UPPER LETHAL TEMPERATURE RELATIONS 0F PARAGNETINA MEDIA WALKER (PLECOPTERA: PERLIDAE) Thesis for the'Degree of M. S. MTCHIGAN STATEUNIVERSITY DENNIS. R; HEIMAN 1968 A 4 . u ‘‘‘‘‘‘ ....... [finals [J LIBRARY 3 El-iichigan Sta ’6 7 ., .. 7‘ a i“ .. . J .3‘ .- ,r_.~"""‘f~ ABSTRACT UPPER LETHAL TEMPERATURE RELATIONS OF PARAGNETINA MEDIA WALKER TPEEEUPTERA: PERLIDAE) By Dennis R. Heiman Continuous water flow and static test apparatus were employed to evaluate the influence of acclimation temperature, size and sex, season, life stage, current velocity, and testing method upon the upper—lethal- temperature of the nymphal stage of the stonefly, Paragnetina media Walker. Lethal temperature was found to be raised by acclimation to higher temperatures throughout the thermal range examined. The rate of gain of heat resistance was approximately 5 C per day. Downard acclimation to colder temperatures was found to be a much slower process. Body size had no significant effect on upper-lethal— temperature during the summer and autumn test periods. Larger nymphs were significantly less tolerant than smaller nymphs during winter and spring. This hetero- geneous response to high temperature was attributed to differences in male and female nymphs and not to the effects of body size. Dennis R. Heiman Nymphs tested in static waters were significantly less tolerant than in comparable tests under flowing water conditions. An increase in current velocity significantly increased heat resistance at all lethal temperatures examined. Upper lethal temperatures showed seasonal differ— ences, independent of acclimation. Stonefly nymphs were least tolerant during the spring, when temperature resistance was influenced by sex, molting condition, and proximity to emergence. Experiments with early instar nymphs indicate that the period immediately following egg hatching is the most temperature sensitive life history stage. The distribution of head capsule measurements indi- cates 3. media requires two years to complete its nymphal growth. Maximum growth period is June to September. UPPER LETHAL TEMPERATURE RELATIONS OF PARAGNETINA MEDIA WALKER (PLECOPTERA: PERLIDAE) By Dennis RIEReiman A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Entomology and the w. K. Kellogg Biological Station 1968 ACKNOWLEDGMENTS I am greatly indebted to my major professor, Dr. Allen w. Knight, Department of Water Science and Engi- neering, University of California, Davis, for his con- tinued interest and encouragement, invaluable suggestions, and financial support throughout this study. I wish also to thank Dr. Gordon E. Guyer, Chairman of the Department of Entomology, Michigan State University, for his encouragement and for financial support in pro- curing necessary equipment and supplies. I am grateful to the members of my guidance committee: Drs. Allen W. Knight; Gordon B. Guyer; Roland L. Fischer; and Niles Kevern, for their critical reading of the manu- script. I should also like to express my sincerest thanks to Mr. Robert L. Lippson for his many helpful suggestions throughout the duration of this study, to Mr. Harold L. Allen for his guidance in the techniques of illustrating, and to Miss Jackeline Fort for her assistance in typing the rough draft of the manuscript. Finally, to my parents, Mr. and Mrs. Royal F. Heiman, a very special debt of gratitude for their continued patience, understanding, and interest throughout. ii This investigation was supported in part by Federal Water Pollution Control Administration Grant WP 01178-01. iii TABLE OF CONTENTS Page ACKNOWLEDGMENTS . . . . . . . . . . . ii LIST OF TABLES . . . . . . . . . . . vi LIST OF FIGURES o o o o o o o o o o o Vii INTRODUCTION . . . . . . . . 1 LIFE HISTORY AND ECOLOGY . . . . . . . . 3 Distribution . . . . . . 3 Collection. . . . . . . A Development . . . . . . . . . 5 Molting. . . . . . . . . . . . l2 Emergence . . . . . . . . . . . 13 METHODS AND MATERIALS . . . . . . . . . 19 Collection and Initial Handling of Stocks . . . . . . . . . . . . 19 Acclimation Tanks . . . . . . . . 19 Thermal Tolerance Apparatus . . . . . 20 Test Procedure . . . 28 Graphic Analysis and Statistical Evalu- ation of the Data . . . . . . . 3O Respiration . . . . . . . . . . 32 RESULTS. . . . . . . . . . . . . . 33 Field Observations . . . 33 Relation Between Lethal and Acclimation Temperatures . . . . . . . . 33 Rates of Gain and Loss of Heat Resistance. . . . . . . . . . 38 Rate of Gain . . . . . . . . . Al Rate of Loss . . . . . . . . . A1 iv Effects of Certain Factors on Thermal Resistance . . . . . . . . . Size and Sex . . . . . Static vs. Flowing Conditions. Effect of Flow. . . . . . . Lethal High Temperatures . . . . . Winter 0 O I O O O O O O 0 Spring 0 O 0 0 0 O O O O 0 summer. 0 O O O O 0 O O O 0 Resistance of Eggs and Newly Hatched Nymphs . . . . . . . . . Respiration. . . . . . . . . . DISCUSSION AND CONCLUSIONS. . . . . Relation Between Lethal and Acclimation Temperatures . . . . . . Rates of Gain and Loss of Heat Resistance . . . . . . . Effects of Size and Sex. . Lethal Temperature as Affected by Test Media and Rate of Flow . . . . Lethal High Temperatures . . . . Seasonal Variations . . . . . . Age and Development . . . . . . LITERATURE CITED . . . . . . . . . . APPENDIX. . . . . . . . . . . . . Page 81 82 88 88 9O 93 96 98 98 10A 108 115 LIST OF TABLES Table 1. Chemical Analysis of Augusta Creek Water Sampled During August, 1968. . . . . 2. Statistical Comparison of Time-Per Cent Curves from Upper—Lethal-Temperature Experiments Using Male and Female Paragnetina media Nymphs. . . . . 3. Comparison of Incipient Lethal Levels for Upper-Lethal-Temperature Experiments Conducted on Paragnetina media Nymphs in Static and Flowing Conditions A. Analysis and Statistical Comparison of Time-Per Cent Curves from Upper Lethal Temperature Experiments with Paragnetina media Nymphs Conducted at High Current Velocity (AO cm/sec) and Low Current Velocity (5.2 cm/sec). 20 C Acclimation Temperature . . . . . . . . 5. Details of Experiments Showing the Effect of Temperature on Oxygen Consumption (pl/g dry wt/hr) of Paragnetina media Nymphs. . . . . . . . . . vi Page 21 A8 56 6O 87 Figure 1. LIST OF FIGURES Page Seasonal Variation in Size Distribution of Paragnetina media Nymphs. Ordinate Refers to the Percentage Composition of Specimens in Each Size Class for Each Month. Sample Sizes: Jan, 202; Feb, 276; Mar, 135; Apr, 2A0; May, 281; Jun, 2A6; Jul, 271; Aug, 205; Sep, 118; Oct, 85; Nov, 102; Dec, 183 . . . 6 Numbers of P. media Nymphs Collected Each Month. Dashed Line Connects the Mean Head-Capsule Width for Each Month. . . 8 Relation Between Head-Capsule Width (mm) and Dry Body Weight (gm) for 3. media Nymphs During Summer (A), Autumn TBS, Winter (C), and Spring (D) . . . . . 10 Paragnetina media Egg and Recently Hatched Nymph. Egg Size, 0.4-0.5 mm. Nymph Size, 0.35 mm Head-Capsule Width . . . 15 Test Apparatus Employed in Determining Upper Lethal Temperature of P. media Nymphs Under a Constant Water Flow . . 23 Test Apparatus Employed in Determining Upper Lethal Temperature of Paragnetina media Numphs in Static Conditions. . . 26 Mean Monthly Water Temperatures Recorded for Dumont Creek in 1967-68. . . . . 3A The Relation Between A8-Hour Upper Lethal Temperature and Acclimation Temperature for P. media Nymphs During Winter, Spring and Summer . . . . . . . . 36 vii Figure Page 9. Rates of Gain and Loss of Heat Resistance in P. media Nymphs. Temperature ShiPt: (A) 10-30 c; (B) 10—20 C; (C) 20-30 C; (D) 30—10 C. Ninety- five Per Cent Confidence Limits for Each LT5O Are Indicated by a Vertical Line . . . . . . . . . . . . . 39 10. Comparison of Time-Per Cent Mortality Curves for Male and Female 3. media Nymphs at 10, 20, and 30 C Acclimation Temperatures. Lines are Labeled with Test Temperature and Date . . . . . AA 11. Comparison of Times to 50 Per Cent Mor- tality of Male and Female P. media Nymphs. Acclimation Temperatures 10 C (A and B) and 20 C (C). Test Dates, (A) A/2A/68—5/31/68; (B) 1/15/68- 1/29/68; (C) 5/1/68—5/31/68 . . . . . A6 12. Time— Per Cent Mortality Curves for P. media Nymphs Tested in Static Conditions at 10, 20, and 30 C Acclimation Temperatures. Lines are Labeled with Test Temperatures (C). The 95 Per Cent Confidence Limits for Each Time to 50 Per Cent Mortality are Indicated by a Horizontal Line. Bracketed Point Indicates Zero and/or 100 Per Cent Mortality. Based on Groups of Animals with Mean Weights from 15.09 to 40.11 mg. (A and B) December; (C, D, E) January; (F, G, H) February . . . . 50 13. Time— Per Cent Mortality Curves for P. media Nymphs Tested in Static Conditions at 10, 20, and 30 C Acclimation Temperatures. Lines are Labeled with Test Temperatures (C). The 95 Per Cent Confidence Limits for Each Time to 50 Per Cent Mortality are Indicated by a Horizontal Line. Bracketed Point Indicates Zero and/or 100 Per Cent Mortality. Based on Groups of Animals with Mean Weights from 3.31 to 26.87 mg. (A) June; (B, C) July; (D, E, F) August; (G, H) October . . . 52 viii Figure Page 14. Comparison of Times to 50 Per Cent Mor- tality for P. media Nymphs Tested in Static and Flowing Conditions. Lines Are Labeled with Acclimation Temper- atures . . . . . . . . . . . . 5A 15. Comparison of Times to 50 Per Cent Mor- tality of 3. media Nymphs Tested Under Conditions of High and Low Current Velocity. Acclimation Temperature at 20 C. The 95 Per Cent Confidence Limits for Each LT50 are Indicated by a Hori- zontal Line . . . . . . . . . . 