite the diversity of form in insect mouthparts, there is little diversity in function, and consequently, in behavior. Most insect mouthparts are used in very similar ways. The apparently discrete cycles described in previous chapters are real, but the descriptions are arbitrary. For instance, the two feeding cycles described for Ephemerella needhami differ only in an arbitrary assessment of where they begin and end, and in the orientation of the mouthparts. But, despite these differences, the basic pattern is: mandibles open, maxillae close, mandibles close, maxillae open, mandibles open, etc. Such basic movements are apparent even when looking at widely divergent mouthparts. The probing movements of an adult mosquito proboscis is basically the alternating movement of the mandibles and the maxillae. Odonate larvae use the labium to seize prey and then ingest it with alternating movements of the mandibles and maxillae; Ephemerella needhami does much the same when it uses its labial palps to align the Cladophora filament with the other mouthparts. Against this rather monotonous background of feeding behavior, the fact that the few insects studied here all fed using multiple feeding behaviors does little to help differentiate organisms on the basis of feeding behavior. There are few distinct feeding behaviors and many organisms can perform more than one. It might be argued that if feeding cycles were examined in extreme detail that real differences might be found; after all, cricket sounds are all produced by a similar mechanism yet are species-specific. The problem with applying this analogy to feeding behavior lies in the fact that sound production is essentially a fixed action pattern (FAP), a behavior produced for the most part without feedback from the sensory structures, and feeding is heavily dependent upon such feedback. In addition, feeding behavior is not related to sexual or reproductive isolation, as is the case for sound production in crickets, and therefore feeding behavior is not subject to direct genetic feedback. Thus, in the real world, feeding behavioral variability is in proportion to the variability of the environment. The diversity of mouthpart structures will continue to be more useful to the taxonomist, particularly the taxonomist faced with a row of preserved specimens. It should be noted though, that most species of aquatic insects have not been studied in detail in regard to their feeding behavior, and it is possible that definite patterns of taxonomic value will emerge in the future.

Ecologists, too, have much to gain from application of this observational protocol to organisms in the environments that they are studying. The information available today is inadequate. Merritt and Cummins (1984) attempted to consolidate the knowledge base of feeding ecology in aquatic insects. Drawing on the extensive and diverse literature, they made assessments for each genus found in North America. Of the three species studied in detail here, the feeding ecology as presented by Cummins et al. (1984) was wrong for two species, and vague for the third. Even taking into consideration the effects of a generic level assignment, the data available in the literature are generally of poor quality. Considering that S. interpunctatum can be the dominant organism in a stream, and that it was assigned (Cummins et al. 1984) not only to the wrong FFG (scraper vs. collector-gatherer), but, by extension, to the wrong trophic level (primary consumer vs. detritivore), the need for accurate assessments of feeding ecology becomes apparent.

The future of the FFG concept will depend largely on its utility to the ecologist. As more and more detailed studies are made of the feeding ecology of aquatic insects, their opportunistic nature will defy most attempts to classify them in a simple, ecologically significant manner. Already, a functionally accurate scheme of classification, such as that presented in Chapter IV, is perhaps too cumbersome to be useful to an ecologist, yet the scheme presented by Merritt and Cummins (1984) is too vague to be accurate and useful. Again, S. interpunctatum illustrates this point. Here is an organism that at a single point in its development can feed from three different food sources - deposits, loosely attached periphyton, and seston. If S. interpunctatum must be assigned to a single category, it would have to be collector-gatherer. This ignores the contribution from filtering to the diet, but, more importantly, the category of collector-gatherer carries little information about what role the organism is playing in the ecosystem. An individual collecting diatoms is at an entirely different trophic level than one collecting detritus. Further, once such complexities are worked out for one stage of an organism's life cycle, we know that many species change feeding habits as they develop; this in turn leads to an entirely new level of complexity. Finally, even such classic, supposedly simple cases such as the Simuliidae, with their apparent specialization for filtering, can feed by browsing the substrate around them.

Ecologists will have to accept the fact that the real world does not behave as simply as a theorist might wish, and they must support attempts to gather more accurate data on the constituent species of the ecosystems they are studying. In the meantime, the data accumulated from this type of study may contribute to an understanding of specific ecosystems comprised of species that have been studied in detail. Perhaps some of the details will help explain the impact of such perturbances as acid rain or organic pollution on an ecosystem, or, at least, key species in that ecosystem.

