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The impact of insect diapause on water balance physiology

Yoder, Jay Alan, Ph.D. The Ohio State University, 1991

UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106

THE IMPACT OF INSECT DIAPAUSE ON WATER BALANCE PHYSIOLOGY

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of the Ohio State University

By Jay Alan Yoder, B.A.

*****

The Ohio State University 1991

Dissertation Committee: David L. Denlinger Approved by Donald E. Johnston Susan W. Fisher Brian H. Smith tyuQ — Advisor Copyright by Jay Alan Yoder 1991 In loving memory of my Grandfather, Jonas M. Yoder, and to the enduring support and strength of my family. I also dedicate this dissertation to the late Professor George W. Wharton, who inspired my interest in arthropod water balance. ACKNOWLEDGMENTS

I am grateful to Dr. David L. Denlinger for his patience, guidance and support throughout my graduate work. I sincerely thank Sir Donald E. Johnston, who provided necessary guidance and fatherly advice and who is one of my greatest inspirations. A special thank you is extended to Drs Susan W. Fisher and Dana L. Wrensch for their continual emotional support and friendship, and to Dr. Brian H. Smith for serving on my dissertation committee. I thank Professor Pappachan E. Kolattukudy for allowing me to work in his laboratory and for his help with the hydrocarbon analyses. I also thank all the graduate students on whose friendship I rely heavily, in particular Ty T. Vaughn, L. Denise Boulet, Eric R. Hoffman, Thane Thay, Astri Wayadande, Brian Gilles, Robert G. and Peter W. Hancock-Kovarik, Donald S. S. Yehling, Zhi Ben Chen, various members of the OSU Zoology Department and our own departmental alumni: Drs Maria E. Casanueva and Marvin D. Sigal. I extend sincere appreciation to my fellow lab colleagues: Chen- Ping Chen, Karl H. Joplin, George D. Yocum, Mei Ling Zhang, and a special thank you to D. Courtney Smith, whose advice has strengthed my character on and off the battlefield. I owe many thanks to Dr. Karen J. Ott and Sandy Sieg, who gave me the

iii incentive to begin graduate work. Mindy, your unchanging faith me is never forgotten. Lastly, I thank the Department of Entomology for supporting my graduate work. VITAE

February 13, 1964, Bom - Dayton, Ohio

1982-1986______B.A., B.A., L.A. University of Evansville, Evansville Indiana

19 8 6 -p resen t_____ .Graduate Teaching and Research Assistant, The Ohio State University, Columbus, Ohio

FIELD OF STUDY

Major field: Entomology (Insect Physiology) TABLE OF CONTENTS

DEDICATION______:______ii

ACKNOWLEDGMENTS______iii

VITA______v

LIST OF TABLES______ix

LIST OF FIGURES______x

CHAPTER

I. INTRODUCTION______1

A. Sarcophaga- A model diapause system______2 B. Endocrinology of diapausing versus nondiapausing pupae of Sarcophaga______3 C Cuticular lipid correlates with insect's water conservation needs______4 D. Embryonic diapause and water relation ______6 E Extatosoma tiaratum, a tropical walking stick possessing two stages of embryonic diapause ______8 F. The chemical and physical properties of water______1 0 G Water balance and basic parameters ______12 H. Early water balance theory ______1 4 I. The basic water balance equation and transpiration m______1 5 J. Water gain by water vapor absorption and metabolic water ______1 6 K Equilibrium water mass and the energetics of mt and ms______1 6 L. Phase barrier Ea and critical transition temperature______1 8 M. Research objectives ______1 9 N. References______2 1

II. WATER BALANCE IN FLESH FLY PUPAE AND WATER VAPOR ABSORPTION ASSOCIATED WITH DIAPAUSE 3 0

A. A bstract 3 0 B. Introduction 3 1 C Materials and Methods 3 2 D. Results 3 7 E Discussion 5 5 F. References 6 1

III. A COMPARISON OF THE WATER BALANCE CHARACTERISTICS OF TEMPERATE AND TROPICAL FLY PUPAE 6 6

A. A bstract 6 6 B. Introduction 6 7 C Materials and Methods 6 8 D. Results 7 0 E Discussion 7 7 F. References 8 1

vii IV. WATER VAPOR UPTAKE BY DIAPAUSING EGGS OF A TROPICAL WALKING STICK 8 3

A. A bstract ______8 3 B. Introduction ______8 4 C Materials and Methods______8 5 D. Results ______„ ______8 9 E Discussion ______101 F. References______107

V. PUP ARIA OF DIAPAUSING FLESH FLIES ARE ENHANCED WITH ADDITIONAL HYDROCARBONS 111

A. Abstract ______1 11 B. Introduction 1 1 2 C Materials and Methods______1 1 3 D. Results ______1 1 9 E Discussion ______1 3 9 F. References______146

VI. EVIDENCE FOR A BRAIN FACTOR THAT STIMULATES SYNTHESIS OF PUPARIAL HYDROCARBONS IN DIAPAUSING FLESH FLIES______1 5 1

A. Results and Discussion ______1 5 1 B. References______159

SUMMARY. 161 7. Net transpirational water loss from eggs of E. tiaratum as early embryos at 20°C (A) and at 25°C (B) and as pharate first instar larvae at 25°C (C) 9 5

8. Percent change in weight in response to long term exposure to three hydrating atmospheres at 20°C for early embryonic eggs 9 7

9. Rates of water vapor exchange for eggs of E. tiaratum as early embryos at 20°C (A) and at 25°C (B) and as pharate first instar larvae at 25°C ( C) 9 9

10. Total ion count (TlC)-gas chromatograms (abundance versus carbon number) of hydrocarbons from empty puparia of nondiapausing (A) and diapausing (B) S. crassipalpis. Both groups were reared at 20°C. indicates the C29 alkane unique to diapause ______129

11. Mass spectrum (abundance versus mass/charge ratio) of C29 indicates a straight-chain alkane ______1 3 1

12. Formation of CO and alkane from [1-14C], [9,10-3H] octadecanal by the 105,000 x g supernatant and resuspended microsomal pellet.______133

13. Radio-gas chromatography by the alkane generated from [1-14C], [9,10-3H]octadecanal by the microsomal preparation______135

14. Effects of the cofactors, ATP, CoA and NADH on the formation of alkane from octadecanal and octodecanoic acid catalyzed by microsomes of flesh fly pupae______1 3 7

xi LIST OF TABLES

TABLE

1. A comparison of wet weight, percent body water, net transpiration rate, 24 h equilibrium weight, CTT, and Ea below and above the CTT for temperate zone and tropical fly pupae______

2. A comparison of wet weight and weight change (%) at saturation for eggs of six species exhibiting different developmental patterns______

3. Quantities of epicuticular hydrocarbon and other lipids extracted from the surface of S. crassipalpis at different developmental stages______1

4. A comparison of net transpiration rate (-kt) at 20°C and quantities of hydrocarbon (pg/empty puparium) for pupae of S. crassipalpis______1 LIST OF FIGURES

FIGURE PAGE

1. Total body water pool expressed in mg or % for nondiapausing (A) and diapausing (B) groups of flesh flies (S. crassipalpis) at different stages of development 4 5

2. Net transpiration at 20°C from nondiapausing (A) and diapausing (B) S. crassipalpis pupae into dry air______47

3. Percent change in weight in response to long term exposure to hydrating (av 0.85) and dehydrating (av 0.33) atmospheres at 20°C for nondiapausing (A) and diapausing (B) S. crassipalpis pupae 4 9

4. Arrhenius plot of In k against K"1 (103) for nondiapausing (A) and diapausing (B) S. crassipalpis pupae 5 1

5. Rates of water vapor exchange for nondiapausing (A) and diapausing (B) S. crassipalpis pupae 5 3

6. The relationship between net transpiration rate in six species or sizes of fly pupae and (A) In wet weight, and (B) 24 h equilibrium weight ______7. Net transpirational water loss from eggs of E. tiaratum as early embryos at 20°C (A) and at 25°C (B) and as pharate first instar larvae at 25°C (C) 9 5

8. Percent change in weight in response to long term exposure to three hydrating atmospheres at 20°C for early embryonic eggs ______97

9. Rates of water vapor exchange for eggs of E. tiaratum as early embryos at 20°C (A) and at 25°C (B) and as pharate first instar larvae at 25°C ( C)______9 9

10. Total ion count (TlC)-gas chromatograms (abundance versus carbon number) of hydrocarbons from empty puparia of nondiapausing (A) and diapausing (B) S. crassipalpis. Both groups were reared at 20°C. indicates the C29 alkane unique to diapause ______129

1 1 . Mass spectrum (abundance versus mass/charge ratio) of C29 indicates a straight-chain alkane ______W1 3 1

12. Formation of CO and alkane from [1- 14 C], [9,10-3 H] octadecanal by the 105,000 x g supernatant and resuspended microsomal pellet.______1 3 3

13. Radio-gas chromatography by the alkane generated from [1-14 C], [9,10-3 H]octadecanal by the microsomal preparation ______135

14. Effects of the cofactors, ATP, CoA and NADH on the formation of alkane from octadecanal and octodecanoic acid catalyzed by microsomes of flesh fly pupae______137

xi CHAPTER I INTRODUCTION The long term maintenance of water balance is a problem faced by all organisms regardless of their habitat. This problem is magnified for terrestrial species which have no access to free water yet must live under desiccating conditions (Hadley, 1981). Such is the case for insect pupae and eggs. Eggs and pupae are closed or cleidoic systems in which only gas exchange occurs and embryogenesis as well as adult development take place without any intake of materials from the environment (Yamashita and Hasegawa, 1985). Such systems appear to be less susceptible to environmental stress and are capable of surviving unfavorable conditions. If diapause is introduced into this system, survivorship is even further enhanced (Yamashita and Hasegawa, 1985). Thus, diapause at these stages enables an insect to survive very challenging environmental stresses. Insects secrete a thin layer of lipid on the outer cuticular surface as protection against desiccation, abrasion, and penetration by microorganisms (Blomquist, 1979 and 1985). It is well documented that complex mixtures of hydrocarbons form the bulk of cuticular lipids though little is known of their role in the water proofing of insect cuticle (Blomquist, 1979). During the 9-10 months of diapause, a pupa (or egg) is vulnerable to the loss of much of its body water, and hydrocarbons appear to participate in detering water loss during this long period of dormancy.