58 16. Time-Per Cent Mortality Curves for 3. media Nymphs Tested in Flowing Conditions at, 10, 20, and 30 C Acclimation Temperatures. Lines are Labeled with Test Temperatures (C). The 95 Per Cent Confidence Limits for Each Time to 50 Per Cent Mortality Are Indicated by a Horizontal Line. Bracketed Point Indicates Zero and/or 100 Per Cent Mortality. Based on Groups of Animals with Mean Weights From 14.65 to Al.20 mg. (A, B, 0) December; (D, E) January; (F, G, H) February . . . . . 61 17. Time-Per Cent Mortality Curves for P. media Nymphs Tested in Flowing Conditions at 10, 20, and 30 C Acclimation Tempera- tures. Lines are Labeled with Test Temperatures (C). The 95 Per Cent Confi- dence Limits for Each Time to 50 Per Cent Mortality Are Indicated by a Horizontal Line. Bracketed Point Indicates Zero and/or 100 Per Cent Mortality. Based on Groups of Animals with Mean Weights from 2.68 to 63.36 mg. (A, B) March; (0, D) April; (E, F, G) May; (H) June. . 63 ix Figure 18. 19. 20. 21. 22. Time—Per Cent Mortality Curves for P. media Nymphs Tested in Flowing Conditions at 10, 20, and 30 C Acclimation Tempera- tures. Lines are Labeled with Test Temperatures (C). The 95 Per Cent Con- fidence Limits for Each Time to 50 Per Cent Mortality are Indicated by a Hori— zontal Line. Bracketed Point Indicates Zero and/or 100 Per Cent Mortality. Based on Groups of Animals with Mean Weights from 2.77 to 20.23 mg. (A and B) June; (C, D) July; (E, F) August; (G, H) October . . . . . . . . . Times to 50 Per Cent Mortality of P. media Nymphs During Winter for 10, 20 and 30 C Acclimation Temperatures. The 95 Per Cent Confidence Limits for Each LT50 are Indi- cated by a Horizontal Line. Based on Groups of Animals with Mean Weights from 1A.65 to A1.2O mg . . . . . . . . . Times to 50 Per Cent Mortality of P. media Nymphs During Spring at 10, 20 and 30 C Acclimation Temperatures. The 95 Per Cent Confidence Limits for Each LT50 are Indi- cated by a Horizontal Line. Based on . Groups of Animals with Mean Weights from 6.15 to 63.36 mg . . . . . . . . . Times to 50 Per Cent Mortality of P. media Nymphs in Three Contrasting Physiological States. Acclimation Temperature at 10 C Effect of Molting on Cumulative Per Cent Mortality of P. media Nymphs. (A, B, D), Test Temperature 33 C, Acclimation Temper- ature 20 C. (C), Test Temperature 33 C, Acclimation Temperature 10 C. Test Dates A/lO-A/IA. Nymphs Tested Either Separ- ately in Two Concurrent Trials (A and C) or Coincidently in the Same Trial (B and D) Page 65 67 7O 73 76 Figure Page 23. Times to 50 Per Cent Mortality of P. media Nymphs During the Summer for 10, 20 and 30 C Acclimation Temperatures. The 95 Per Cent Confidence Limits for Each LT50 are Indicated by a Horizontal Line. Based on Groups of Animals with Mean Weights from 2.68 to 20.23 mg . . . 79 2A. Times to 50 Per Cent Mortality of Newly Hatched P. media Nymphs Acclimated at Room Temperature (Approximately 2A C) . . 83 25. Mean Respiratory Rates of Nine P. media Nymphs Tested in Conditions of Slowly Rising Water Temperatures. Vertical Lines Indicate + One Standard Error. Weight Range 6.I6 to 11.86 mg. . . . . 85 xi INTRODUCTION Temperature is an intrinsic ecological factor deter- mining to a great extent, the abundance, the duration of the life cycle and distribution of many aquatic organisms. Few cases have been recorded of heat death in fishes or macroinvertebrates in the natural environment. Never- theless, information on their upper thermal tolerance be- comes increasingly important with the advent of more and larger electric-power-generating plants, particularly those with atomic reactors. Present estimates indicate that about one-sixth of all the water in the nation wide annual runoff, some 1,200 billion gallons per day, will be needed by 1980 to cool the country's steam electric stations. Thus, some 200 billion gallons of cooling water will be needed daily to meet projected needs (Sport Fishing Institute Bulletin, September, 1968). The present study was conducted to learn more of the upper lethal temperature relations among aquatic insects. The stonefly, Paragnetina media Walker was selected as the test organism. Hart (1947) divided the quantitative study of the lethal effect of temperature on organisms into three phases: (1) establishing in the laboratory a method of l assessment that permits prediction of lethal effects in the field; (2) application of this method to the description of the species; and (3) relating this laboratory description to the distribution and success of the species in nature. The use of P. media in a study of thermal tolerance is justified in that the temperature of the streams and rivers in which it occurs is subject to modification by alterations in the surrounding land, such as tree removal or by the installation of power generating plants along its banks which discharge large amounts of heated effluent. In a discussion of water temperature criteria to protect aquatic life, Mihursky and Kennedy (1967) stated: It is perhaps superfluous to indicate tempera- ture research is necessary on many more species. To focus on this need is the fact that no single species has been subjected to multivariate studies for all life history stages. Also, of the almost 1,900 fish species listed in the American Fisheries Society's list of fishes from the United States and'Canada (1960) less than 5% have been specifically examined for their response to temperature (even less for macroinvertebrates)--quite a small figure upon which to base water temperature requirements for the en- tire United States. 0 LIFE HISTORY AND ECOLOGY Concurrent with conducting a laboratory evaluation of environmental parameters it is essential to conduct field studies on the general biology and life—history patterns of the selected test organism. Laboratory sur— vival data can have ecological significance only by interpreting in light of information on environmental conditions. There have been few detailed life-history studies of species of PleCOptera in North America. Notable ex- ceptions include Holdsworth (19A1), Smith (1913) and Wu (1923). Distribution Paragnetina media is common and widely distributed throughout the state of Michigan. Recorded distribution includes nearly every county with suitable habitat. P. TEEEE.15 surpassed only by Perlesta placida Hagen in the number of streams in Minnesota from which the species has been collected (Hardin and Mickel, 1952). Field studies in Wisconsin further indicate that P. media is an important component of the northern stonefly fauna and that northwestern Illinois probably represents the extreme southern edge of its distribution in the central states (Frison, 1935). Collection During the months from October 1967 to September 1968 detailed observations and growth estimates were re— corded for P. T2912 in several streams close to the laboratory, in particular Dumont Creek. P. mgdia nymphs are commonly found associated with riffle areas having a rubble streambed. The nymphs were taken primarily under the rock substrate, although they were also found among logs, sticks, leaf packets, and a variable assortment of stream debris. The numphs were taken most frequently in the fast-flowing riffle areas but they also inhabit the quiet sections of the stream. The only other Plec0ptera collected from Dumont Creek were Isoperla signata and Taeniopteryx sp. Other associated fauna includes: Stenonema sp. (Heptageniidae); Baetis sp. (Baetidae); Hydropsyche sp. (Hydropsychidae); Gomphus sp. (Gomphidae) and the amphipod Hyalella azteca Saussure. Stonefly collections were generally made two or three times per month, with the frequency of sampling and number taken being dependent on the need for test organisms in the laboratory. The sample methods were not quantitative, but care was taken to collect all the nymphs from each hand screen sample in an attempt to obtain nymphs representing all available size categories. Development Figures 1 and 2 show seasonal variation in size distribution. The distribution of head—capsule measure— ments indicates that P. T2933 requires two years to com— plete its nymphal growth (Figure 2). The stoneflies exhibited maximum growth during June, July, August and September. By the end of October the year—class II nymphs have nearly reached maximum growth as indicated by head capsule width. The unexpected size distribution recorded for October reflects the small sample taken. Year-class I nymphs were completely over- looked during October, November, and December, and the numbers of very small stoneflies are not adequately repre- sented in the growth histograms. Very small nymphs were collected in January, however, and their rate of develop- ment was recorded for the remainder of their life cycle (Figure 2). Growth as indicated by head capsule width was minimal during the winter and spring months for the year—class II nymphs. This was not the case with total body weight, however. Figure 3 shows the relationship between head capsule width and dry body weight for each season. Little difference was noted in the summer and autumn data, but the apparent alteration in the slope of the Figure l.——Seasonal variation in size distribution of Paragnetina media nymphs. Ordinate refers to the percentage composition of specimens in each size class for each month. Sample sizes: January, 202; February, 276; March, 135; April, 2A0; May, 281; June, 2A6; July, 271; August, 205; September, 118; October, 85; November, 102; December, 183. "/0 of TOTAL Nooobfiifil T! ‘IIIVIvtvvvv O Y I T TTTT‘rTYTTYT—fiTYY a 'YITITTT 5 TfT Tfi— JAN FEB .5 MAR APR MAY JUNE 05 IO I3 20 25 30 35 40 {5 50 55 60 65 HEAD CAPSULE WIDTH (mm) AUG SEPT OCT 05 IO I5 20 25 30 35 £0 45 50 15 £0 £5 Figure 2.