Overall, the promise for this type of study appears strong. It is a powerful tool in several disciplines, yet it requires a relatively modest level of support. Future directions include looking at representatives of all current FFGs and all orders of aquatic insects, and, eventually, a systematic study of all aquatic species. Advancements in video technology will make such study much easier. As more and more species are studied, it will become possible to begin to construct an overall assessment of feeding in aquatic insects. Eventually, the methods developed in the course of this study will prove more useful than the knowledge of the detailed feeding ecology, behavior, and morphology of 3 species, and contribute to our overall understanding of the aquatic environment.

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LIST OF REFERENCES

North American Benthological Society Bibliography

Mayfly Central

Ephemeroptera Galactica - Bibliography

 

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Vita

Dave McShaffrey was born on September 7, 1959 (Labor Day) in Akron, Ohio. After his distinguished childhood there, his parents moved to Malvern, Ohio. Dave possessed a keen homing instinct even then, and followed them. He attended St. Edward's Central Catholic High School, where he graduated in 1977, first in a class of nine. Perhaps dealt a fatal blow by his tenure there, the school closed the next year.

Dave entered the Honor's Program at the University of Akron, and graduated from that institution 4 years later with a B.S. (no pun intended) degree in Biology-Aquatic Ecology; his senior honors project was "Benthic macroinvertebrates as indices of water quality in the Sandy Creek at Malvern, Ohio". He continued on at the University of Akron, with some graduate work at Ohio State's Stone Lab, and received his M.S. degree in 1983. His thesis was "The ecology and distribution of the Chironomidae (Diptera) in Carroll County, Ohio, U.S.A.", and his advisor was Dr. John Olive.

Leaving Akron, much to the relief of all concerned, he came to Purdue University, where the work you have just read (or will read) was conducted under the direction of Dr. W.P. McCafferty.

Updated Resume

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Dedication

This dissertation is dedicated to a long line of biologists and teachers who guided me to this point: Phillip Hoschar, Linda Dunn, Roger Keller, Scott Orcutt, John Frola, Lazarus Macior, Don Ott, Bob Flanders, Jim McNair, Delmar Broersma, Ann Spacie, and, of course, John MacDonald. Special appreciation is due to John Olive and Pat McCafferty for their guidance and counsel.

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ACKNOWLEDGMENTS

This study would not have been possible without the inspired genius of John Keltner and the equipment he assembled. The Aquatics Group at Purdue - Pat McCafferty, Arwin Provonsha, Dan Bloodgood, John Keltner, Bob Waltz and Yeon Jae Bae , contributed much useful advice. Special thanks to Arwin for his drawings, and Dan for drawings, field work, and construction of equipment, some of which had something to do with entomology.

The Entomology Department at Purdue has been most supportive. Dr. Eldon Ortman, Department Head, has always managed to provide financial support. John and Jo Anne MacDonald were kind beyond reason, and John and the rest of my committee have always provided sound counsel. Due to reasons beyond my control and comprehension, Bob Flanders and Jim McNair were not able to see this project through to the end, but I do appreciate their early support. Delmar Broersma and Ann Spacie were kind enough to step into a program already well along and help me finish it. Terry McCain and Carl Geiger answered many computer-related questions. Sandy Lindsey, Gwen Boutte, Shelly Van Allen, and Cathy Rooze, as well as the rest of the office staff, cut through a lot of red tape in my behalf.

Drs. Charles Bracker and Dan Hess made the JSM 840 SEM available, and Dick Bentlage runs the best EM suite in the world. The NSF payed for the 'scope via grant # PCM 8400-133.

Friends like Mary Catherine Hastert, Dominick Casadonte, Anita Kassuba, Mark Sanders, and everyone in BIO-GRAD INC. kept me going. My parents, Don and Lucy, have encouraged and supported me through years and years and years of school. I can't thank them enough for the loving environment they provided.

And, of course, special thanks to Annn Delleur for drawings, prints, distraction and so very much more. I never would have gotten this thing done without her support and help. And, yes, she does spell her name Annn.