Sarcophaga-A Model Diapause System Flesh flies belong to the family Sarcophagidae, and some of the most widely studied members of this family are in the genus Sarcophaga. These flies overwinter in pupal diapause and are distributed world-wide. Reports of twenty-two species from both the temperate zone and the tropics indicate a consistent reliance upon pupal diapause within this taxonomic group (Denlinger, 1981a). Due to adverse climatic conditions, the fly may delay further development (diapause) while in the pupal stage until favorable conditions warrant continued development. This genus is an appropriate group of insects to study water balance physiology because of established rearing techniques (Denlinger, 1972) and vast knowledge of diapause physiology. Despite the wealth of literature on pupal diapause in temperate flesh flies: morphology (Fraenkel and Hsiao, 1968), phenology and environmental signals (Denlinger, 1972), clock mechanisms (Saunders, 1982), neuroendocrine regulation (Denlinger, 1981a), metabolic aspects (Denlinger, 1981b; Adedokun and Denlinger, 1985), geographic differences (Denlinger, 1979), genetic components (Henrich and Denlinger, 1982), cold-hardiness (Adedokun and Denlinger, 1984; Lee et al., 1987b), response to cold (Chen et al., 1987a and b) and heat (Joplin and Denlinger, 3 1990), aspects of water balance physiology represent a heretofore neglected dimension of diapause physiology.

Endocrinology of Diapause versus Nondiapausing Pupae of Sarcophaga Lipid metabolism in insects is affected by a number of neuroendocrinological, physiological and environmental influences (Blomquist, 1985). Just prior to the onset of pupal diapause the prothoracic glands of both diapause- and nondiapause-destined individuals actively produce the surge of ecdysone that triggers pupariation (in flies) and pupation (Denlinger, 1985). In individuals not programmed for diapause, a second surge of ecdysone is released and leads to initiation of adult development. However, individuals programmed for diapause fail to release the second peak of ecdysone, and ecdysteroid titre drops to levels that are undetectable with bioassay techniques (Walker and Denlinger, 1980). Among the Diptera, the efficacy of 20-hydroxyecdysone (20-HE) in breaking pupal diapause has been demonstrated in numerous sacrophagid species (Denlinger, 1985). Armold and Regnier (1975b) suggest that 20-HE stimulates hydrocarbon formation. In flesh flies, juvenile hormone (JH) appears to play an important functional role prior to the onset of diapause, during diapause as well as the beginning of adult development (Denlinger, 1985). Though JH will shorten diapause, it does not invoke an immediate termination of diapause in Sarcophaga (Fraenkel and Hsiao, 1968; Zdarek and Denlinger, 1975; Denlinger, 1979). The behavioral strategies of migration and diapause require prior accumulation of lipid reserves (Downer and Mattews, 1976; Adedokun and Denlinger, 1985), and as the expression of the behavior is often associated with depressed JH titres, it is apparent that JH may be intimately involved in determining and preparing for such behavior. Denlinger (1985) found that simultaneous application of 20-HE and JH produced a shorter duration of diapause than either hormone applied separately.

Cuticular Lipid Correlates with Insect's Water Conservation Needs Cuticular hydrocarbons vary both qualitatively and quantitatively throughout the lifecycle of the flesh fly, Sarcophaga bullata, and the quantity of cuticular hydrocarbon correlates with the water conservation needs of the insect (Armold, 1975a). Diapausing pupae of the tobacco hornworm, Manduca sexta, (Bell et al.,\915) secrete three times as much surface wax as nondiapausing pupae. The extra thickness of the wax layer apparently protects the insect from desiccation and the authors speculate that the deposition of additional wax may result from hormonal changes accompanying entry into diapause (Blomquist, 1985). Goodrich (1978) has examined the cuticular lipids of puparia and adults of the sheep blow fly, Lucilia cuprina (Wied.). These studies indicate significant qualitative and quantitative lipid differences between these two stages. The environments occupied by pupae and adults are very different, and differ in such a way as to suggest cuticular lipids may vary with the environmental need for water conservation. Armold et al. (1969) found that terrestrial adult stoneflies, Pteronarcys colifornica, had more surface lipid than the immature aquatic naiad. A similar situation occurs in adult flesh flies, Sarcophaga bullata. Early instar larvae, which occupy moist habitats, had relatively little cuticular hydrocarbons compared with pupae and newly emerged adults which are subjected to much drier conditions (Armold and Regnier, 1975). In the Bertha army worm, Mamestra configurata, diapausing pupae had a thicker wax and a higher content of hydrocarbons than nondiapausing pupae (Hegdekar, 1979). Hadley and Schultz (1987) found that the quantity and quality of epicuticular hydrocarbons amongst three species of tiger beetles correlated with water loss with respect to overall quantity and enhanced sp2 hybridization. Collectively, these studies suggest that a strong correlation exists between the quantities of extracted cuticular waxes and net transpiration in insects. Moreover, the greater amount of cuticular lipid often occurs when there is a greater need for water conservation. Armold (1975) found two periods of rapid lipid accumulation in S. bullata during ontogeny. Rapid accumulation occurred post­ adult eclosion and during the first two days of puparium formation, thus at the time of pupation (±1 day), deposition of lipid within and on the puparium is complete. In Sarcophaga crassipalpis, four days of short day length late in embryonic development and early larval life are adequate for pupal diapause induction (Denlinger, 1971). The onset of pupal diapause in S. crassipalpis is preceded by a series of preparative steps not observed in larvae destined for continuous development (Adedokun and Denlinger, 1985). This study demonstrates that diapause is an "all or none" response dependent upon the programming the individual received during its early developmental history. Such a preparative event discussed in this thesis is the accumulation of lipid, predominately hydrocarbon, on the inner wall of the puparium. The additional amount of lipid apparently reduces water loss and imparts unique water balance characteristics to pupae which enable them to survive many months (9-10) in diapause.

Embryonic Diapause and Water Relations Compared with larval, pupal and adult diapause, embryonic diapause exhibits characteristic features (Yamashita and Hasegawa, 1985). Environmental signals received by the mother are expressed in the eggs, as in pupal diapause (Henrich and Denlinger, 1982); however, egg diapause of the Australian cricket Teleogryllus sp. is completely paternal (Masaki, 1960). A second feature of embryonic diapause is a unique endocrine system different from the endocrine hierarchy during post-embryonic life. This system utilizes a new hormone (diapause hormone) that has different chemical properties and biochemical actions. Although pupal diapause termination is controlled by ecdysteroids (Denlinger, 1985), termination of embryonic diapause has not been demonstrated to be under hormonal control (Yamashita and Hasegawa, 1985). Embryonic diapause does not appear to be characterized by any special biological advantage, but is a reflection of the evolutionary history in which diapause occurred at this stage (Hinton, 1981). Ando (1972) classified insects with egg diapause into three groups according to the water required for embryonic development, (1) the egg contains enough water at the time of oviposition for embryogenesis (e.g. silkworm, Bombyx mori, gypsy moth, Lymantria dispar). (2) the egg absorbs liquid water before diapause for post-diapause development (e.g. crickets, Teleogryllus emma, Scapsipedus aspersus (Masaki, 1960), grasshoppers, Melanoplus bivittatus (Lees, 1955), Chorthippus brunneus (Moriarty, 1960 a and b). (3) the egg absorbs liquid water after diapause termination for the completion of embryogenesis (e.g. crickets, Loxoblemus sp., Pteronemobius fascipes, Pteronemobius taprobanensis, Pteronemobius ohmachii (Masaki, 1960); grasshoppers, Austroiceter cruciata (Lees, 1955), Melanoplus differentialis (Lees, 1955); locusts, Locustana pardalina (Lees, 1955), Dociostaurus maroccanus (Lees, 1955); chrysomelids, Atrachya memtriesi (Ando, 1972), Diabrotica virgifera (Krysan, 1978); dragonflies, Lestes congener, Lestes disjunctus, Lestes unguiculatus (Sawchyn and Gillot, 1974 a and b). For eggs belonging to group 3, uptake of liquid water is required for diapause termination and resumption of development. Uptake of water vapor from atmospheric air has never been demonstrated in insect eggs (Hinton, 1981), even at humidities near saturation (98% R.H.) (Shulov, 1952). Other factors known to terminate embyonic diapause are photoperiod (Lees, 1955;Williams, 1969; Bedford, 1970), and temperature (Hogan, 1960; Masaki, 1962).