--Numbers of Paragnetina media nymphs collected each month. Dashed line con- nects the mean head-capsule width for each month. IO INDIVIDUALS Emergence ‘ Z 4 ' Lu 2 :Y ‘ 1 OCT nliillnllllllllllillllllllllflllllllilllllllll11111111111111111111114 Q '0. Q '0. O. “7 O '0. O. In. 0. ‘0. Q In. IR :9 w m If) ‘7 v r0 :0 N N — - o (tum) HiOIAA EI—IOScIVD OVBH SEP AUG JUL JUN MAY APR MAR FEB JAN DEC NOV I968 I967 10 Figure 3.-—Relation between head—capsule width (mm) and dry body weight for P. media nymphs during summer (A), autumH (B), winter (C), and spring (D). I0 IO P 100 I0 7.0 60 r 50 > 4.0 b 3.0 - (LUUJ) HIOIAA ll ‘Io 7.0 6.0 . 5.0 ~ 4.0 ~ 30 » 2.0 ~ 1.0 » IO 70 6.0 » EI—IOS dVD OVBH 5.0 . 4.0 ~ 30 » 2.0 ~ 1.0 » DRY WEIGHT (mg) l2 regression (Figure 3) during the winter and spring months is the result of an increase in total body weight. Head- capsule width measurements and personal observations on various body proportions during this period showed little or no increase. An increase in body weight is particularly evident during the spring, just prior to emergence, which may account in part for the noticeable distention of the nymphal body at that time. A large percentage of this weight increase can undoubtedly be attributed to the development of reproductive tissue in both male and fe- male nymphs. No external morphological alterations were apparent during this period of developing reproductive organs in the nymph. A bimodal p0pu1ation in the year—class II nymphs in December to May became evident in the course of plotting size distributions. Examination of emerging adults con- firmed the suspicion that these two p0pulations were the result of distinct size differences between male and female nymphs. Head—capsule widths for males and females were respectively 3.0-A.5 and A.6—6.0 mm. Similar findings have been reported for the stonefly PhasganOphora capitata Pictet (Alward, personal communication, 1968) and the mayfly Epeorus pliuralis Banks (Minshall, 1967). Molting As with all arthropods, growth in stoneflies is accompanied by molting. Occasional molts were observed 13 in nymphs collected from June to December. The cessation of growth in body dimensions during the mid—winter months resulted in an extensive intermolt period. The exo- skeleton became very hard with an attendant unicolorous dark shading. Accompanying the warming trend in water temperatures, most of the year—class II nymphs began to molt near the end of March and throughout April. Con- siderable change in the general pattern and texture of the exo-skeleton resulted from this molt. The nymphal bodies, following molting, were very soft and brightly colored with contrasting light and dark patterns. Emergence Emerging adults were observed during the last week of May in both 1967 and 1968. In the laboratory, adults emerged several days earlier when the nymphs were main- tained at 30 C. Males emerged before the females, for only adult males were collected during the week following the first emergence. Frison (1935) states that the adults of P. TEEEE are diurnal and congregate on vegetation near rivers in which the nymphs live. Mating takes place during the day. The adults emerge from nymphs which leave the water at night. Male and female stoneflies mated readily in glass rearing cages. A large egg mass formed at the genital opening of the female within one hour of copulation. The extruded egg mass (estimated at 200 to 500 eggs) 1A was removed from the female and placed in a shallow dish containing small stones and well-aerated stream water. Eggs collected before they had become too dry affixed readily to the first solid object they touched-—by means of a sticky gelatinous base and several fine strands sup- porting the egg (Figure A). The first hatching occurred after a 30 day incu- bation period at room temperature (2A-26 C). P. mgggg eggs contain a circular aperture near the top, creating an opening from which the nymphs escape as from a "flip— top box." Figure A shows a nymph of P. mgg$§_within 5 days of hatching. These tiny animals, though very active at this time, were completely helpless when dislodged from the substrate. Numerous cast skins were observed, indi- cating that the nymphs underwent intensive molting soon after hatching. Little growth was observed in laboratory reared-nymphs, and most died after several weeks. Attempts failed to locate either eggs or newly hatched nymphs in the field. Therefore, the year—class I animals plotted for July (Figure 2) were those reared in the laboratory. Year—class I nymphs were collected in August, and their developmental rate can be followed in the histograms. The variable size range of small nymphs taken in the field collections indicates an extended hatching period, or delayed nymphal growth after hatching, or 15 Figure A.--Paragnetina media egg and newly hatched nymph. Egg size, 0.A-0.5 mm. Nymph size, 0.35 mm head-capsule width. l6 o #00 ' .4 c . 9 etc! I C ‘ 17 both. The emergence period extending over several weeks suggests that hatching may also occur over an extended period. It appears that temperature is one of the factors influencing the initiation of stonefly hatching as well as triggering emergence. Brink (19A9) pointed out that the emergence of many species of Plecoptera occurred pro- gressively later in colder zones in Sweden. The main reason he gave was the slower growth in colder waters, which necessitates a longer larval life. Ide (1935), in studying the distribution of several species of mayflies in a stream, found that emergence started earlier and ended later near the source of the stream than at the lower levels. He postulated that the emergence period ended earlier downstream because the temperatures there soon became lethal for any nymphs that had not yet emerged. Only the egg stage could resist the high summer tempera- tures; nymphs from eggs hatching too early would be killed by the high temperatures in late summer or early fall. Thus, hatching usually started later at the lower stations than at the source, so that emergence also started later. The importance of considering the general biology and life history of the test organism becomes apparent in attempting to evaluate the effects of an environmental parameter such as temperature. Detailed information on 18 the life history of P. E3333 enabled this investigator to differentiate between male and female nymphal popu- lations, define periods of maximum growth, and delimit temperature sensitive physiological states such as pre- molt, pre—emergence, and egg hatching. METHODS AND MATERIALS Collection and Initial Handling of Stocks The test organism used was collected from Dumont Creek (T2N R13W S6) near Allegan, Michigan, a shallow stream, averaging less than 0.5 meter in depth, and characterized by swift water flowing over a loosely cemented rock and cobble bed. The stream receives a considerable quantity of allochthonous material from surrounding farm and woodland drainage. During the late summer months, dense growths of Cladophora glomerata be- come attached to much of the bottom substrate. Most of the nymphs were collected with a hand screen, while others were collected by hand picking of rocks. The animals were transported to the laboratory in a lO-liter polyethylene pail. Air and water temperatures were recorded in the field with a mercury thermometer at the time of each collection. Acclimation Tanks In the laboratory, the stoneflies were separated into one liter polyethylene bottles and supplied with 19 20 rock and bark substrates. Approximately 100 animals were placed in each flask. Sufficient dissolved oxygen was provided each bottle by means of air stones and compressed air. The bottles so provisioned were placed in constant temperature water baths. The baths were maintained at 10, 20 and 30 C (i 0.5 C) by electrical water heaters and a portable cooling unit. The accli- mation temperatures were chosen for their close approxi- mation to the normal thermal range of the test organism. Test animals retained in the laboratory longer than four weeks were discarded. Although test animals were not fed during acclimation or experimental evaluation, nymphs held for three weeks showed no significant loss of heat tolerance. A 10 to 20 per cent mortality due to cannabilism was indicated by a decrease in the number of test organisms during acclimation and the presence of partial body segments. Both acclimation and testing were conducted in Augusta Creek (TlS R9W S21) water transported from the stream to the laboratory in five—gallon polyethylene con- tainers. P. EEQEE is relatively abundant in this stream. A chemical analysis of Augusta Creek water is given in Table 1. Thermal Tolerance Apparatus Initially, the technique used in determining upper thermal limits was static water bioassay. P. media 21 TABLE l.