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LIST OF FIGURES

  1. Figure 1. Areas of investigation
  2. Figure 2. Observation cell
  3. Figure 3. Ventral view of the mouthparts of Stenacron interpunctatum
  4. Figure 4. Diagrammatic cross-section of mouthparts of Stenacron interpunctatum
  5. Figures 5-9. Stenacron interpunctatum mouthparts
    1. 5. Labium of S. interpunctatum.
    2. 6. Maxillae of S. interpunctatum.
    3. 7. Hypopharynx of S. interpunctatum.
    4. 8. Mandibles of S. interpunctatum.
    5. 9. Labrum of S. interpunctatum.
  6. Figures 10-15. Stenacron interpunctatum, brushing feeding cycle
    1. 10. Labium and Maxillae, resting position.
    2. 11. Labium and Maxillae, Stage 1 of initial cycle.
    3. 12. Labium and Maxillae, Stage 2.
    4. 13. Labium and Maxillae, Stage 3.
    5. 14. Maxillae and Mandibles, Stage 3.
    6. 15. Maxillae and Hypopharynx, Stage 1 of subsequent cycle.
  7. Figure 16. Tank cell used in feeding experiments
  8. Figure 17. Plan view, surface of glass microscope slide
  9. Figure 18. Oblique view, surface of glass microscope slide
  10. Figure 19. Ventral view of mouthparts of Rhithrogena pellucida
  11. Figure 20. Diagrammatic cross section of mouthparts of Rhithrogena pellucida
  12. Figures 21-25. Rhithrogena pellucida mouthparts
    1. 21. Labium of R. pellucida.
    2. 22. Maxillae of R. pellucida.
    3. 23. Hypopharynx of R. pellucida.
    4. 24. Mandibles of R. pellucida.
    5. 25. Labrum of R. pellucida.
  13. Figures 26-32. Rhithrogena pellucida, labial brushing cycle
    1. 26. Resting Position.
    2. 27. Stage 1 of first cycle.
    3. 28. Stage 2 of first cycle.
    4. 29. Intermediate position of Stage 3 of first cycle.
    5. 30. Final position of Stage 3 of first cycle: Labium.
    6. 31. Final position of Stage 3 of first cycle: Maxilla.
    7. 32. Stage 1 of second and all subsequent cycles.
  14. Figure 33. Pectinate-stout setae, Rhithrogena pellucida .
  15. Figure 34. Mandibular molae and epipharynx of Rhithrogena pellucida
  16. Figure 35. Lateral view of heptageniid larva in feeding position
  17. Figures 36-41. Setal types, Simple to Pinnate
    1. 36. (2536) Simple-hairlike seta.
    2. 37. (2434) Simple-triangular seta.
    3. 38. (2921) Pilose seta.
    4. 39. (2535) Pinnate-hairlike setae.
    5. 40. (3085) Pinnate-hairlike setae.
    6. 41. (2365) Pinnate-hairlike setae.
  18. Figures 42-47. Setal types, Pinnate to Bipectinate  
    1. 42. (2648) Pinnate-plumose setae.
    2. 43. (3016) Pinnate-plumose setae.
    3. 44. (3055) Pinnate-lanceolate setae.
    4. 45. (2533) Pectinate-hairlike setae.
    5. 46. (2423) Bipectinate-stout setae.
    6. 47. (2853) Bipectinate-hairlike setae, Types I and II.
  19. Figures 48-52. Setal types, Bipectinate to Palmate  
    1. 48. (2880) Bipectinate hairlike seta, Type III.
    2. 49. (2426) Furcate-stout setae.
    3. 50. (2918) Bifurcate setae.
    4. 51. (2917) Pinnate-plumose setae.
    5. 52. (2428) Palmate setae.
  20. Figures 53-58. Stenacron interpunctatum mouthparts, Labrum to Mandibles
    1. 53. (2526) Labrum and maxillae.
    2. 54. (2529) Diatom on Simple-hairlike seta.
    3. 55. (5456) Right mandible.
    4. 56. (5457) Left mandible.
    5. 57. (5465) Left mandible, apical projections.
    6. 58. (5458) Left mandible, mola.
  21. Figures 59-64. Stenacron interpunctatum mouthparts, Mandibles
    1. 59. (5459) Left mandible, mola.
    2. 60. (5460) Left mandible.
    3. 61. (5466) Right mandible, apical projections.
    4. 62. (5463) Right mandible, mola.
    5. 63. (5461) Right mandible, mola.