Extatosoma tiaratum, a Tropical Walking Stick Possessing Two Stages of Embryonic Diapause The tropical phasmid Extatosoma tiaratum poses a similar economic threat as phasmids Didymuria violoscens (Leach), Podacanthus wilkinsoni Macl. and Ctenomorpedes tessulatus. These species have occurred in plague numbers and have caused extensive defoliation of mountain eucalyptus forests in south­ eastern Australia (Hadlington and Shipp, 1961; Readshaw, 1965). The life histories of these species are similar, but life cycle duration is variable. Oviposition occurs during late summer and autumn; eggs hatch during the spring of the same year or the year following oviposition. Individuals reach the adult stage and begin reproduction in mid-summer. The eggs of these insects undergo two diapauses during their development (Hadlington and Shipp, 1961) and morphogenesis is regulated such that hatching occurs during the most favorable periods of the year. Voy (1954 a and b) studied development in eggs of Clonopsis gallica (Charp.), a phasmid inhabiting south-eastern France and found that they also underwent two diapauses before completing development. The first diapause, which occurs at an early but undetermined stage of development, is referred to as early- embryonic, while the second diapause occurs as a pharate first instar larva. It appears that temperature influences diapause and embryonic behavior of these phasmid eggs. Exposure to low temperatures is necessary for diapause, while the second diapause depends on higher temperatures for the completion of embryogenesis (Hadlington and Shipp, 1961). Termination of early embryonic diapause in eggs of P. wilkinsoni requires periods of cold exposure for the second diapause to proceed. The intensity and duration of cold exposure during the early-embryonic stage determines whether morphogenesis will be initiated (Readshaw, 1965). Eggs producing 2 and 3 year individuals have both stages of embryonic diapause and react to the environment in a similar way as P. wilkinsoni (Hadlington and Shipp, 1961). C. tessulatus eggs have only one diapause, which occurs in the stage of the pharate first instar larvae (Hadlington and Shipp, 1961). The temperature range most effective for diapause termination varies from one species to another (Hogan, 1960) and appears to be related to the climatic zone occupied by each. For eggs of these 10 tropical walking sticks, like other diapausing insects (Lees, 1955), the warmer the climate of occurrence, the higher are the optimum temperatures required for diapause termination (Readshaw, 1965).

The Chemical and Physical Properties of Water Water in insects serves many functions (Wharton and Arlian, 1972) as it does in all organisms. Water's chemical and physical properties provide an excellent milieu for the chemical and physical machinery essential to all life (Henderson, 1913). Extensive hydrogen bonding among water molecules in the liquid phase is responsible for high values of specific heat (4 J-g'^C '1), high freezing point with significant heat of fusion (333.6 J’g"1), and high heat of vaporization (2372 J*g_1) at the boiling point (Eisenberg and Kaufman, 1969). Water’s high dielectric constant (78) maintains electric potentials across cell membranes and promotes ionization of dissolved electrolytes due to a stressed sp3 bond angle (a) of 104.52° (normal sp3a=109° 28'). Water's low viscosity, (0.890 centipoise), enables ions to diffuse rapidly and produce vital electrical potentials associated with many functions, e.g. nervous activity and muscular contraction. The dipole moment of water (1.76 Debye units) interacts with hydrophilic and hydrophobic sites on macromolecules to determine the three- dimensional configurations responsible for their function. All these properties of water provide thermal stability for cellular activities (Kohn, 1965). 11 Water as a solvent for aqueous solutions maintains its various colligative properties such as freezing point depression, boiling point elevation, osmotic pressure and vapor pressure lowering (Andrews, 1976). These colligative properties depend on the ratio of solvent molecules to total molecules in solution. In an ideal solution, this ratio corresponds to the activity (aw) of the water present. Activity of liquid water (aw) is the same as the activity of water in the vapor phase (av). Aw is a ratio that compares the vapor pressure of water in an aqueous solution (aq.) to the vapor pressure (V.P.) of pure water at the same temperature; aw's range is from 0 (no water) to 1 (pure water), equation 1 (Wharton, 1985).

(1) aw = V.P. H20 (aq.)-V.P. H20 ° -1

The concentration of water in the air is expressed as relative humidity (R.H.). The activity of water in the vapor phase (av) is described as the concentration of water in the atmosphere (R.H.) compared to the concentration at saturation, equation 2.

(2) av = R.H.-IOO’1 1 2 The difference, av-aw, between air and insect determines whether the insect’s net water exchange with surrounding air. If av-aw>0, water is gained; if <0, water is lost. The cellular contents of higher animals and plants have aqueous activities between 0.990 and 0.997. Such activities enable organisms to survive in a broad range of habitats (Wharton, 1985); therefore, an aw in the 0.99 range is required for optimum metabolic rate and homeostasis. As such, insects must continuously balance their aw (0.99, Wharton, 1985) against a characteristically lower atmospheric av to maintain a water activity in the 0.99 range.

Water Balance and Basic Parameters Insects have developed many adaptations to conserve their water content and replace water lost. Water concentration in insect cells is high, but the main water reserve is the hemolymph (Wharton, 1985). Maintenance of adequate body water levels (water balance) is of primary importance to insects because they have large surface area to volume ratios. The arthropod integument surface has a layer of high lipid content (Kuhnelt, 1928) that apparently impedes water loss (Wharton, 1985). In addition, an internal respiratory system, communicating with external air via small valve-controlled spiracles, contributes to water conservation. Water can be obtained by drinking, ingestion of moist food, as a product of metabolic activity and absorption of 1 3 vapor from atmospheric air (Edney, 1977). For pupae or eggs, the maintenance of water balance is a problem because they are incapable of countering water loss with gain by drinking or feeding. The study of water balance represents an interaction between biotic and abiotic factors. Abiotic factors (temperature, humidity, and pressure) have predictable affects on the rates of water flux involving the organism’s physical surfaces and structures (Crank, 1956). Biotic factors include adaptations such as cuticular water proofing processes (Hadley, 1982), active water uptake mechanisms (Beament, 1964), and behavioral responses (Lees, 1969). Regulation of water balance for any arthropod requires characterization of basic water balance parameters: wet and dry weight, water mass, and percent body water. The quantity of water (% body water) in an insect is expressed as the ratio of water weight (m) to total weight (f) times 100 or water as percentage of total weight, equation 3 (Wharton, 1985).

(3) Percentage m = 100 (f-d)-f'1

Water mass (m) is the difference between total weight (f) and dry weight (d). Mean water content of insects is about 70% (Edney, 1977) and range from 45% to 90% (Rapoport and Tschapek, 1967). Insects and mites can lose more than 50% of their water mass and 14 still survive, but others can tolerate only a 20% loss (Arlian and Veselica, 1979).

Early Water Balance Theory Observing water loss from insects in desiccating environments is the most common method of gaining water balance information (Wharton, 1985). Many life processes expend significant amounts of water, e.g. respiration, excretion, secretion, growth and reproduction. External respiration, exchange of respiratory gases between the respiratory system and environment occurs at the expense of water loss if the ambient air av is less than the aw of the insect's body water. Early water balance experiments used gravimetric methods for detecting changes in water mass (m). Net flux rates for water loss during dehydration was described by Fick's diffusion equation (4) (Edney, 1977).

(4) J = -DS08X'1

This law assumes that a constant net flux (J) of water occurs across a permeable boundary separating two aqueous compartments. Fick's law requires that net flux (J) between the two compartments be equal to the negative product of a concentration gradient (8c‘Sax'1) and a diffusion coefficient (D). The negative sign denotes that net flux proceeds along a 1 5 concentration gradient and indicates a net decrease in the organism's water mass.

The Basic Water Balance Equation and Transpiration m An organism's water balance results from simultaneous water flux into and out of the organism. To maintain water balance in its natural habitat, an organism's water loss (by excretion, defecation, respiration, secretions, reproduction products and integumental transpiration) is balanced by water gain (by drinking, feeding, metabolism and water vapor absorption). Change in water weight results from the difference between rate of water movement from insect to air (transpiration, mx) and rate of water movement from air to insect (absorption, ms). This difference reflects changes in water mass (m) and is described by the general water balance equation (5) (Wharton, 1985).

(5) m = ms - mx

In dry air (av 0.00) ms=0, thus transpiration m (mx)=m, and a constant percentage of the water mass (m) is lost in unit time. Only under this condition, when ms=0 is water content at any time t (mt) equal to water mass at time 0 (mo) X e"kt, where k is percent lost in unit time and t is time elapsed between mo and mt. The instantaneous rate of loss at any time t is equal to kt, equation 6 (Wharton, 1985). 1 6

(6) mt = mo e-kt, or In mt/mo = -kt

Water Gain by Water Vapor Absorption and Metabolic Water To counter water loss by transpiration m (mx), its competing flow, absorption m (ms), must also be considered (eqn. 5). For fasting terrestrial arthropods inhabiting desiccating envrionments, water sorption from the atmosphere may be the only avenue of water gain (Knulle, 1967; Wharton and Devine, 1968; Arlian and Wharton, 1974; Arlian, 1975 a and b). The rate of water vapor absorption can be derived from water vapor exchange with respect to ambient av (Machin, 1984). Metabolic water also contributes to water gain and is available to insects as a by product of oxidative catabolism of carbohydrates and fats (Wharton, 1985). The oxidation of lg of carbohydrates produces over 0.5g water and releases 4.2 kcal of energy; fat forms over lg of water/gram with the release of 9.5 kcal of energy. Per gram, fat is more efficient for metabolic water production, but per calorie, more water is obtained from carbohydrate oxidation. Aerobic catabolism of carbohydrates forms lg of metabolic water and releases 8.4 kcal while lg of fat releases 9.5 kcal (Wharton, 1985). 1 7 Equilibrium Water Mass and the Energetics of mj and ms When ms=niT (eqn. 5) a 0% change in total water weight is observed and the equilibrium water mass is designated as moo. If moo is subtracted from both sides of equation 5, weight changes describe transpiration into moist air, equation 7 (Wharton, 1985).