——Chemica1 analysis of Augusta Creek water sampled during August, 1968.1 pH 8.2 Total alkalinity A.6 meg/1* CO3 Alk. 0.0 meq/l HCO3 Alk. A.A6 meq/l Boron 0.0 meq/l Chloride 0.15 meq/l Sulfate 0.A5 meq/l Nitrate 0.06 meq/l Sodium 0.11 meq/l Calcium 2.80 meq/l Magnesium 1.83 meq/l Potassium 0.0A meq/l 1 Provided in part by the Department of Water Science and Engineering, Chemical Laboratory, University of California, Davis. *Milliequivalent occurs in streams of considerable current velocity, however, and it was therefore conjectured that water flow had a significant effect on its thermal resistance. Consequently, both static and flowing bioassays were employed and the results compared. Six laboratory streams were constructed for use in obtaining upper lethal temperature information in a flowing water system. Wooden troughs, 120 x 10 x 9.5 cm, 22 treated with onert epoxy paint, were provided with a sub— strate of gravel and rock (approximately 8 cm in diameter). A centrifugal pump transported test water from a catch reservior (ten—gallon aquarium) to the upper end of the artificial stream, producing a continuous and uniform system of water recycling (Figure 5). Wire screen (#20 mesh) was provided at each end of the trough to stabilize the substrate and prevent the ani- mals from migrating or drifting from the test area. Constant temperature was maintained within the test apparatus with an immersion heater and bimetal or mercury thermoregulator coupled to a temperature controller in— corporating a sensitive relay, power relay, and a control— circuit transformer. With this arrangement, bath tempera- ture was controlled within : 0.1 C. .Aeration was accomplished during recycling by agitation of the water. No attempt was made to control lighting. Flow rate was regulated with an adjustable ball— valve in the output line of the centrifugal pump. Surface velocity of water flow in the test apparatus was determined with a cork float 1.5 cm in diameter. Mean current velocity was approximated by taking the average of five trials (time elapsed for the float to traverse the stream bed) and multiplying by a correction factor of 1.33 for water depth (Welch, 19A8). 23 Figure 5.-—Test apparatus employed in determining upper lethal temperature of P. media nymphs under a constant water flow. 2A 25 Rate of water flow was calculated from Embody's formula: wdal where: r = rate of flow (cm3/sec) w = average width of channel (cm) d = average depth (cm) 1 = length of channel (cm) a = constant, depicting bottom type (in this case equal to 0.8 for loose rock and gravel) t = average time required for float to traverse channel (sec) Water current velocity was maintained at 31.9 cm/sec (flow rate = 792 cm3/sec) for all lethal-temperature evalu- ations unless otherwise stated. The static water test apparatus and conditions were as follows: the animals were placed in 600-ml glass beakers provided with small rock and gravel substrate. The beakers were immersed in a water bath (five—gallon aquarium) and the temperature maintained within i 0.1 C by electric immersion water heaters. Compressed air was diffused throughout each beaker by means of air stones (Figure 6). The test apparatus generally performed quite satis- factorily. Centrifugal pumps containing bronze impellers, used in the early phase of the study, were found to create toxic conditions for the test organism. They were re- placed with pumps having a stainless steel shaft and 26 Figure 6.--Test apparatus employed in determining upper lethal temperature of Paragnetina media nymphs in static conditions. I\.) N ’. .9. s *8 T ‘C . ._ . ‘_ 'fi 0 9 4 e n In . . . 0.). ‘.‘G,‘¢ ‘0 ‘5‘ ‘. ‘ '9 , , .. ..-...»x;'.{/7;f;/.v‘?.7""’ v_‘a,-,-.'a_v2/. . ' f . . ' .- ' I. - l‘ ' . fl'r' ‘ ‘ I ' a ' _ . ‘ ' .. r . . 1 . 1‘ ‘ " 28 impeller. In several control runs made thereafter for periods up to three weeks, the nymphs remained healthy and active throughout. Florke g£_§l. (1960), in a study of metabolism and heat resistance, found that an accumulation of 0.02 mg Cu per liter from a coil in the water bath greatly accelerated mortality in the gudgeon. These examples illustrate the importance of any toxic substances in the water during bioassays. The electronic relay unit coupled to a mercury thermoregulator was found to be more dependable than the bi-metal thermoregulator for maintaining constant temper- atures over considerable periods of time. Dissolved oxygen was determined frequently by the Micro—Winkler Method, Azide Modification, using 0.001 N Na2S2O3 with replicate samples in both the static and flowing bioassay evaluations. The concentration of dis- solved oxygen in both sets of experiments was maintained near 100 per cent saturation. Oxygen stress was there- fore assumed to be nonexistent in effect on upper lethal— temperature levels. Test Procedure Acclimated stoneflies were introduced into test apparatus maintained at the same temperature used for acclimation. Then the water temperature was raised from the acclimation to the test level during a tempering period of approximately 30 minutes. This procedure 29 reduced shock or "heat rigor" that resulted if the temper— ature change was too rapid. Heat rigor was particularly evident in tests under static conditions. The 30—minute tempering period was initially assumed not to alter the acclimation temperature to any appreciable extent. This assumption was later confirmed in experiments testing the rate of gain and loss of heat resistance. This method presumably approximates natural conditions more closely and yields a better estimate of actual lethal temperature than does introducing the test organism abruptly from acclimation temperature to test temperature. This method should also be superior to the gradually raising of temper- ature until death ensues, a method which ignores the time factor in resistance. The organisms were inspected at frequent intervals, the time of inspection being dictated by experimental conditions. At each inspection, dead animals were removed and the time lapse recorded. The criterion of death was cessation of all movement and failure to respond to mechanical stimulation. This was determined to be a good approximation of death when numerous test animals deemed "dead" developed no sign of recovery when placed in a well aerated water chamber for 2A hours at room temperature. Numerous measurements were made on dead animals removed during the test period for assessment of size classes. Such measurements included head capsule width 30 (taken directly posterior to the compound eye), pronotum length and total body length (anterior margin of the head to the apex of the abdomen). Oven dry weights were determined by placing the test organisms in a drying oven at 10A 0 for A8 hours, desicat- ing for two hours and weighing to the nearest 0.1 mg. Graphic Analysis and Statistical Evaluation of the Data The measure of resistance was time to 50 per cent mortality at a given constant temperature. Results were analyzed by the methods of Litchfield (19A9). In this procedure, resistance times are determined by plotting accumulative percentage dead at the end of successive observation intervals against time of exposure. A line is fitted by inspection to the points, and the LT50 (lethal time for 50 per cent of the test population) is read from the graph. The variability of a test population is shown by the slope of the line. It is denoted numeri- cally by the slope function, S, and is defined as +LT l/2(LT8u/LT /LTl6); the greater the value of the 50 50 slope function, the more variable the response of the test population. The number of animals and the slope function are used in conjunction with a series of nomographs to obtain 95 per cent confidence limits for the LT50. These confi- dence limits are calculated by multiplying and dividing the estimate of the parameter (in this case the LT50) by 31 a factor, f. This procedure corresponds to adding the standard error to and subtracting it from the logarithm of the parameter. In other words, the factor, f, is equal to the antilogarithm of the standard error (Litchfield, l9A9). The equations are solved to obtain f by means of a nomograph. This method also allows for the comparison of two curves to determine whether they deviate significantly from parallelism. If the two curves are parallel within experimental error, they can then be compared for signifi— cant difference. Considered to represent indefinite survival was 10,000 minutes (approximately 7 days). In no instance in this study did 50 per cent mortality occur when tests were extended beyond this point, occasionally for as long as three weeks. This appears to be a fairly accurate estimate of the level of resistance to the direct lethal effects of high temperatures, later mortality would be considered the result of a complex of factors. The relation between test temperature and LT50 was plotted on semilogarithmic grid paper, and the points were fitted with a straight line by inspection. The point at which this line intercepted 10,000 minutes marked the incipient lethal level (ILL)--the level of the environmental identity (in this case temperature) beyond which the organism can no longer live for an indefinite 32 period of time (Fry, 19A7). Thermal resistance is the measure of the ability of an organism to withstand a temperature above the ILL. Respiration The effect of temperature on oxygen consumption of P. mgdgg nymphs was evaluated with a Gilson Medical Electronics refrigerated Differential Respirometer, Model GR 1A. The procedures for oxygen consumption evaluations and calculations followed those set forth by Umbreit g£_§P. (1959) and the Gilson Operating instruction manual (Gilson Medical Electronics, Middleton, Wisconsin). RESULTS Field Observations Figure 7 shows the mean monthly water temperature of Dumont Creek for the periods of October to December (1967) and January to September (1968). Summer water temperatures for Dumont Creek averaged 20 to 25 C, with a maximum of 28 0 recorded on August 20, 1968. As ex- pected, spring and autumn included periods of rapidly changing water temperature. Winter temperatures were quite stable, generally averaging between 0.5 and 2.0 C. An extensive covering of anchor ice was observed on the stream bed in January, 1968. No serious effects on the p0pulation of P. TEEEE were apparent, for large numbers were collected immediately following release of the ice. Relation Between Lethal and Acclimation Temperatures Figure 8 shows the relation between A8-hour upper lethal temperature and acclimation temperature. It is evident that lethal temperature was raised by accli- mation to higher temperatures throughout the thermal range examined. 