AN ABSTRACT OF THE THESIS OF Boyd Jay Hanson for the degree of Doctor of Philosophy in Fisheries presented on January 6, 1983 Title: Lipid Content and Fatty Acid Composition of Aquatic Insects: Dietary Influence and Aquatic Adaptation. Redacted for privacy Abstract approved: K.W. Cuins The effect of diet on the lipid content and fatty acid composition of aquatic insects was examined in a series of laboratory feeding experiments, through collection of species of various dietary types from natural populations, and through field introductions of insects into habitats with varying dietary resources. Dietary influences on growth and biochemical composition of the caddisfly Clistoronia magnifica were examined with a variety of diets including wheat, microbially conditioned alder leaves, and wheat plus alder. Larval growth of late instar C. magnifica was slower and resulting pupae were smaller and had less lipid on alder alone than on the diets with wheat. Increased temperature negatively affected the growth of insects on the itadequate alder diet, but not that of larvae receiving wheat. Biochemical analyses of foods and insects indicated that the higher growth rates of wheat-fed larvae resulted from the storage of lipids derived from the wheat. It appears that a source of carbohydrate for the synthesis of storage lipid is a major requirement for late instar C. magnifica. Lipid content and fatty acid composition was determined for representatives of 58 aquatic genera from 7 orders and 6 functional feeding groups. The majority of the insects had total lipid contents of 10% - 20% of total dry weight, and fatty acid compositions generally similar to those reported for related terrestrial species. An important exception was the presence in all individuals of the 20-carbon polyunsaturates, arachidonic and eicosapentaenoic acids, in quantities up to 7.2% and 24.7%, respectively, of the total fatty acids. The presence of relatively large amounts of these acids could be an adaptation to the aquatic environment, possibly related to membrane function. C. magnifica can apparently synthesize these acids from 18-carbon polyunsaturates, an ability not previously reported for insects. Fatty acid composition differed among functional feeding groups; most notably for the dietarily derived polyunsaturatess. The fatty acid patterns generally support the usefulness of the functional group concept as a tool in the study of the dietary ecology of aquatic insects. Insects introduced into natural habitats with varying food resources showed growth and lipid differences which supported the results of the laboratory feeding experiments. LIPID CONTENT AND FATTY ACIDCOMPOSITION OF AQUATICINSECTS: DIETARY INFLUENCE AND AQUATIC ADAPTATION by Boyd Jay Hanson A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Completed January 6, 1983 Commencement June 1983 APPROVED: Redacted for privacy Professor of 'Fis1eries in charge of major Redacted for privacy Head of Depact t'?isries and Wildlife Redacted for privacy Dean of Graduat Date thesis is presented January 6, 1983 Typed by Marcy Davis and Lisa Harris for Boyd Jay Hanson ACKNOWLEDGEMENTS Although the ideas expressed in this thesis are those of the author, they reflect the cumulative influences of many friends and associates.My major adviser, in addition to providing support and friendship and staying out of my way in center field (most of the time), has allowed me great latitude in my research and in my program, an advantage many doctoral students never enjoy. Dr. N.H. Anderson taught me how to spell and rear Clistoronia magnifica and provided valuable criticism of my research and writing. The other members of my committee, Drs. J.D. Hall, K. Croinack, and C.D. Mclntire were resources of guidance and assistance. R.R. Lowry and A.S. Cargill II appear as co-authors on manuscripts within the body of the thesis.The cooperation and advice of Bob Lowry provided the means for a neophyte biochemist to learn the techniques and enter the literature which made this thesis possible. Roney Cargill provided lipid analyses of the diets, and amino acid analysis of the diets and insects discussed in Chapters III and V. But more importantly, he greatly enriched this research through many hours of discussion, openly sharing his ideas and critically evaluating mine. The influence of his ideas and suggestions pervades the thesis. The Stream Team at O.S.U. is the atmosphere one would hope to find in an academic environment. The variety of approaches, opinions, and areas of expertise has been extremely stimulating. I thank the Departments of Animal Science and Agricultural Chemistry for the use of their facilities for biochemical analyses. The Computer Center provided computer time f or data anlyses. This research was supported by U.S. National Science Foundation Grants DEB-8022634 and DEB-8112455 and by U.S. Dept. of Energy Contract DE ATO6 79EV 1004. I especially thank my wife and family who willingly sacrifice as we pursue our goals. TABLE OF CONTENTS I. INTRODUCTION 1 II. DIETARY EFFECTS ON LIPID AND FATTY ACID COMPOSITION OF CLISTORONIA MAGNIFICA (TRICHOPTERA:LIMNEPHILIDAE) 3 Abstract 4 Introduction 5 Materials and Methods 7 Results and Discussion 14 Conclusions 23 III. LIPID CONTENT AND FATTY ACID COMPOSITION OF AQUATIC INSECTS 38 Abstract 38 Introduction 39 Materials and Methods 42 Results and Discussion 45 Acknowledgements 55 IV. POLYUNSATURATED FATTY ACIDS IN AQUATIC INSECTS: AN ADAPTATION TO THE COLD FRESHWATER ENVIRONNENT 78 V. CONCLUSION 86 VI. BIBLIOGRAPHY 89 VII. APPENDICES 101 AppendixI 101 AppendixII 109 AppendixIII 112 AppendixIV 115 AppendixV 117 AppendixVI 128 AppendixVII 130 AppendixVIII 139 AppendixIx 141 LIST OF FIGURES Figure Page II.]