(7) mt-moo = (mo-moo )e"kt

Net transpiration (eqn. 6) proceeds as an exponential loss of the exchangeable water mass (mt-moo) in unit time (eqn. 7). Transpiration m (mx) is restricted to molecules possessing sufficient free energy to penetrate the liquid-vapor phase barrier that exists between insect and atmosphere (Toolson, 1980) thus, mx=aw. Absorption m (ms) involves a phase barrier from vapor to liquid; therefore, sorption is driven by the free energy of water vapor (Wharton, 1985), thus ms=av. The free energy of the phase barrier must be overcome by both condensing molecules: water molecules absorbed and transpiring molecules. Only a percentage of water molecules possess sufficient energy to exceed that of the phase barrier and pass through. Although ms=av, the amount of vapor absorbed is constant (eqn. 8, Wharton, 1985) but small compared to the total available vapor for absorption per ambient

(8) ms = km0 (av-aw_1) 1 8 Phase Barrier Ea and Critical Transition Temperature The energy ( activation energy, Ea) required by a water molecule to pass through the liquid-vapor phase barrier between the insect and its surroundings is derived from an Arrhenius plot of In k versus T'1. The slope of a regression line passing through the points of In k and T"1 is equal to -Ea*R_1, equations 9 and 10 (Seethaler et al., 1979; Toolson, 1980).

(9) In k = -Ea-R^-T"1 + In A

(10) -Ea = [( tjlnki- ti-n-‘) X ( t,2-( ti)2-n'1)"'] R

Calculation of the Ea is possible only for a temperature range (i) over which the rate of loss (k) of the amount (n) of water equals the reciprocal of absolute temperature (T_1). A simultaneous change in activation energy with absolute temperature designates a new range (i) and a critical transition temperature (CTT) is found at the intersection of the slopes defining the two activation energies (Seethaler, et al., 1979). The CTT indicates the temperature at which epicuticular lipids undergo a phase change resulting in dramatic water loss (Edney, 1977). For all arthropods under fasting conditions, the primary components of water exchange are water lost by net transpiration and water gained by vapor sorption (Wharton, 1985). 19 Research Objectives In this study on water balance in flesh fly pupae and insect eggs, I have defined the following objectives: 1. To determine water balance parameters of, water mass and percent body water of nondiapausing and diapausing pupae of the flesh fly Sarcophaga crassipalpis. The dehydration tolerance limit is also determined. Several water balance characteristics are measured: net transpiration rates, water vapor absorption from subsaturated air, critical transition temperature (CTT) with accompanying activation energies (Ea) and rates of water vapor uptake. The water balance characteristics of diapausing pupae are evaluated and mechanisms of water conservation are discussed. Comparative observations are made with a northern population of a closely related species, S. bullata (Massachusetts strain). 2. To compare water balance parameters of temperate fly pupae, S. crassipalpis, S. bullata, and Musca domestica with flies originating from the tropics: Peckia abnormis and Sarcodexia sternodontis. The correlation between net transpiration rate at different temperatures and size is evaluated in addition to the capacity for water vapor absorption and determination of the CTT. The utility of activation energy (Ea), from which the CTT is derived, is evaluated as an indicator of habitat preference and suitability for a species. 3. To examine the water vapor absorption capacity in eggs of the tropical walking stick Extatosoma tiaratum during both stages 20 of its diapause. Water balance parameters and flux rates are determined. Insect eggs belonging to three different orders and two different developmental statuses (nondiapause or diapause) are also tested for their ability to absorb atmospheric water vapor. 4. To correlate water balance characteristics observed in diapausing flesh fly pupae of S. crassipalpis with quantitative and qualitative changes in hydrocarbons of the puparia. The site of hydrocarbon deposition within the puparium is determined. Also, a class characterization of other lipids present within the puparium is determined. Quantification of epicuticular lipids and hydrocarbons are determined throughout ontogeny for both nondiapausing and diapausing flies. 5. To determine which hormone is responsible for the extra puparial hydrocarbon associated with the diapause of Sarcophaga. The efficacy of brain extracts, 20-hydroxyecdysone, juvenile hormone and cyclic hormones were evaluated in their capacity to elavate levels of hydrocarbon in nondiapausing pupae to diapausing levels. Pupal net transpiration rate is also used an indicator of extra hydrocarbon production. 21

REFERENCES

Adedokun, T. A. and Denlinger, D. L. (1984) Cold hardiness: a component of the diapause syndrome in pupae of the flesh flies, Sarcophaga crassipalpis and S. bullata. Physiol. Ent. 9 :3 6 1 -3 6 4 .

Adedokun, T. A. and Denlinger, D. L. (1985) Metabolic reserves associated with pupal diapause in the flesh fly Sarcophaga crassipalpis. J. Insect Physiol. 31:229-233.

Ando, Y. (1972) Egg diapause and water absorption in the false melon beetle, Atrachya menetriesi Faldermann (Coleoptera:Chrysomelidae). Appl. Ent. Zool. 7:142-154.

Andrews, F. C. (1976) Colligative properties of simple solutions. Science 194:567-571.

Amano, H. and Chant, D. A. (1978) Mating behavior and reproductive mechanisms of two species of mites. Appl. Ent. Zool. 6:102- 104.

Arlian, L. G. (1975a) Dehydration and survival of the European house dust mite, Dermatophagoides pteronyssinus. J. Med. Entomol. 12(4):437-442.

Arlian, L. G. (1975b) Water exchange and effect of water vapour activity on metabolic rate in the dust mite, Dermatophagoides. J. Insect Physiol. 21:1439-1442.

Arlian, L. G. and Veselica, M. M. (1979) Water balance in insect and mites. Comp. Biochem. Physiol. 64A:191-200. 22

Arlian, L. G. and Wharton, G. W. (1974) Kinetics of active and passive water exchange between the air and a mite, Dermatophagoides farinae. J. Insect Physiol. 20:1063-1077.

Armold, M. T., Blomquist, G. J. and Jackson, L. L. (1969) Cuticular lipids of insects III. The surface lipids of the aquatic and terrestrial life forms of the big stonefly Pteronarcys callifornica. Newport Comp. Biochem. Physiol. 31:685-692.

Armold, M. T. and Regnier, F. E. (1975a) A developm ental study of the cuticular hydrocarbons of Sarcophaga bullata. J. Insect Physiol. 21:1827-1833.

Armold, M. T. and Regnier, F. E. (1975b) Stimulation of hydrocarbon biosynthesis by ecdysone in the flesh fly Sarcophaga bullata. J. Insect Physiol. 21:1581-1586.

Beament, J. W. L. (1964) Active transport and passive movement of water in insects. Adv. Insect Physiol. 2:67-129.

Bedford, G. O. (1970) the development of the egg of Didymuria violescens and determination fo the stage at which first diapause occurs. Aust. J. Zool. 18:155-169.

Bell, R. A., Nelson, D. R., Borg, T. G. and Caldwell, D. L. (1975) Wax secretion in nondiapausing and diapausing pupae of the tobacco hornworm, Manduca sexta. J. Insect Physiol. 21:1725-1729.

Blommers, L (1977) Studies on the mating behavior of beetles and their tropical habitats. Appl. Ent. Zool. 7:142-154.

Blomquist, G. J. and Dillwith, J. W. (1985) Cuticular lipids. In: Comparative Insect Physiology, Biochemistry and Pharmacology, Kerkut, G. A. and Gilbert, L. I. (eds.) Pergamon Press, Oxford. 4:117-150.

Blomquist, G. J. and Jackson, L. L. (1979) Chemistry and biochemistry of insect waxes. Prog. Lip. Res. 17:319-345. 23

Chen, C-P., Denlinger, D. L. and Lee, R. E. Jr. (1987a) Cold-shock injury and rapid cold hardening in the flesh fly Sarcophaga crassipalpis. Physiol. Zool. 60 (3) 297-304.

Chen, C-P., Denlinger, D. L. and Lee, R. E. Jr. (1987b) Responses of nondiapausing flesh flies (Diptera:Sarcophagidae) to low rearing temperatures: Developmental rate, cold tolerance, and glycerol concentrations. Ann. Ent. Soc. Amer. 80:790-796.

Crank, J. (1956) The Mathematics of Diffusion. Oxford University Press, London.

Denlinger, D. L. (1971) Embryonic determination of pupal diapause in the flesh fly Sarcophaga crassipalpis. J. Insect Physiol. 17:1815-1822.

Denlinger, D. L. (1972) Induction and termination of pupal diapause in Sarcophaga (Diptera:Sarcophagidae). Biol. Bull. 142:11-24.

Denlinger, D. L. (1979) Pupal diapause in tropical flesh flies: environmental, and endocrinologcial regulation, metabolic rate and genetic selection. Biol. Bull. 156:31-46.

Denlinger, D. L. (1981a) Hormonal and metabolic aspects of pupal diapause in Diptera. Ent. Gen. 7:245-249.

Denlinger, D. L. (1981b) The physiology of pupal diapause in flesh flies. In: Current Topics in Insect Endocrinology and Nutrition. Bhaskaran, G. et al. (eds.) Plenum, New York, pp.131-160.

Denlinger, D. L. (1985) Hormonal control of diapause. In: Comparative Insect Physiology, Biochemistry and Pharmacology. Kerkut, G. A. and Gilbert, L. I. (eds.) Pergamon Press, Oxford. 11:1353-412.

D osse, B. V. (1975) Life history and life tables of resistance for the predatory mite Appl. Ent. Zool. 7:5-12. 24

Dosse, B. V. (1977) Biological studies on the predator mite with the effect of prey and non-prey substances Appl. Ent. Zool. 7 :2 3 -2 7 .

Downer, R. G. H. and Mattews, J. R. (1976) Patterns of lipid distribution and utilisation in insects. Amer. Zool. 16:733- 745.