33 3A Figure 7.-—Mean monthly water temperature recorded for Dumont Creek in 1967-68. 35 ——-1 0 05 C U fie O: F—« LllllllglllllllJllJllllJlilllllllJl In In 0 no In '0 8 N N — Q (0) EHOIVHBdWBI HEIIVM FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC JAN MONTHS 36 Figure 8.--Relation between A8—hour upper lethal temperature and acclimation temperature in P. media nymphs during winter, spring, and summer. 38 The rate of increased temperature tolerance with a rise in acclimation temperature was greatest in spring, and minimal in summer. This may reflect difficulty in attaining downward acclimation. The diagonal line on the right side of the panel is a construction line marking the locus of points where acclimation and lethal temper- atures are the same. The point of intersection of this line, indicating the level at which lethal temperature can no longer be raised by acclimation, corresponds with Fry's (19A7) ultimate upper incipient lethal temperature (35.3 0). Rates of Gain and Loss of Heat Resistance Experiments were conducted on four separate dates to determine the rates of gain and loss of heat resistance. Nymphs were collected from the stream during the period of near minimal water temperatures and slowly acclimated to 10 0. They were then immediately introduced to the 30 C acclimation bath. Groups of 8 to 10 were selected for determination of LT50's at various intervals thereafter, and tests continued until no change occurred in LT50. The selected test temperature was 35 C. From previous results, it was known that this temperature would result in rapid mortality of P. mgglg nymphs acclimated at 10 0, whereas nymphs acclimated to 30 C could tolerate this temperature for approximately A8 hours. The results are presented in Figure 9. 39 Figure 9.--Rates of gain and loss of heat resistance in P. media nymphs. Temperature shift: (A) 10-30 C; (B) 10—20 C; (C) 20-30 C; (D) 30-10 C. Ninety-five per cent confi- dence limits for each LT50 are indicated by a vertical line. A0 332 8.5a 20:42:84 0v 0N 05 00.x ma 295323004 0v 00m 1000. I OOON 000m OS 00 1 . OON A 00m 000. . OOON i 0000 (”IL“) 091') (U'u‘) 0911 :5 QOEMQ 20.553004 Om d 25 OOEma ZOFQEDUUA‘ 00m \H 000 b—QA—t . 00m . 00m . OOO. . 000m . 000m OOn . OOO_ . 000w g 0000 (mm) 0911 (”A”) 091'! Al Rate of Gain The acclimation temperature of P, mngg was raised from 10 to 30 C in approximately 120 hours (Figure 9A). The maximum level of resistance was maintained for 300 hours, when this series of experiments was terminated. In Figure 9B, acclimation was raised from 10 to 20 C in A5 hours, an apparent rate of 5 degrees per day, whereas in series 90 the rise was from 20 to 30 C in slightly less than A0 hours, an increase of 5.8 degrees per day. The rate of gain of heat resistance for P. ESEEE was found to be approximately 5 C per day. Decrease in resistance with prolonged acclimation did not significantly alter the LT50. Rate of Loss Figure 9D shows the rate of loss of heat resistance when animals acclimated to 30 C were introduced to the 10 0 bath. Resistance decreased only slightly during 39 days of holding. LT50's for tests at l and 39 days were not significantly different at the 0.05 level of significance. Exact rates were not determined, but it is concluded that downward acclimation is much slower than upward acclimation. Effects of Certain Factors on Thermal Resistance Size and Sex The factors of size and sex are inseparable in the present study with stonefly nymphs as it was impossible to A2 differentiate between male and female P. EEQEE on the basis of morphological characteristics. In the process of recording growth increments, however, it became evi- dent that two distinct and separate pOpulations were discernible (see Life History and Ecology section, Figure 2). By sexing adults it was concluded that the bimodal population was in fact a result of sex differ- ences with the larger nymphs being females and the smaller being males. Sex could thus be determined fairly accurately on the basis of nymphal size class. As mentioned in the Methods section, survival time was recorded for each animal along with measurement of body size. During the summer, autumn, and early winter periods, inspection of test results of individual experi— ments, and analysis of the correlation coefficient from regressions of body size and order of death (Snedecor and Cochran, 1967), failed to show any significant effect of size on resistance time. In these and all subsequent lethal temperature experiments, care was taken to select an equal number of representatives from all,size classes so that if any such effect was operating it would not alter mortality distributions seriously. Near the end of the winter test period, time-per cent curves began to indicate that size was affecting thermal resistance, though inconsistently. For this reason, a number of experiments were conducted in which A3 nymphs of two distinct sizes (and consequently different sexes) were tested separately and concurrently at the same temperature and with the same thermal histories. The results are presented in Figures 10 and 11, and their statistical comparison is given in Table 2. The hypothesis tested was: (a) H : b = b o m f (b) H1: bm # bf where bm = time-per cent curve for males, and bf = time- per cent curve for females. With 10 C acclimation (Figure 11A) male stoneflies showed significantly greater tolerance than females at test temperatures of 32-3A C but not at 35-37 C. All the nymphs in Figure 11A had recently molted, except those at 32 C. This could account for the unexpected loss of heat toler- ance at 32 C. In Figure 11B, males showed significantly greater tolerance than females at 3A C but not at 35 C. The time-per cent curves for 33 C were not parallel and could not be compared statistically, though a trend was evident toward greater tolerance by males at the lower test temperatures. The tests at 20 C acclimation were not compared statistically because of the "split probit" response exhibited in the time-per cent curves (Figure 10). The results show similar trends, the males being more tolerant at the lower test temperatures. The test at AA Figure 10.—-Comparison of time—per cent mortality curves of male and female P. media nymphs at 10, 20, and 30 C acclimation tempera— tures. Lines are labeled with test temperature and date. MORTALIT Y (°/oI _" "—7“ f' "T 7 "—_ —T_—__I 0 C . II‘I { . , . "2 33 to 9... """If ""7 - a} , I' ,0 at; ——— rr-Im r IImQ.‘ / I ’.'.'I‘ IS" IIIII ‘ PM ’0 | «OI 5" ,m —_ FfMAI r “no-I a ‘3 I «am I (vmw 9' / / ..-/ I ,LLALA.‘_A__‘_—L_A “JJ 6 N a—v-wT—v-Y- v ' ' 'fi ' a \a\ ' Io' no mm 0000 «u nnoo I0 C 3" I 20 C ”I / 'I IV!» I 9!! / L J" I b (2,”! f I I ”I .d _‘ I I) b" h ‘ ~ I ca 10» , . I 70 I " I I . 50‘ 'b I 5°. (no: so» — FEMALE (Iago) I" I SOI- — FEMALE (lace) L ------ MALE (Small) I f 3 I ------ MALE (smol) 0' / 4 I IOI- .' I I . I / .' I I" 2) ‘ I 2' .' / . , r . . '0 ALL—WWW (X) DC!) m D 00 000 m j 20 C I) .0 v I ’0» I 90 .1 I I a + ”I I 70+ I so) I soI — FEMALE (Iorqe) . r I r ----- - MALE IsmIII /.' IO» vol 1", I /’ .I . W—A A A AAAAAA A AA A AA- A *4 l0 IO a” one » l : 30 c 3. U I I0 c u» [/3 I‘M) I ”I: I ,.' . 32 L I P 60‘ 90 8‘ I ”I I .‘ I b m .- roI . aoI E I ' 7 _ .' 50‘ P so — FEMALF (Iago) .4" I 307 —- FFMAI E (Icon) 01 ‘ I . . .. . MAI f HMO") '1‘ I I MN P (“"0”) . . to) ,1 "o I lot / v A //. I I / z n > " I I» / I .H). NEW“ A _t.._a l ..¢...A_A_4_“Ama___L__A_A._A_A‘AA__ *b—FHJ FXPOSURE (mun) 46 Figure ll.--Comparison of times to 50 per cent mortality of male and female E, media nymphs. Acclimation temperatures 10 C (A and B) and 20 C (C). Test dates, (A) u/2u/68-5/31/68; (B) 1/15/68-1/29/68; (c) 5/1/68—5/31/68. lfl 0000. 009 EC: Om .5 oo. 008. 000. N 2.68.? 59: 82V “33%: oIIo 2.69-9 £9; 82: 532 v-5; L S awn mm 52 oo. o. / (I 1 I I 1/ AEEowév £9; 82: mag/Gal // ”559-8 59: 085 34.2 9---; V” A Eeoo .3 £9; 89: 532mm 1 AEEmv-Om £93 89: mugs). 