. Larval growth and mean pupal weights for Clistoronia magnif lea from four dietary treatments. 32 11.2 Mean amounts of total fatty acids, other lipid components, protein, and other body constituents of C. magnifica from four dietary treatments. 34 11.3 Mean amounts of the eight major fatty acids in total body lipid of C. magnifica pupae from four dietary treatments, and initial larvae. 36 AV.l Changes in total insect dry weight, and weights of lipid and fatty acids for C. magnifica reared from larval week 7 to pupation on one of four diets. 122 A. Alder 123 B. Alder plus supplement (wheat, beginning at 124 week 13). C. Alder plus wheat 125 D. Wheat 126 AV.2 Relative fatty acid composition of pupal C. magnifica reared on one of four diets. 127 AIX.1 Plot of relative retention times of fatty acid methyl esters vs. number of carbons. 143 LIST OF TABLES Table Page 11.1 Characteristics of food associated with feeding and growth of aquatic macrolnvertebrates. 27 11.2 Summary of feeding experiments conducted for the present study. 28 11.3 Relative growth rate, consumptive index, relative nitrogen change, and relative lipid change of 5th instar Clistoronla magnifica on a variety of diets 29 11.4 Relative growth rate, relative lipid change, and relative fatty acid change of C. magnif lea larvae in four dietary treatments at 15.6°C. 30 11.5 Sources and important metabolic functions of the predominant fatty acids of C. magnifica. 31 111.1 Lipid contents, as a percentge of wet weight or dry weight, of aquatic insects. 56 111.2 Fatty acid composition of aquatic insects, as percentage of total fatty acids. 59 111.3 Mean percentage lipid and fatty acid compositon of aquatic insects collected for the present study. 63 111.4 Mean fatty acid composition of aquatic genera from seven insect orders and terrestrial genera from three orders. 70 111.5 Mean composition of fatty acids in functional feeding groups for aquatic Trichoptera and Diptera. 72 111.6 Predicted classification of aquatic insects using functions derived by discriniinant analysis. 74 111.7 Significance of multivariate pairwise differences for order-functional group cells. 76 IV.1 Mean composition of polyunsaturated fatty acids in aquatic genera of seven insect orders. 84 IV.2 Mean composition of polyunsaturated fatty acids in functional feeding groups for aquatic Trichoptera and Diptera. 85 Page Table AI.1 Summary ofweights of C. magnificaat start and conclusionof initial introductionexperiment. 107 AI.2 Generalizedaverage weight of latelife stages of C. magnifica when reared at 15.6°C. 108 AII.1 Mean fattyacid composition, lipidcontent, and insect weight ofC. magnifica reared under four laboratoryor field condlitions. 111 AIII.1 Mean fattyacid composition, lipidcontent, and dry weightof Hydatophylax sp. introducedinto streams oftwo riparian types. 114 AIV.1 Nitrogen content of a variety of dietsgiven to C. magnifica and nitrogen, lipid, and cholesterol contents of insect larvae given thediets for 21 days. 116 AV.1 Fatty acid composition of C. magnificareared on one of four diets. A. Alder 118 B. Alder plus supplement (wheat after 6weeks) 119 C. Alder plus wheat 120 D. Wheat 121 AVI.1 Fatty acid composition of normal C. magnificaand of those with emergence difficulties. 129 AVII.1 Mean fatty acid compositions for insects ineach order-functional group cell. 131 AVII.2 Predicted order-functional group membershipof individual insects based on discriminantfunctions derived from a sampled portion of data. 132 AVIII.1 Relative fatty acid composition and lipid content of Lepidostonia unicolor and Allocosmoecuspartitus prepupae and pupae from coniferousand open sections of Mack Creek, HJ. Andrews ExperimentalForest. 140 AIX.1 Fatty acid composition of routinely preparedand hydrogenated methyl esters of two individuals each of Clistoronia magnifica and Hesperoperla pacifica. 142 LIPID CONTENT AND FATTY ACID COMPOSITION OF AQUATIC INSECTS: DIETARY INFLUENCE AND AQUATIC ADAPTATION INTRODtJCT1ON The lipids of aquatic insects have been generally ignored by both aquatic entomologists and insect biochemists, despite their important roles in processes studied by each group. Several recent reviews indicate the current interest in the feeding ecology of aquatic insects (Anderson and Cummins 1979; Anderson and Sedell 1979; Cuminins and Klug 1979; Wallace and Merritt 1980).