Edney, E. B. (1977) Water balance in land arthropods:zoophysiology and ecology. Vol 9. Springer-Verlag, New York.

Eisenberg, D. and Kaufman, W. (1969) The Structure and Properties of Water. Oxford University Press, Oxford.

Fraenkel, G. and Hsiao, C. (1968) Morphological and endocrinological aspects of pupal diapause in a flesh fly Sarcophaga argyrostoma. J. Insect Physiol. 14:707-718.

Goodrich, B. S. (1970) Cuticular lipids of puparia of the Australian sheep blowfly Lucilia cuprina (Wied). J. Lipid Res. 11:1-6.

Hadley, N. F. (1981) Cuticular lipids of terrestrial plants and arthropods: a comparison of their structure, composition and water-proofing function. Biol. Rev. 56:23-47.

Hadley, N. F. (organizer) (1982) Water exchange and water balance in Arthropods (A Symposium). J. Exp. Zool. 222 (3):203-349.

Hadley, N. F. and Schultz, T. D. (1987) Water loss in three species of tiger beetles (Cicindela): correlations with epicuticular hydrocarbons. J. Insect Physiol. 23:667-682.

Hadlington, P. and Shipp, E. (1961) Diapause and parthenogenesis in the eggs of three species of Phasmatodea. Proc. Linnean Soc. of New South Wales. 86 (3):268-279.

Hegdekar, B. M. (1979) Epicuticular wax secretion in diapausing and nondiapausing pupae of Bertha armyworm. Annals of the Ent. Soc. of Amer. 72:13-15. 25

Henderson, L. J. (1913) The Fitness of the Environment Beacon Press, Boston.

Henrich, V. and Denlinger, D. L. (1982) A maternal effect that eliminates pupal diapause in progeny of the flesh fly Sarcophaga bullata. J. Insect Physiol. 28:881-884.

Hinton, H. E. (1981) Biology of Insect Eggs. Pergamon Press, Oxford, p p .1125.

Hogan, T. W. (1960) The onset and duration of diapause in eggs of Aceta commodus (Walk.) (Orthoptera) Aust. J. Biol. Sci. 1 3 :1 4 -2 9 .

Hoy, C. W. (1969) Hydrocarbon biosynthesis in the Sarcophagidae: a phylogenetic study. Appl. Ent. Zool. 5:12-14.

Hoy, C. W. (1990) Water balance in the tropical beetle as a model for predator-prey relationships. Appl. Ent. Zool. 7:12-14.

Joplin, K. H. and Denlinger, D. L. (1990) Developmental and tissue specific control of the heat shock induced 70 kDa related proteins in the flesh fly, Sarcophaga crassipalpis. J. Insect Physiol. 36(4):239-249.

Knulle, W. (1967) Physiological properties and biological implications of the water vapour sorption mechanism in larvae of the oriental rat flea, Xenopsylla cheopis Roths. J. Insect Physiol. 13:333-357.

Kohn, P. G. (1965) Tables of some physical and chemical properties o f water. In: the State and Movement ofWater in Living Organisms. Fogg, G. E. (ed.) Cambridge University Press.

Krysan, J. L. (1978) Diapause, quiescence, and moisture in the egg of the Western corn rootworm, Diabrotica virgifera. J. Insect Physiol. 24:535-540. 26

Kuhnelt, W. (1928) Uber den Bau des Insektskelettes. Zool. Jahrb. Abt. Anat. Ontol. Tiere. 50:218-278.

Lee, R. E. Jr., Chen, C-P., Meacham, M. H. and Denlinger, D. L. (1987) Ontogenetic patterns of cold-hardiness and glycerol production in Sarcophaga crassipalpis. J. Insect Physiol. 33:587-592.

Lees, A. D. (1955) The Physiology of Diapause in Arthropods. University Press, Cambridge.

Lees, A. D. (1969) The behavior and physiology of ticks. Acarologia. 11 (3):397-410.

Lees, A. D. (1973) The morphology of ticks and their relationships with humidity Appl. Ent. Zool. 7:142-154.

Lees, A. D. (1974) The history of phylogenetics in the Sarcophagidae. Appl. Ent. Zool. 7:14-15.

Machin, J. (1984) The study of atmospheric water absorption. In: Measurement of Ion Transport and Metabolic Rate in Insects. Bradley, T. J. and Miller, T. A. (eds.) Springer-Verlag, New York, pp 69-99.

Machin, J. (1990) The study of vapor absorption by arthropods and their eclological significance. Appl. Ent. Zool. 4:10-15

Masaki, S. (1960) Thermal relations of diapause in the eggs of certain crickets (Orthoptera:Gryllidae). Bull. Fac. Agric. Hirosaki Univ. 6:5-20.

Masaki, S. (1962) The influence of temperature on the intensity of diapause in the eggs of the Emma field cricket (Orthoptera:Gryllidae). Kontyu 30:9-16.

Moriarty, F. (1969a) Water uptake and embryonic development in eggs of Chorthippus brunneus Thunberg (Saltroia:Acrididae). J. Exp. Biol. 50:327-333. 27

Moriarty, F. (1969b) Egg diapause and water absorption in the grasshopper Chorthippus brunneus. J. Insect Physiol. 15:2069-2074.

Rapoport, E. H. and Tschapek, M. (1967) Soil water and soil fauna. Rev. Ecol. Biol. Soc. 4:1-58.

Readshaw, J. L. (1965) A theory of phasmatid outbreak release. Aust. J. Zool. 13:475-490.

Readshaw, J. L. (1966) The stick insect and the life histories describing the influences of the reproductive success of their progeny. Appl. Ent. Zool. 2:5-13.

Readshaw, J. L. (1967) Humidity relations in the stick insect, Extatosoma tiaratum and its ecological parameters. Appl. Ent. Zool. 2:5-13.

Readshaw, J. L. (1972) The ecological significance of the stick insect in the surrounding air under equilibrium conditions. Appl. Ent. Zool. 4:5-13.

Readshaw, J. L. (1973) The stick insect and the reproductive consequences of the progeny strains. Appl. Ent. Zool. 4:5-13.

Saunders, D. S. (1982) Insect Clocks (2nd ed.) Pergamon Press, Oxford.

Saunders, D. S. (1983) The reproductive success of the predatitory mite and their humidity relationships with other arthropods. Appl. Ent. Zool. 4:5-13.

Sawchyn, W. W. and Gillot, C. (1974a) The life history of Lestes congener (Odonata:Zygoptera) on the Canadian prairies. Canad. Ent. 106:367-376.

Sawchyn, W. W. and Gillot, C. (1974b) The life histroy of three species of Lestes (Odonata:Zygoptera) in Saskatchewan. Canad. Ent. 106:1283-1293. 28

Seethaler, H. W. Knulle, W. and Devine, T. L. (1979) Water vapor intake and body water (3HOH) clearance in the housemite (Flycyphagus domesticus). Acarolgia. 21:440-450.

Shulov, A. (1952) The development of eggs of Shistocerca gregaria (Forskal) in relation to water. Bull Ent. Res. 43:469-476.

Toolson, E. C. (1980) Thermodynamics and kinetic aspects of water flux through the arthropod cuticle. J. Therm. Biol. 5:1-6.

Toolson, E. C. (1981) The insect cuticle as a model system for vapor absorption kinetic studies. Appl. Ent. Zool. 7:14-15.

Toolson, E. C. (1982) The arthropod integument: new approaches for evidence of a cuticular phenomena Appl. Ent. Zool. 4:5-13.

Voy, A. (1954a) Sur Pexistence de deux categories d'oeufs dans la ponte globale du phasme (Clonopsis gallica Charp.). Comptes Rendus de I'Academie des Sciences a Paris. 238:625-627.

Voy, A. (1954b) Biologie et croissance chez le phasme femelle (Clonopsis gallica Charp.). Bulletin Biologique de la France et de la Belgique. 58:101-129.

Voy, A. (1955) (.'entrance d'une preditorie sur les environments des France et les relations d'une humidite unique. Appl. Ent. Zool. 4 :5 -1 1 .

Voy, A. (1957) L'evidence d'une characteristique des humidities sur la developpement des insects. Appl. Ent. Zool. 12:5-13.

Walker, G. P. and Denlinger, D. L. (1980) Juvenile hormone and moulting hormone titres in diapausing and nondiapausing destined flesh flies. J. Insect Physiol. 26:661-664.

Wharton, G. W. (1985) Water balance of insects. In: Comprehensive Insect Physiology, Biochemistry and Pharmacology. Kerkut, G. A. and Gilbert, L. I. (eds.) Pergamon Press, New York. 14:565- 601. 29

Wharton, G. W. (1986) Water relations in the arthropoda: water sorption hydraulics. Appl. Ent. Zool. 4:53-54.

Wharton, G. W. and Arlian, L. G. (1972) Utilization of water by terrestrial mites and insects. Insect and Mite Nutrition. North Holland pp. 153-165.

Wharton, G. W. and Devine, T. L. (1968) Exchange of water between a mite, Laelaps echidnina, and the surrounding air under equilibrium conditions. J. Insect Physiol. 14:1303-1318.

Williams, C. M. (1969) Photoperiodism and the endocrine aspects of insect diapause. Symp. Soc. Exp. Biol. 23:285-300.

Yamashita, O. and Hasegawa, K. (1985) Embryonic Diapause. In: Comparative Insect Physiology, Biochemistry and Pharmacology. Kerkut, G. A. and Gilbert, L. I. (eds.) Pergamon Press, Oxford. 11:407-430.