9 1-; on an hm mm vn .nn on hm mm BHHLVHEdWEL i831 (CD) H8 .m0.0 QOQQPQGOOG .HO HQ>$Q$ .Amzmfiv Ufimfiwnopfiqd empomhmp Hm.fi mm.H mm mmxfi empomhma :N.H mo.a empgmoom mo.H mH.H am mm\H empomnmp mm.a Hm.H empamoom mo.H mo.H am mH\H empamoom m:.H oo.H empgmoom m.H mm.a mm mH\H empomnmp mm.H mm.a empamoom m.H oH.H mm. Hm\m empomhmp :0.H mm.H empgmoom mm.H :O.H mm s\m empomhmp ao.m :m.m empamoom mw.H mo.H :m :m\: empamoom om.H Hm.H empgooom :m.a :0.H mm mxm empgmoom :m.a sm.H empamoom sm.H mm.H mm o\m umpgmoom mH.H :H.H *Umugmoom mm.fi wo.H pm ©\m *a9 u ”a VMWMWMWV wwwmwmv Emmammma Ammmmfimav Ammmwmv abww mpma u m mm mm a u m m pmme w .Amm>950 ammo meImEflp mo mflmzamcm how xfiwcmmqw mmmv Hmnasmc weave mcfipmcmmmwm mHmEmm vzm mama wcflms mpcmsfihmmxm mmSpmanEmpIHmnpmalhmggs Eopm mm>h50 pcmo pmalwefip mo somfithEoo HQOfipmempmnl.m mqm<9 ’49 33 C in Figure 110 was complicated by the close proximity to emergence. At high test temperatures the ensuing death pro- cesses are apparently so rapid that the expression of separate male and female tolerance levels is suppressed. Since no significant differences in resistance of different size groups were found in the summer and autumn, the differences found during winter and spring are probably attributable to physiological prOperties associated with sex, and not with the size of the organism. Static vs. Flowing Conditions As stated earlier, the static bioassay method was replaced as the test procedure by a flowing bioassay apparatus. Static tests were conducted for comparative purposes during the summer and winter months, and the results are shown in Figures 12 and 13 (detailed graphic analysis of the time—per cent curves is given in the Appendix). Figure 1“ compares static bioassay results with results in flowing water. The animals were considerably more resistant to temperature effects in the flowing system than in the static. Comparison of concurrent tests by the method of Litchfield (19H9) confirmed this. The differences were significant at the 0.05 level in all tests compared. The trend was similar for estimated ILL's (Table 3). 50 Figure 12.--Time—per cent mortiality curves for 3. media nymphs tested in static conditions at 10, 20, and 30 C acclimation temper- atures. Lines are labeled with test temperatures (C). The 95 per cent confi— dence limits for each time to 50 per cent mortality are indicated by a horizontal line. Bracketed point indicates zero or 100 per cent mortality. (A, B) December; (C, D, E) January; (F, G, H) February. Based on groups of animals with mean weights from 15.09 to “0.11 mg. 51 g CHEF EOE EXPOSURE (mun) 50 Figure 12.--Time-per cent mortiality curves for 3- media nymphs tested in static conditions at 10, 20, and 30 C acclimation temper- atures. Lines are labeled with test temperatures (C). The 95 per cent confi— dence limits for each time to 50 per cent mortality are indicated by a horizontal line. Bracketed point indicates zero or 100 per cent mortality. (A, B) December; (C, D, E) January; (F, G, H) February. Based on groups of animals with mean weights from 15.09 to “0.11 mg. 51 flab >tu_<._. EOE EXPOSURE (man) 52 Figure l3.—-Time—per cent mortality curves for P. media nymphs tested in static conditions at 10, 20 and 30 C acclimation tempera- tures. Lines are labeled with test temperatures (C). The 95 per cent confi- dence limits for each time to 50 per cent mortality are indicated by a horizontal line. Bracketed point indicates zero or 100 per cent mortality. (A) June; (B, C) July; (D, E, F) August; (G, H) October. Based on groups of animals with mean weights from 3.31 to 26.87 mg. 53 20C r PI! [I DILI‘III‘ .Ll’ll'llblblill'I-m u m m w m o 2 20C fi>>b 20C 30c C C m F u- n m w w o 2 I101/1 I 3 A/ I, I‘llm 3 x I. ./ p l/ A k / 'I I/’ l A If 9; . I. b, ,4 d/A . . u.// ,,/ A 1.7/1 . . / lu/x/ l /N/ < / 4 4/. i . k c m B I n m m w n a u 8 >§h§ IOC EXPOSURE (rm) 5“ Figure 1A.-—Comparison of times to 50 per cent mortality for 3. media nymphs tested in static and flowing conditions. Lines are labeled with acclimation temperatures. TEST TEMPERATURE (C) 38 37 36 35 34 33 32 E 39 38 37 36 35 34 33 32 WINTER LT 50 (min) — o——o FLOW ..-_--. STATIC l “% IO IOO IOOO IOOOO 30 L 30, SUMMER * 200.‘ _ |O&. — o——«> FLOW ~----A STATIC A “x“. 0 DO IOOO IOOOO 56 TABLE 3.--Comparison of incipient lethal levels for upper-lethal-temperature experiments conducted on Paragnetina media nymphs in static and flowing conditions. ILL (0) @001 Winter Summer emp (C) Static Flow Static Flow 10 30.6 32.2 33.0 33.0 20 32.1 32.8 32.7 33.3 30 33.5 33.6 32.8 33.7 The animals appeared less able to adapt to higher temperatures when tested in static conditions. The nymphs would often succumb to heat rigor during the rise from acclimation temperature to test temperature. Even when death did not ensue immediately, the nymphs were frequently unable to regain normal muscular coordination. This resulted in excessive test variability as indicated by the rather large value of S in some of the statistical curves. Under flowing-water conditions the nymphs retained normal mobility until near death. These results indicate a significant difference in thermal resistance between the static and flowing-water experiments. Consequently, further tests were conducted with the same apparatus but regulating the rate of flow. 57 Effect of Flow Figure 15 presents the results of lethal temper- ature experiments at current velocities of 5.2 cm/sec (flow rate = 171 cm3/sec) and 40.0 cm/sec (flow rate = 1312 cm3/sec). Table A gives the graphic analysis and statistical comparison. The results indicate that thermal tolerance was significantly greater for the higher current velocity at all test temperatures. Lethal High Temperatures Upper—lethal—temperature experiments were conducted at acclimation temperatures of 10, 20, and 30 C for all seasons from October, 1967, to September, 1968. Time- per cent curves for these experiments are presented in Figures 16-18 with detailed graphic analysis of the time- per cent curves given in the appendix. Replicate tests were conducted when the test results appeared questionable. LT50's obtained for similar seasons and under the same conditions were combined whenever possible. Winter Figure 19 presents the relation between test temperature and median resistance time for December, January, and February. The LT50's for this period are aligned along a straight line which passes within their 95 per cent confidence limits. This is partially a result of the uniform effect of acclimation temperature 58 Figure l5.--Comparison of times to 50 per cent mortality of P. media nymphs tested under conditian of High and low cur- rent velocity. Acclimation temperature at 20 C. The 95 per cent confidence limits for each LT50 are indicated by a horizontal line. 59 E75 02%. 0000. 000. 00. O. (I Games NE 26.7.. 264m 9:-.. I 8mm>co 00$ Bod FmE I L _ Nm mm in m r") (O) EHHLVHBdWEi 183i mm hm 60 .mmpscfie oovoH SH mpfifimphos pcmo pom om pom pom UHQ** .mo.o mocmpdmoom mo Hm>oq* .Amnmav pamfimnopfiqfi mm.a AnzmmIBOsz ooze mm m.m ** 0.0: mH~.H Aomwmlooomv oom: m.m ompomhmp 2m mH.m Aoammalnmmmv comm 0.0: Hm.H Ammmummmv ems m.m ompomhmg mm 0H.H Anmmmlmoamv ozmm 0.0: mom.a Ammznmamv mwm m.m pmpommmp mm Hm.H Amzmalmmmv omoa 0.0: Hm.a Amoaummv as m.m ompommmb um mm.a AmmHImHHV ozfi 0.0: Amp n ma ”om m Acaev mpfiefla csoo on Aomm\eOV pcmo pod mm Ugo omeq damp pmoe mpfiooam> unoppso H.®L:pmthEmp coapmefifioom 0 ON .AommNEo mhmv mpfloowm> pampLSQ 30H paw Aomw\Eo ozv mpfiooam> pcmhpzo swan um Umpozvcoo mnmEz: mHUoE mcfipwcwmgwm Sufi: mucmefipmmxm mLSpmmeEmp Iamnpmaupwdgs Eomm mm>a:o pcmo pmaloeflp no comfimeEoo HmOHpmempm cam mHmmec¢|l.z mqm<9 61 Figure l6.—-Time—per cent mortality curves for 3. media nymphs tested in flowing conditions at 10, 20, and 30 C acclimation temperatures. Lines are labeled with test temperatures (C). The 95 per cent confidence limits for each time to 50 per cent mortality are indicated by a horizontal line. Bracketed point indicates zero and/or 100 per cent mortality. Based on groups of animals with mean weights from 14.65 to A1.2O mg. (A, B, C) December; (D, E) January; (F, G, H) February. 3‘ IO M“& 35 I000 36 37 35 .h.-‘....... )7 A .A..... _ 62 I000 7 ,VLA- L A “L I00 100 IOC _l,_. ,t --,.... IO 90 'OC‘C >F3 f I E . 10; If] I c II," o , I I I (I D J I H I / I ’ ‘ . 0 1/ J 0/ I -_-,,. .,E_._,,.i __-,__ - i.“ ,_.____._.-_.A+._.J - k . L , [6 I6: 766;: IGCIOO .0 _ I66 ‘ ‘ ”*mu—P * I&m EXPOSURE Umn) 63 Figure l7.—-Time-per cent mortality curves for 3. media nymphs tested in flowing conditions at 10, 20, and 30 C acclimation temperatures. Lines are labeled with test temperatures (C). The 95 per cent confidence limits for each time to 50 per cent mortality are indicated by a horizontal line. Bracketed point indicates zero and/or 100 per cent mortality. Based on groups of animals with mean weights from 2.68 to 63.36 mg. (A, B) March; (C, D) April; (E, F, G) May; (H) June. 614 L 4 i 3/, .I../I/ o/ /W/ .f/I/l/ ~W _ m H c r L! 5 PItLI',I' Lwl Ir[.Ll[nllm m w m w m- m 2 2 /; I m a ,--/ i 4i B .l 4/! I. A M/ I II .IIPI- /// I- . III”: 35 )7 '/ / I I . I .- I ‘000 “v inf/I A i I Ill I I‘JI‘IIE. A “NJ/{OJ 0 I?! .. i («F 0 .II- -I m IA.’ i . 7,- u / I 4r m / Am a v. I fax-II- 0 5h, _ w -I/ 4r,- 0 I . , bf, - - i 1.!I q 0 . 4/ I! . II» M . J Am 20C Ioooo 1 3 30v >H_I_H34Hm02 loco 00 I" . 30C H Hg il-Uhj I000 low 30C I.L . (I I ..-v||rlr| (mm) EXPOSURE "“".~ .‘ ,_ ‘O‘Ju 67 Figure l9.--Times to 50 per cent mortality of 3. media nymphs during winter for 10, 20, and acclimation temperatures. The 95 per cent confidence limits of each LT50 are indi- cated by a horizontal line. Based on groups of animals with mean weights from 1U.65 to ”1.20 mg. 68 3E: om: O. Om mm._.Z_>> ON 0. mm mm #0 mm mm hm mm (3) BHHLVHEdWELL .LSEIJ. 69 on thermal resistance, with the actual influence of acclimation on lethal temperature being most accurately manifested during this period. The minimum temperature found to result in 50 per cent mortality during the winter test period was 33 C following 10 C acclimation. The ILL was estimated for each acclimation temperature, and the maximum temperature at which 50 per cent of the animals could survive in- definitely was found to be 32.2 C (Fig. 19). The relation between lethal temperature and acclimation temperature (Fig. 8) indicates that this incipient lethal level would be slightly less if the animals were acclimated to the actual environmental temperature, which ranged from 1 to M C during this period. Spring Response to abnormally high temperature was found to be more variable in the spring months (March, April, May). Figure 20 presents the results of upper-lethal- temperature experiments conducted in the spring. Three separate and distinct physiological states could be assigned to the nymphs during the spring period: 1) Prior to Spring Molt (March l-April 1H). Ecdysis is important in the physiology of stoneflies be- cause of the rapid growth which accompanies molting. Cast skins were observed occasionally in the field and in the acclimation baths throughout most of the year. 70 Figure 20.