    6. 64. (5464) Right mandible, mola.
  22. Figures 65-70. Stenacron interpunctatum mouthparts, Maxillae and Hypopharynx
    1. 65. (0902) Left maxillary palpus and left mandible.
    2. 66. (2908) Galealacinia, palmate setae.
    3. 67. (2530) Galealacinia.
    4. 68. (2534) Galealacinia.
    5. 69. (2875) Maxillary palpus, distal segment.
    6. 70. (5422) Hypopharynx.
  23. Figures 71-76. Stenacron interpunctatum mouthparts, Hypopharynx to Labium
    1. 71. (2904) Lingua.
    2. 72. (2901) Superlingua.
    3. 73. (5427) Labium.
    4. 74. (2800) Paraglossa.
    5. 75. (2801) Paraglossa.
    6. 76. (2803) Paraglossa.
  24. Figures 77-80. Stenacron interpunctatum mouthparts, Labium
    1. 77. (2852) Labial palpus.
    2. 78. (5433) Labial palpus.
    3. 79. (3059) Labial palpus.
    4. 80. (2855) Labial palpus.
  25. Figures 81-86. Rhithrogena pellucida mouthparts, Labrum to Mandibles
    1. 81. (2695) Labrum.
    2. 82. (2433) Labrum.
    3. 83. (5518) Right mandible.
    4. 84. (5517) Left mandible.
    5. 85. (5514) Left mandible, apical plate.
    6. 86. (5477) Left mandible, mola.
  26. Figures 87-92. Rhithrogena pellucida mouthparts, Mandibles
    1. 87. (2925) Left mandible, mola.
    2. 88. (2926) Left mandible, mola.
    3. 89. (2429) Left mandible, mola.
    4. 90. (2430) Left mandible, mola.
    5. 91. (5519) Right mandible, apical plate.
    6. 92. (5474) Right mandible.
  27. Figures 93-98. Rhithrogena pellucida mouthparts, Mandibles to Hypopharynx
    1. 93. (5472) Right mandible, mola.
    2. 94. (5513) Left mandible.
    3. 95. (2239) Right maxilla.
    4. 96. (2240) Right maxilla.
    5. 97. (5523) Hypopharynx.
    6. 98. (5524) Hypopharynx.
  28. Figures 99-104. Rhithrogena pellucida mouthparts, Hypopharynx to Labium
    1. 99. (3289) Hypopharynx.
    2. 100. (5525) Superlingua.
    3. 101. (2696) Labium.
    4. 102. (2920) Paraglossa.
    5. 103. (2447) Labial palpus.
    6. 104. (2914) Labial palpus.
  29. Figures 105-107. Rhithrogena pellucida mouthparts, Labium
    1. 105. (2278) Labial palpus.
    2. 106. (2647) Paraglossa.
    3. 107. (3025) Paraglossa.
  30. Figures 108-109. Ephemerella needhami mouthparts
    1. 108. Lateral view.
    2. 109. Ventral view.
  31. Figure 110. Diagrammatic cross-section of the mouthparts of Ephemerella needhami
  32. Figures 111-114. Schematic diagram of the maxillae and mandibles of Ephemerella needhami during feeding
    1. 111. Resting position, all cycles.
    2. 112. End of Stage 2 of the Maxillary Brushing Cycle.
    3. 113. End of Stage 3 of the Maxillary Brushing Cycle.
    4. 114. Beginning of Stage 3 of the Mandibular Biting Cycle.
  33. Figures 115-118. My other Ephemerella needhami mouthparts
    1. 115. Distal end of galealacinia.
    2. 116. Bipectinate-hairlike filtering setae.
    3. 117. Right mandible, posterior view.
    4. 118. Right mandible, distal end, dorsal view.
  34. Figures 119-124. Ephemerella needhami and Cladophora
    1. 119. Ephemerella needhami larva clinging to Cladophora.
    2. 120. Dorsal abdominal spines of Ephemerella needhami.
    3. 121. Cladophora clumps in natural habitat.
    4. 122. Cladophora filaments with and without epiphytes.
    5. 123. Epiphyte community on Cladophora.
    6. 124. Cladophora filament and associated epiphytes taken from the gut of an Ephemerella needhami larva.
  35. Figure 125. Number of epiphytes found per mm2 of Cladophora filament
  36. Figure 126. Width of randomly selected Cladophora filaments
  37. Figure 127. Gut contents of Ephemerella needhami larvae

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Modified 8/28/96