Zdarek, J and Denlinger, D. L. (1975) Action of ecdysteroids, juvenoids, and non-hormonal agents on termination of pupal diapause in the flesh fly. J. Insect Physiol. 21:1193-1202. CHAPTER II

WATER BALANCE IN FLESH FLY PUPAE AND WATER VAPOR ABSORPTION ASSOCIATED WITH DIAPAUSE

Abstract

We report the water balance characteristics for diapausing and nondiapausing pupae of the flesh fly, Sarcophaga crassipalpis. The challenge of maintaining water balance is particularly acute for pupae that spend nine to ten months in diapause, without access to drinking water. While diapausing pupae can tolerate a loss of up to 24.5% of their total body water content (67.3%), they have also acquired several other physiological attributes that have enhanced their capacity for maintaining water balance. Net

transpiration rates for diapausing pupae (0.008%* h"1) are far

lower than rates for nondiapausing pupae (0.023%*h '1). In

addition, diapausing pupae can counter water loss with their ability to absorb water vapor from lower humidities (ca. av 0.58 at 20°C) than nondiapausing pupae (ca. av 0.74 at 20°G). The high critical transition temperature for diapausing pupae (39°C, compared to 30°C for nondiapausing pupae) suggests that

30 3 1

epicuticular lipids have been modified to restrict water loss during diapause.

Introduction

One of the most formidable challenges for an insect during diapause, especially a diapause that occurs during the egg or pupal stage, is the maintenance of water balance. In many cases diapause persists for nine to ten months, and during this long period a diapausing egg or pupa has no ability to replenish its water supply by drinking or feeding. This dilemma is further exacerbated by the large surface area : volume ratio characteristic of most insects. Under these circumstances, how is water balance achieved? While it is widely acknowledged as a problem (Lees, 1955; Denlinger, 1986; Tauber et al. 1986), few experiments have examined water balance characteristics of diapausing insects. In this paper we investigate water balance in diapausing and nondiapausing pupae of the flesh , fly, Sarcophaga crassipalpis, and describe the attributes of diapause that enable the pupa to survive for many months without desiccating. Water balance is a function of both water loss and water gain (Wharton, 1985). We determine these rates in diapausing and nondiapausing pupae and demonstrate that diapausing pupae absorb atmospheric water vapor at a much lower av (% R.H./100) than pupae not in diapause. Water vapor absorption allows both 32

types of pupae to recruit water from subsaturated atmospheres to maintain water balance. The effect of temperature on water flux is examined, and our experiments also seek to evaluate the role of the puparium in maintaining water balance.

Materials and methods

Experimental Animals Colonies of Sarcophaga crassipalpis Macquart and Sarcophaga bullata Parker originating from Urbana, Illinois (40°N), and Lexington, Massachusetts (42°N) respectively, were maintained in the laboratory as described (Denlinger, 1972). To ensure a high incidence of pupal diapause, adults lacking a diapause history were exposed to short daylengths (LD 12:12) at 25 ± 1°C, and larvae and pupae were reared at a LD 12:12 at 20 ± 0.5°C. Nondiapausing individuals were generated by exposing adults to long daylength (LD 15:9) at 25 ± 1°C; larvae and pupae were reared at LD 12:12, 20 + 0.5°C. Larvae used in the experiments were in the wandering phase of the third (final) larval instar, two days after departure from the food. The day of pupariation was used as a developmental landmark to stage pupae and pharate adults. All adults used in this study were two to four day old virgin females that were deprived of food and water for 24 h to minimize effects of ingestion, excretion, defecation and reproduction on weight changes (Arlian and Eckstrand, 1975). 33

Experimental Conditions Experiments at 25°C were performed in an environmental room ( + 1°C), and experiments at other temperatures utilized environmental chambers ( ± 0.5°C). Ambient water vapor activities (av) were maintained in sealed desiccators using glycerol- distilled water solutions (Johnson, 1940) or by using anhydrous

CaSC>4 (Drierite) to generate av 0.00. All humidities were verified weekly with a Taylor hygrometer (av ± .03) (Thomas Scientific, Philadelphia). Within the desiccators, pupae were placed on a steel mesh grid upheld with a porcelain plate. Individual adult female flies and third instar wandering larvae were placed in separate 1.5 cm3 polypropylene Eppendorf tubes perforated by 12-1 mm diameter holes as described by Lighton and Feener (1989) and weighed individually. No change in weight was observed for empty tubes during the experiment. No excretory material was observed in the tubes and no excretory material was deposited in the tubes by the stages of flies used in these experiments. All components of the system were predesiccated at av 0.00 for 24 h before use. Body Water Pools, Dehydration Tolerance and Net Transpiration Rate To determine body weight, individuals were weighed on an electrobalance (Cahn 25, Ventron Co.). Initial weight at day 0 was recorded as wet weight, and dry weight was determined after drying the fly over anhydrous CaS 0 4 at 50°C until constant weight. 34

Water mass (m) was defined as the difference between wet weight and dry weight. The quantity of water in the insect was expressed as the ratio of water mass (m) to total wet weight X 100, or water as a percentage of total weight (Wharton, 1985). Dehydration tolerance for diapausing pupae was calculated by placing groups of pupae at 30°C over anhydrous CaS0 4 . Subgroups were removed each day and weighed. The percent change in body weight was determined as the ratio of the difference between the weight at time 0 and at any given time t to the original weight X 100, or percent change in body weight from the original wet weight. Diapause was terminated in the pupae by exposing them to a closed system of hexane vapor for 2 hours (Denlinger et al. 1980), and pupae were held at LD 15:9, 25°C until eclosion. The limit of dehydration tolerance was defined as the minimum amount of water loss that prevented 50% eclosion. To calculate net transpiration (integumental and respiratory water loss), the insects were weighed, placed at av 0.00 for a specified time, and reweighed. The ratio of the water mass at time t (mt) to original mass at time 0 (mo) represents water lost over the time interval with respect to initial water mass. In the case of transpiration into dry air, water content at any time t (mt) is equal to water mass at time 0 (mo) X e"^1, where k is percent water lost in unit time and t is time elapsed between mo and mt (Wharton, 1985) (equation 1): 35

(1) mt = mo e"^t, or In mt/mo = -kt

The observed change in weight of water results from the difference between rate of movement of water from insect to air, (mx), and rate of movement of water from air to insect (ms). This difference is the rate of change of water mass (m) (Wharton, 1985) (equation 2).

(2) m = ms - m i

If ms < m-r, then m < 0 and water mass decreases per unit time. If m s> niT, then m > 0 and water mass increases in time. When ms = m i, m = 0, the pupa is in water balance. At av 0.00, ms is zero and thus mT equals water moving out or m, and transpiration proceeds so that a constant rate (k) of water mass (m) is lost in unit time (eqn. 1). The instantaneous rate of loss at any time t is equal to the product kt. Water loss obeys kinetics governing first order rate laws in which loss is dependent on a single component and is expressed in %‘h'1. The first order rate constant (%*h"1) is equal to the negative slope of an exponential regression between In (mt/mo) and elapsed time (Wharton and Devine, 1968; Devine and Wharton, 1973). An insect's water balance is evaluated by determining net water loss in addition to its competing flow, net absorption (Wharton, 1985). 36

Uptake Kinetics, Critical Transition Temperature Rates of uptake were determined as described by Machin (1984). Briefly, net weight changes (mg) at hydrating vapor activities were corrected for cuticular losses (mg*h_1) at vapor activities to which water was lost. The slope of the regression through the points of water vapor exchange (m g’ h*1) on av describes the rate of uptake (rng’h '1 • Aav_1). Pupae were synchronized physiologically by predesiccation at av 0.00, 20°C for 24 h. Uptake rates were determined 24 h after exposure to the experimental av. The critical transition temperature (CTT) is the temperature at which epicuticular lipids undergo a phase change resulting in dramatic water loss (Edney, 1977). The CTT was estimated for predesiccated (av 0.52, 20°C for 24 h) nondiapausing and diapausing pupae. Rates of net water loss (In k) were determined at various temperatures as previously described and plotted as a function of the reciprocal of absolute temperature (T_1). The slope of the plot of In k against T"1 is equal to -EaR_1 (Seethaler et al. 1979; Toolson, 1980) (equations 3 and 4),

(3) In k = -EaR^T"1 + In A

(4) Ea = -[( tiln ki- tin '1) X ( tV ( ti)2 i f 1) '1] R 37

where Ea is the energy of activation, R is the gas constant, and A the frequency factor; t represents temperature over range i with respect to the loss rate (k) of the amount (n) of water. A simultaneous change in Ea with absolute temperature (T_1) designates a new temperature range (i), and the CTT is defined at the intercept of the slopes describing the two activation energies (Wharton, 1985). A new temperature range was selected when the correlation coefficient describing the relationship between net transpiration (In k) and temperature (K_1(103)) was < 0.95. All water balance parameters were compared with analysis of variance (ANOVA), using Sokal and Rohlfs (1981) test for equality of slopes of several regressions.