-—Times to 50 per cent mortality of 3. media nymphs during spring for 10, 20, and 35 C acclimation temperatures. The 95 per cent confidence limits of each LT50 are indicated by a horizontal line. Based on groups of animals with mean weights from 6.15 to 63.36 mg. 71 Elev Om: 0000. 000. OO. O. .34 q . _ u q 1 q J q a . q q a 4 &. ’0 vv I Ml. we V I . OZEQW W. mam; v V I o ow o. It I mm mm wm mm mm hm mm (3) EHfllVHEdWEl _LSE_L 72 During the winter and early spring months very little growth was evident (see Life History and Ecology section, Fig. 2) and consequently an extensive intermolt period occurred at this time. As this intermolt period ap- proached termination the nymphal exoskeleton became very hard and darkened. It was at this time that the nymphs reached maximum vulnerability to high temperatures. On April in, nymphs acclimated at 10 c and tested at 31 C showed an LT50 of 7,200 minutes. This was the minimum temperature found to result in 50 per cent mor- tality. The trend of thermal resistance for test temper- atures 31-3u C in Figure 21 gives an estimated ILL of 30.2 C. In Figure 20 a straight line fitted by inspection to all the test temperatures for the spring period (31-37 C) at 10 C acclimation gives a somewhat higher estimation of the ILL because of increased resistance in tests conducted earlier in the spring. Heat resistance decreased continually until the time at which the Spring molt takes place. Attempts to acclimate the nymphs at 30 C resulted in a high percentage of mortality at this time. Results from other tests indicate that, in static conditions of accli- mation, the ILL was below 30 C. Nymphs in the pre-molt condition exhibited con- siderable body swelling prior to and accompanying the death from high temperature. This phenomenon gave rise to the suspicion that the osmoregulatory process was breaking down. This hypothesis is discussed in a later section. 73 Figure 2l.--Times to 50 per cent mortality of 3, media in three contrasting physiological states. Acclimation temperature at 10 C. 7U- EEV Om-_-I_ 0000. 000. 00. o. to MJ / Cw , §__a<-_eeo§ So: mac-mm oil-o .- .../ . 0,9», geese-egg mozmommzm man-.2 .121 » 6.62-9 :53 850.2 552 7----.. - on mm 8, vm mm mm hm (D) BafllVHBdWBL .LSBJ. 75 2) After lst Spring Molt (April l5—May 15). Rising water temperature was accompanied by extensive molting throughout April. The newly molted nymphs ex— hibited a significant increase in tolerance to high temperature. Figure 22 compares the effects of high temperature on nymphs in two contrasting physiological states. These data indicate that the nymphs were significantly more resistant after molting. Further evidence is shown in Figure 21, where the ILL was esti— mated at 32.6 C, compared with 30.2 C for nymphs in the pre-molt condition. Abnormal body swelling was not observed in newly molted nymphs dead from exposure to increased water temperature. 3) Prior to Emergence (May l6-June 1). Near the middle of May the nymphal body becomes slightly swollen and the wing pads become very dark. This period is accompanied by a decrease in thermal resistance (Fig. 21). Response to high temperature is extremely variable at this time, often resulting in a "split probit" in mor- tality distribution (Fig. 10). This response is indi- cated by a horizontal section in the regression. Such a split is generally interpreted as two mechanisms of death, both in response to high temperature in the pre— sent study. Temperature resistance at this time appears to be governed directly by proximity to emergence, with 76 Figure 22.--Effect of molting on cumulative per cent mortality of 2. media nymphs. (A, B, D) test temperature 33 C, acclimation temper- ature 20 C; (C) test temperature 33 C, acclimation temperature 10 C. Test dates A/lO-A/lu. Nymphs tested either separately in two concurrent trials (A and C) or coincidently in the same trial (B and D). 77 EE: mmamdem 0000. 000. 00. o. I N So: mmfiq o - o 50.2 mmohmm . - o. :9: \ :08 occau occa .. i--. ....... e3 36 \ - on .32 on. - boss; #05 i on . I ox. L - om G 1mm 88. 80. I om. o. \ I .\ o .o. . - x. 502 3E4 0 en \. 502 MES-hm . i - on . Os A I o I A AL L l .1 L . o . . ~ ._.|_O_2 mmkmd o .502 “Eon—mm o .0. - On I I1 . on IIIIIIIIIII I, llllll A \ .0. «30: 09 3E3 80m . . Om m . mm L L 0000. 000. 09 O_ 23; S. \\ I N 3222.. ox. mm \ - .592 EMF-id 0 F492 MEG-1mm o - o. \. i \ 4 cm I . on i ON I 00 mm (°/o) All—IVLHOW 78 nymphs very near to emergence being less tolerant. Stonefly nymphs subjected to water temperatures up to 30 C successfully completed their life cycle whereas temperatures in excess of 30 C resulted in inability to emerge from the nymphal stage. Nymphs are apparently more tolerant during emergence than in the pre-molt condition (Fig. 21). Summer The months of June, July, and August are charac- terized by increased water temperature and rapid stone— fly growth. It is during this period that the nymphs acquire their greatest level of heat resistance (Fig. 23). Few tests were conducted in the autumn months, but the results were similar to those in the summer. This might be accounted for by the very slow rate of downward accli- mation of stonefly nymphs and the fact that environmental water temperatures were decreasing at this time. For this reason results for summer and autumn are combined. Table II (Appendix) compares the ILL for each accli- mation temperature during winter, spring and summer. No attempt was made to compare individual time-per cent curves, since the differences in tolerance are quite clear-cut and computation of the probability of their significance would be superfluous. A comparison of the ILL's does indicate that the trends are real and indicates a temperature tolerance that is maximum in summer, mini— mum in spring, and intermediate in winter. 79 Figure 23.-~Times to 50 per cent mortality of 2. media nymphs during summer for 10, 20, and 30 C acclimation temperatures. The 95 per cent confidence limits of each LT50 are indicated by a horizontal line. Based on groups of animals with mean weights from 2.68 to 20.23 mg. 80 Leevomen 080. 80. oo. o. OWN; .- 6. J MN. vv , «.- mmszam - we be e. - on ON mm mm vm mm on Km mm mm (3) BHDLVHEdWBL .LSELL 81 Resistance of Eggs and Newly Hatched Nymphs On June 1, 1968, flasks containing E. media eggs fertilized and deposited in the laboratory were placed at l, 10, 2M, 27, and 30 C. This was done in an effort to determine the effect of temperature on egg hatching rate and survival. This experiment was rather unsuc- cessful since the 10 and 27 C eggs were destroyed by Saprolegnia sp. and the l C eggs by dessiccation. 0n July 1, 1968 a large number of nymphs were hatched at 24 C. Eggs at 30 C hatched on July 2 but nymphs died in the next several days. Laboratory reared 3. media nymphs were tested within 1 to 3 weeks of hatching to assess the effects of high temperature on the survival of young stoneflies. Static lethal temperature experiments were con- ducted in lOO-ml glass beakers. The animals were ex— tremely difficult to transfer from the rearing bath to the test bath because of their very small size. Often the nymphs were unable to grasp the small stones provided in the test bath, and therefore floated helplessly to the surface where they were caught, unable to break the sur- face film and return to the substrate. Despite con- siderable difficulty in the testing procedure, the data obtained were sufficient to give some insight on the thermal resistance of very young stonefly nymphs. 82 Time-per cent curves were plotted, and LT50's estimated, for test temperatures 30 to 3” C. Because of the uncertainty and variability of the results, interpretations must be considered tentative, and no detailed graphic analysis was attempted. The results of the tests (Fig. 2“) indicate that nymphs are more susceptible to high temperature when newly hatched than in the later stages of their life cycle. Figure 2A gives an estimated ILL of approximately 29 C. If the trend is real, some degree of tolerance is apparently lost immediately after hatching for eggs hatched at the 30 C rearing temperature. Respiration Figure 25 and Table 5 present the effects of slowly rising water temperature on the respiratory rate of 3. media nymphs. The data represent the mean rates of 9 animals from the size class 6.16-ll.86 mg dry body weight. Rates were ascertained over a 20-hour period of increas- ing water temperature: one-hour at each temperature with one-hour intervals for adaptation to the next- highest level. 3. media nymphs appear to remain metabolically active up to the lethal death point. A rather sharp in- crease in 002 is observed at 33 C, which might possibly indicate an "overshoot" in oxygen consumption resulting from stress from the approaching incipient lethal level. 83 Figure 2u.—-Times to 50 per cent mortality of newly hatched P. media nymphs acclimated to room temperature (approximately 2“ C). (C) TEST TEMPERATURE 35 34 33 32 3| 3O 81-l _1 l 1 11111 IOO IOOO LT 50 (min) 85 Figure 25.