Results Water Pool Changes in the total exchangeable water pool through development for nondiapausing and diapausing flies are shown in Figure 1. Throughout development there is a general decrease in body water mass (m) between stadia (ANOVA, P<0.05). When the total body water mass is expressed as a percent adjusted to original weight, no significant difference was observed between pupae and adult flies; larval values however, remained significantly different from pupae and adults (ANOVA, P<0.05). Throughout development, the total available water pool is not significantly different between nondiapausing and diapausing flies. 38

For nondiapausing and diapausing pupae, dry weight is a positive correlate of body water mass (R>0.89), and significantly different from zero (F>519.27, df=399, P<0.0001). Dehydration Tolerance Limit Preweighed diapausing pupae (6 weeks in diapause at 20°C) were transferred to av 0.00, 30°C. The dehydration tolerance limit was defined as the point where 50% of the subgroup (each N=30) failed to eclose. This point was reached when pupae lost 24.5 + 2.5% of their original body mass (initial wet mass range = 128.5 mg - 133.1 mg). Net Transpiration Rates In an atmosphere where the water content is zero, water lost from the pupa's water mass is described by a first order kinetic relationship of exponential decay (eqn.l). Nondiapausing pupae held at av 0.00, 20°C lost water at a rate of 0.023%* h’1 which is significantly faster than diapausing cohorts that lost water at a rate of 0.008%‘h'1 (F=5.447, df=89, P<0.01, Fig. 2). When held at av

0.00, 20°C, third instar larvae not destined for pupal diapause lost water at a rate of 0.43%•h'1. Adult females that emerged from nondiapausing pupae exhibited a water loss rate of 1.10%* h"1. Transpiration rates for third instar larvae and adult females were not significantly different between the nondiapause and diapause groups. All flies were synchronized physiologically by 39 predesiccation at av 0.52, 20°C for 24 h so that weight changes equal the weight of water lost (Wharton, 1985). Water Retention at Hydrating Vapor Activities Percent change in weight was documented for 14 days at 20°C for diapausing pupae (predesiccated av 0.52, 20°C, for 24 h) at hydrating (av 0.85), and dehydrating (av 0.33) atmospheres (Fig. 3B) to determine how long pupae could retain absorbed water and to examine the possibility that infradian O2 consumption cycles (Denlinger et al. 1984) might alter water balance physiology during diapause. Examination of nondiapausing pupae under the same humidity regimes demonstrated that water loss increased with decreasing vapor activity and that no water was gained in nondiapausing pupae at av 0.85 (Fig. 3A). After being held at av 0.85 for 24 h, diapausing pupae gained 0.6 ± .1% while nondiapausing pupae lost 2.0 + .1% of their original weight. At av 0.33, diapausing pupaie lost 0.3 ± .1% of their original weight as nondiapausing cohorts lost 3.2 + .1% within 24 h. For diapausing pupae held at a hydrating av (av 0.85) the initial water vapor absorbed was maintained above 0% weight change and extended over the 14 day time period. The percent change in weight observed for both types of pupae in different humidities demonstrates that total body water loss is reduced in diapausing pupae. The smooth rate of weight change in diapausing pupae suggests that there is no connection between water flux and the 40 infradian cycles of O2 consumption that have been observed during pupal diapause. Mechanism of Water Gain To test the possibility that water gain at hydrating vapor activities might be due to production of metabolic water, a group (N=90) of diapausing pupae was held in a hydrating atmosphere (av 0.85) , then dehydrated for 12 h (av 0.33). A subset of the group (N=45) was dried to constant weight, while remaining pupae were rehydrated (av 0.85) for 12 h. Following rehydration the group was dried to constant weight, and dry weights compared (Knulle, 1967). If metabolism contributes to water gain, oxidation of fatty acids should decrease residual dry weight . Residual dry weights of diapausing pupae were not significantly different following hydration-dehydration (mean dry weight 35.66 ± 2.18 mg), and hydration-dehydration-rehydration (mean dry weight 34.95 ± 1.72 mg) av regimes, thus indicating that the water gain in hydrating atmospheres cannot be attributed to the production of metabolic water. A subgroup of HCN-killed diapausing pupae (N=45) held at a hydrating atmosphere (av 0.85, 20°C) lost 1.4 + .1% of its original weight over a period of five days (Fig. 3B). Control pupae maintained a net gain of 0.6 ± .2% over the elapsed time under the same conditions. The water loss observed when HCN- killed diapausing pupae (predesiccated av 0.52, 20°C, for 24 h) were held at a hydrating vapor activity suggests their reliance on oxygen consumption for water absorption and thus suggests the 41 possibility of an active process or that water loss (Fig. 1) is under spiracular control. Role of the Puparium

Blocking different regions (anterior, middle, and posterior third) of the puparium of diapausing pupae (each N=45) with wax did not alter the ability to gain water at av 0.85, 20°C. Amounts of gain did not differ significantly from control pupae (net gain of 0.62 + .15%). The only significant blockage occurred when the entire puparium was covered with wax (net gain of 0.03 ± .05%) (F=5.407, df=89, PcO.Ol). This suggests that the entire puparium is sufficiently porous to permit water entry. To examine the role of the puparium in restricting water loss, head capsules were removed from a subgroup (N=45) of 6 week old diapausing pupae (predesiccated av 0.52, 20°C, for 24 h) that were placed at av 0.60, 20°C. Open puparia (head capsules removed) lost water at a rate of 3.0 X 10'4 %‘h'1 which was significantly greater than the loss (2.2 X 10'4 %‘h"1) observed with intact puparia held under the same conditions (F=5.431, df=89, P<0.01). Critical Transition Temperature The critical transition temperature (CTT) for nondiapausing pupae was 30.2 + 2.1°C, and in diapausing cohorts the CTT was 39.1 ± 1.4°C (Fig. 4). Differences between nondiapausing and diapausing pupae were significant (F=4.369, df=89, P<0.025). Activation energies, which are used to derive the CTT calculation, were 0.25

± .14 kcal'mol*1 for nondiapausing pupae in the low temperature

range and 4.95 ± .11 kcal’m ol'1 at higher temperatures. These activation energies differed significantly from those of diapausing

pupae: activation energies of 0.09 ± .02 kcal’mol'1 and 3.40 ± .19

kcal'm ol'1 respectively (Fctt=3.243, df=89, P<0.05). CTT values for nondiapausing and diapausing pupae killed with HCN were nearly identical to values observed in viable pupae (29.8 + 1.6°C and 40.2 + 2.3°C respectively). Uptake Kinetics Rates of uptake, determined from the slope of a regression

of water exchange (mg^h'1) on av (Machin, 1984), were estimated

at different temperatures for pupae that were predesiccated at av 0.00, 20°C for 24 h. At 20°C, nondiapausing pupae exhibited a net

uptake rate of 3.49 ± .09 mg’h'^Aay"1 which differed significantly from diapausing pupae at the same temperature (1.76 + .13 mg*h'

^Aay'1) (F=3.549, df=89, P<0.05, Fig. 5). At 25°C, nondiapausing pupae exhibited an uptake rate of 8.44 + .07 mg’h'^Aay'1 that differed from diapausing pupae (5.32 ± .17 m g'h'^Aay'1) (F=4.427, df=89, P<0.025). At 30°C, the estimated CTT for nondiapausing pupae, the rate of uptake declined to 6.67 + .18 mg^h'^Aay'1 while diapausing pupae had an uptake rate of 7.40 ± .09 mg’h^’Aay'1 43

(F=3.147, df=89, P<0.05). At 42°C, a temperature slightly above the estimated CTT for diapausing pupae, the rate of uptake declined to

6.50 + .08 m g'h'^Aay'1. Below the CTT, rate of uptake increased with temperature; once within the range of the CTT however, the rate of uptake dramatically declined. With respect to temperature, rates of uptake between nondiapausing and diapausing groups were significant at PcO.Ol. A perpendicular line drawn from the intercept of the regression line describing the rate of uptake and 0 water vapor exchange to the x axis (Figure 5) defines the minimum av at which water can be absorbed under these conditions. This calculation demonstrates that diapausing pupae can absorb water vapor from lower vapor activities (ca. av 0.58) than nondiapausing pupae (ca. av 0.74) at 20°C. For both types of pupae, those predesiccated at av 0.52, 20°C for 24 h exhibited different changes in weight at av 0.85 (Fig. 3) than those predesiccated at av 0.00 (Fig. 5). Pretreatment at av 0.00 removes a greater percentage of body water than av 0.52 (because ms = 0, eqn. 2), thus rehydration occurs at a higher rate after exposure to a lower av. Both pretreatments however demonstrate the capacity of diapausing pupae to absorb water vapor from lower vapor activities than nondiapausing pupae. Comparative Observations in Sarcophaga bullata Water balance parameters were also examined for a more northern population of a closely related species, S. bullata, under 44

the same set of conditions as described for S. crassipalpis. The total water pool for nondiapausing and diapausing S. bullata pupae (66.8%) is not significantly different from the available water in S. crassipalpis pupae. As in S. crassipalpis, net water loss rates for

nondiapausing pupae of S. bullata (0.018%* h'1) differed

significantly from those in diapause (0.006%* h*1) (F=5.281, df=89,

P<0.01). For both types of pupae, an interspecific difference exists between the two sarcophagid species (Fnd=5.141, df=89, P<0.01; Fd=5.267, df=89, P<0.01). Though water loss rates for nondiapausing and diapausing pupae vary interspecifically, the rate-ratio of nondiapause : diapause remains constant (ca. 3.0). The water vapor absorption and CTT values for both nondiapausing and diapausing pupae ofS. bullata lie within the respective ranges seen in S. crassipalpis. Activation energies from which the CTT is derived are significantly lower for the more northern species (F=3.067, df=89, P<0.05). Activation energies for

nondiapausing pupae were 0.14 ± .06 kcal’mol'1 below the CTT

and 3.55 + .12 kcal’m ol'1 at higher temperatures. Diapausing pupae had energies of 0.073 ± .004 kcal’m ol'1 at low temperatures and 2.27 ± .09 kcal’moT1 above the CTT (F=4.891, df=89, P<0.025). Fig. 1. Total body water pool expressed in mg or % for nondiapausing (A) and diapausing (B) groups of flesh flies (S. crassipalpis) at different stages of development. L-third instar wandering larvae, 2 days after leaving food (N=100); P-day of pupariation (N=200); A-adult virgin females, 2-4 days after eclosion (N=50). Data points represent the mean of N individuals. Vertical error bars lie within the confines of the symbols used on the graph. Water Mass (mg) □ 100 0 2 1 140-1 100 120 140 0 4 80 0 - 80 60 40 0 - 60