——Mean respiratory rates of nine Paragnetina media nymphs tested in conditions of slow y rising water temperatures. Vertical lines indicate + one standard error. Weight range 6.16-ll.86 mg. 8V mePEMn—EMH B mm on em mm mm _.m on mm mm 86 .. - d u u q q u u I O X - 82 m 3 - N - 8! MW m , 232.6 :72 .26.. 35%. L - com. PI. 2222.53 .28.. 23%... W . u d . . u u - 8- w u u N - . m m - 88 m1 m m .w _ u 1 08” p u u - M . . . u M m u - 8% A U.- 1 Id TABLE 5.--Details of experiments showing the effect of temperature on oxygen consumption (ul/g dry wt/hr) of Paragnetina media nymphs. Temperature Oxygen consumption (C) n K Range Sf 28 9 1588.0 3476.0-726.6 308.0 29 9 1293.7 1969.8-1033.3 102.1 30 9 11165.7 2061.4-1183.2 98.6 31 9 1690.1 2519.5-1183.2 150.1 32 9 1963.4 3093.0—1508.6 178.0 33 9 2725.6 “558.1-206l.5 280.“ 39 8 2625.1 3951.2—205u.6 213.1 35 8 3168.3 ““73.1-2606.5 209.7 36 8 3UO3.6 4971.6-2120.7 320.0 38 8 3300.0 U328.9-2725.0 180.1 It is also apparent that at the thermal death point the respiratory rate had not decreased, which demonstrates that "heat death" does not imply that all the constituent tissues are necessarily dead. DISCUSSION AND CONCLUSIONS Relation Between Lethal and Acclimation Temperatures The limits of an animal's tolerance zone are not "fixed" but depend to some extent upon the condition of the animal (Andrewartha and Birch, 195“). Temperature effects are greatly modified by the past thermal history of an individual; hence, acclimation state should be qualified. Acclimation, in regard to temperature adaptation, refers to the animal's compensation for a persistent change in environmental temperature, usually in the laboratory (Prosser, 1962). The mechanisms involved in acclimation have been reviewed and discussed by several workers (Bullock, 1955; Fry, 1957; Prosser, 1962). Generally, as acclimation temperature increases, lethal temperatures progressively increase. Examples of this phenomenon are numerous, and only a few selected examples are presented here. Fry (1992) reported that in the goldfish, Carassius auratus L., a 3 C increase in acclimation temperature was axxzompanied by a rise in lethal temperature of about 1 C. 88 89 A considerably lesser rate of change was obtained for yearling speckled trout, Salvelinus fontinalis Mitchill, which required a 7 C change in acclimation temperature to produce a l C change in lethal temperature (Fry ee_ai., 1956). When acclimation temperature was raised from 10 to 20 C the 2A-hour lethal temperature was increased by 1.9 C in Gammarus fasciatus Say, 1.3 C in Asellus intermedius Forbes, 0.5 C in Gammarus pseudolimnaeus Bousfield, and apparently not at all in Hyalella azteca Saussure (Sprague, 1963). Mcleese (1956) showed that the lobster, Homarus americanus Milne-Edwards, tested in water containing dissolved oxygen of 6.“ mg/l, raised its lethal temperature 3 C when the acclimation temperature was increased from 10 to 20 C. The lethal temperature determined for the crayfish, Orconnectes rusticus (Girard), was increased approximately 1 C for each 10 C increase in acclimation temperature (Spoor, 1955). Lethal temperature in the giant scallop, Placopecten magellanicus (Gmelin), increased 1 C with a 5 C rise in acclimation temperature (Dickie, 1958). An exception to the general rule is presented by Matutani (1961). He found that the COpepOd, TigriOpus Japonicus Mori, had a greater heat resistance when acclimated at 5 C than at 10 C. This inconsistencyfwas ascribed to the inactivation of aerobic metabolism by cold, the effects of which remain long after the temper- ature is raised. 90 It has been shown, primarily in fish (Fry 23451;: 19U2; Brett, 19AM), that there is an upper limit to the temperatures at which acclimation takes place, and there is a corresponding limit on the temperature tolerance which can be induced by acclimation. This phenomenon was not demonstrated in P. media since acclimation was not attempted beyond 30 0. Information on other species, however, indicates that, for P. media, the straight line extending to the point where acclimation temperature equals lethal temperature (Fig. 8) would actually "plateau" if acclimation temperatures were above 30 C. Because acclimation temperature was increased more readily than decreased, the period of December, January, and February is most representative of the effect of acclimation in 3, media. An increase of 0.75 C in lethal temperature for each 10 C rise in acclimation temperature is somewhat moderate when compared with values for other species. Typically, over most of their biokinetic ranges fish will show an increase of about 1 C in upper lethal temperature for each 3 C change in acclimation temperature (Gilson, 1955). Rates of Gain and Loss of Heat Resistance In evaluating rate of acclimation to changing temper- ature, the thermal history of a species under consideration must be established, then the resistance to either a high or a low temperature can be followed by moving either up 91 or down the temperature scale. Difficulty in comparing rates of acclimation among different species results from the fact that no common factor has been established, either in the method of approach or in the range and level of temperature used. The method employed in this investigation, used originally by Loeb and Wasteneys (1912), is essentially the tracing of average mortality at a given high temperature until mortality ceases. It has been shown that organisms tend to acclimate upward to high temperatures faster than they acclimate downward to lowered temperatures. This is important from an ecological point of view because it means that animals will not be acclimated as quickly to decreasing temperatures in autumn as to increasing temperatures in spring. It also means that acclimation to daily changes in temperature will proceed at different rates, adjusting more rapidly to temperature increases. In consequence, the animals will be acclimated to a higher level than the mean daily temperature. a. intermedius increased its acclimation by A C per day whereas H. azteca required 2 days to acquire a new level of heat resistance after being moved from a 14 C acclimation bath to a 20 C bath (Sprague, 1963). The acclimation of male d. fasciatus was increased by 5 C in one day, whereas females took twice as long (2.5 C per day) in adjusting from 15 to 20 C (Sprague, 1963). 92 Downward acclimation was not determined precisely in these animals. Dickie (1958) indicated that the giant scallOp acclimates upward by 1.7 C per day but may take up to 3 months to lose this acclimation. Spoor (1955) found that the heat resistance of g. rusticus increased quite rapidly,one day\ '71 {IV .C 'r- A E E 1. L. o 1 .~ A v v I. .Je rt I 1 r \\ 1) L!- ‘T‘/\ 9.; (Ci hi" -«—I Q: E (L) :H \_.J O :2 I/‘\ - E? L) Exp. e 0 me Accl. (Hr) Accl. n C Date 1.30 r'i C1,.) OW (Y1 -—10 jf‘. \O L(\ I / (1.125‘ . > 7 r. 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H. 3 O (X‘- F“ r4 (" , - (v I". I C «- 0 I [4‘ p4 I v ,1 {l‘ . N \C o \\ Om.m CH. 2 ('4 OH CH H 71 OH OH L.“ OH Om :m Om x.) (\fi >m 2m Hm mm .Hm nm I:\ M mm :m HN C‘\ m HN a" L.’\ H) OH OH mH NH OH HH ON Om (3 n1 \- 1!. am Om Om (,3 (\J ON ON ON ON Om OM ON >O\HH\OH NO\ON\OH mw\4N\h mw\mH\N mm\am\m mw\mN\w mw\mH\o TABLE VIII.-—Details of upper lethal temperature experi— ments with Paragnetina media nymphs in winter (December, January, February). Values in parentheses following test temperature indicate number of replicates. LT50 and 95% Accl. Test Confidence Temp. Temp Limits (C) (C) (min) 10 37(1) 58 (U2-80) 10 36(1) 1ND (105-186) 10 35(2) 5MB (“ES-703) 10 34(1) 1450 (1224—1718) 10 33(2) 3600 (2069—6368) 10 32(1) ' * 20 37(1) 190 (160-226) 20 36(1) “90 (uu3_5u1) 20 35(3) 1200 (1061-1358) 20 34(3) 3817 (2986—5000) 20 33(1) 6700 (5630-7973) 20 32(1) * 30 38(1) l60 (131-195) 30 37(l) HMO (UO6-u77) 30 36(1) 770 (602-986) 30 35(3) 2660 (2297-3103) 30 3U(2) 7250 (5738-9471) 30 33(2) * *50 per cent mortality did not occur in 10,000 minutes. 128 TABLE IX.--Details of upper lethal temperature experiments with Paragnetina media nymphs in spring (March, April, May). Values in parentheses following test temperature indicate number of replicates. Accl Test LT50 and 95% T Confidence emp Temp Limits (C) (C) (min) 10 37(1) 79 (64.2-97) 10 36(1) 158 (114-218) 10 35(1) 570 (460-707) 10 34(1) 1570 (1330-1853) 10 33(2) 2600 (1812-3450) 10 32(2) 4650 (3184-6794) 10 31(1) 7200 (3789-13680) 20 38(1) 62 (26—149) 20 37(1) 140 (120-165) 20 36(3) 341 (293-400) 20 35(2) 1145 (746-1782) 20 34(2) 2055 (1031-4205) 20 33(4) 3900 (2385-6582) 20 32(1) * 30 38(1) 150 (124-182) 30 37(1) 290 (228-368) 30 36(1) 870 (740-1022) 30 35(1) 2700 (2093-3483) 30 34(1) 4280 (3755-4879) 30 33(1) * *50 per cent mortality did not occur in 10,000 minutes. 129 TABLE X.--Details of upper lethal temperature experiments with Paragnetina media nymphs in summer (June, July, August). values in parentheses following test temperature indicates number of replicates. LT50 and 95% #2:; $285 Coggggggce (c) (C) (min) 10 36(1) * 450 (338-589) 10 35(1) 1010 (737-1384) 10 34(1) 3800 (3234-4465) 10 33(1) * - 20 38(1) 88 (64.7-120) 20 37(1) 220 (173-279) 20 36(2) 700 (621-791) 20 35(4) 1475 (1234-1770) 20 34(3) 5283 (4102-6807) 20 33(2) * 30 39(1) 55 (41.7—72.6) 30 38(1) 120 (92-156) 30 37(1) '365 (329-405) 30 36(2) 969 (872—1077) 30 35(4) 2345 (1957—3032) 30 34(2) 6750 (4842-9504) 30 33(2) * *50 per cent mortality did not occur in 10,000 minutes. "47771147;417777174“ 293 030