- - -

eeomna Stage Developmental A P A P L B i. 1 Fig. r 90 r 70 -7 0 -8 70 60 80 60 90 (0 cn ca ca

(mg'd*1) of exponential decay (eqn. 1). Common regression line (R>0.99) fit to the mean of 45 pupae. 48

0.00-f

- 0.01 -

- 0.02 -

-0.03 -

-0.04 0 2 4 Time (days)

Fig. 2 Fig. 3. Percent change in weight in response to long term exposure to hydrating (av 0.85) and dehydrating (av 0.33) atmospheres at 20°C for nondiapausing (A) and diapausing (B) S. crassipalpis pupae (predesiccated av 0.52, 20°C, 24 h). Weight change was also monitored in a group of diapausing pupae killed with HCN. Data points represent the mean of 45 pupae. Change in Weight (%) 10 -4.0 - -4.0 •0.5- ■ ■ ' 1.5 0.0 - 0.5 1.0 8.0 1.0 6.0 2.0 . 0 - - - - -■ 0 me days) s y a (d e im T 5 i. 3 Fig. v .5 C killed HCN 0.85 av 1

0 1 5 u 0.33 au w 0.33 3w 0.85 u a y 0.85 3y 50 Fig. 4. Arrhenius plot of In k against K'^IO3) for nondiapausing (A) and diapausing (B) S. crassipalpis pupae (predesiccated av 0.52, 20°C, 24 h). The slope of the lines gives the mean activation energy for each temperature range. The intersection of the two lines is the CTT. Data points represent the mean of 45 pupae; vertical error bars signify 95% confidence limits. Water Loss (%*h 0.00 0 0 0.30- 0.40- 0.00 0 0.40- 0.60- 0.80- 1 1 . . . 0 2 10 . . 0 2 0 0 0 2 5 0 5 0 5 0 5 ° 0 5 45 40 35 30 25 20 15 - - - * * .0 .0 .0 3.1 0 3.20 3.30 3.40 1.7 4.95x - 16.37 • 08 • .0 R 0.99 = R 3.40x • 10.87 at e r tu ra e p m e T i. 4 Fig. 0.97 08 • 0.25x • 0.87 - 03 - 0.09x - 0.32 - 1 13) (103 "1 K 52 53

Fig. 5. Rates of water vapor exchange for nondiapausing (A) and diapausing (B) S. crassipalpis pupae (predesiccated av 0.00, 20°C for 24 h). The slope of the line at hydrating av’s indicates the mean rate of uptake (mg’h'^A av'1). Data points represent the mean of 45 pupae; vertical error bars signify 95% confidence limits. Water Vapor Exchange (mg»h -0 .2 0 H 0 .2 -0 - 0.00 0 0.40- 0.60- 0.801 0.20 0.00 0 0.40- 0.60- 0.80- 1 1 . . . . 0 2 0 2 0 0 20-1 0.00 ------

0.20 1— 10 + 1.76x + 1.03 - 25 + .9 R 0.95 = R 3.49x + 2.54 - .0 0.60 0.40 i. 5 Fig. av 0.99 0.80 1.00 55

Discussion This study demonstrates that diapause has a profound effect on several aspects of water balance. In diapausing flesh fly pupae, net transpiration rates are far lower than in nondiapausing pupae, water vapor absorption occurs at lower humidities, and a high critical transition temperature associated with diapausing pupae suggests the presence of an epicuticular barrier that is unique to diapause. Water content of insects can range from 45 to 90% (Rapoport and Tschapek, 1967), but for most insects mean body water content is about 70% (Edney, 1977). The percent body water in flesh fly pupae (67%) is thus close to this reported mean value, as is the tsetse fly pupae (71%) as reported by Bursell (1958). Some insects and even small mites can lose more than 50% of their water masses and still survive (Wharton, 1985), but others tolerate only about a 20% loss (Arlian and Veselica, 1979). Across a broad size range of Glossina species, Bursell (1958) reports that tsetse fly pupae can withstand a loss of about 28% of their body water. The dehydration tolerance limit is slightly less (24%) for diapausing pupae of S. crassipalpis. The low net transpiration rate observed in diapausing flesh fly pupae (0.008%*h '1) makes a major contribution to the conservation of the body water pool for the long duration of diapause. For flesh fly pupae not in diapause, the rate of water 56

loss (0.023%* h '1) approximates that of tsetse fly pupae(Glossina morsitans) at a comparable developmental stage (Bursell, 1958). In tsetse pupae, Bursell (1958) found that the puparium confers resistance to desiccation. This is also observed in diapausing flesh fly pupae. Throughout the nine to ten months of diapause, a pupa is vulnerable to the loss of a large percentage of its body water. To counter this loss, pupae can absorb water against the aw (0.99) of their hemolymph. In tsetse, a species that lacks the capacity for diapause, Jack (1939) and Bursell (1958) observed no uptake of water by pupae held in saturated air beyond that which could be accounted for by the hygroscopic properties of the puparium. Some water gain in flesh fly pupae may be attributed to the hygroscopic properties of the puparium as proposed by Bursell (1958), but the vapor activity at which water absorption occurs in both diapausing and nondiapausing pupae is below vapor saturation and presents a dramatic case of water movement against a large atmospheric gradient. Water vapor absorption at subsaturated atmospheres is often assumed to be an active process because it ceases at death (Edney, 1977). Killed diapausing flesh fly pupae lost water when exposed to a vapor activity that hydrated viable pupae; however, killed pupae are not definitive controls for active uptake because spiracular closing mechanisms will be inoperable, thus much of the water may be lost from respiratory surfaces. The possibility that 57

water gain may be due to passive chemisorption and physical adsorption of water vapor is a likely scenario because predesiccated pupae increase in mass only by a small amount when transferred to a higher av, the uptake is not maintained, and a new equilibrium water content is approached after 1 - 2 days. But, the amount of water gained by adsorption decreases with temperature (Glasstone and Lewis, 1960), and the rates of water gain we observe increase with temperature (until the CTT), thus implying that water is absorbed and contributes to sorption (ms, eqn. 2). HCN-killed diapausing pupae show no evidence of first- day passive absorption that could account for the water gain (even though quite small) observed in living pupae held at the same humidity. Furthermore, no significant (passive) absorption was observed in pupae transferred from dry air to humidities below 'hydrating' humidities. Thus, these data indicate that passive absorption cannot completely account for the total water gained from subsaturated air, but the methods we have used in these experiments are not fully adequate to distinguish between active and passive processes. Water vapor absorption is usually restricted to a specific body region (Noble-Nesbitt, 1970; Machin, 1975; O'Donnell, 1977; Rudolph and Knulle, 1982). To identify the site of absorption, different body regions can be coated with molten wax that once solidified, forms an obstruction to vapor uptake. Only when puparia of diapausing pupae are completely covered with wax do 58 they lose their ability to absorb water. This implies that the puparium, the inert third instar exocuticle that encapsulates the pupa, is permeable to water over its entire surface. Whether there is a specific site of uptake on the pupa has not been determined. Metabolic water is available to insects as a by-product of oxidative catabolism of primarily carbohydrates and fats (Wharton, 1985). Fat constitutes the main energy reserve of flesh fly pupae (Adedokun and Denlinger, 1985) and tsetse pupae (Buxton and Lewis, 1934). Bursell (1958) reports that the fat content of pupae held at 0% R.H. is not statistically different from pupae held at 98% R.H. and concluded that metabolic water does not increase to compensate for water loss at lower humidities. The same conclusion can be drawn from flesh fly pupae following one cycle of dehydration-rehydration. The critical transition temperature (CTT) is the point above which transpiration rates dramatically increase with temperature, and CTT's in insects range from 30°C to 60°C (Edney, 1977; Lighton and Feener, 1989). The temperature-dependency function of water loss fits a Boltzmann temperature function. An Arrhenius equation predicts the proportion of water molecules permitted to escape the cuticle as necessary energies approach a given temperature (Williams and Williams, 1967). A change in cuticular permeability denotes a new activation energy (kcal’m ol'1) indicated by a change in the slope of the Arrhenius plot (Needham and Teel, 1986). Though the basis for the CTT remains controversial (Edney, 1977; Toolson, 1978; Seethaler, et al. 1979; Gilbey, 1980; Monteith and Campbell, 1980; Machin, 1980), most workers agree that the CTT denotes an important transition in the epicuticular lipids, an important barrier to transpirational water loss. The fact that the CTT of diapausing pupae is 9°C higher than in nondiapausing pupae implies that pupae in diapause require a higher temperature to induce a phase change in these epicuticular lipids. This study suggests that the high CTT's for diapausing flesh fly pupae and their low rates of water loss may be related to increases in quantity or qualitative changes in epicuticular lipids, as demonstrated in other diapausing pupae, the tobacco hornworm, Manduca sexta (Bell, et al. 1975) and the Bertha army worm, Mamestra configurata (Hegdekar, 1979). Our recent experiments have verified that such changes are indeed associated with the diapause of Sarcophaga (unpublished observations). The 2-3 fold increase in cuticular hydrocarbons we observe in association with diapause is likely to require a higher temperature to elicit a phase transition, i.e. the CTT should be higher. According to reaction energetics, a large Ea, as shown in this study, corresponds to low collision frequencies of water molecules, thereby partitioning more water into the gaseous phase and increasing the ease of water escape. As shown by these data, at higher CTT values (implying a quantitative or qualitative change in epicuticular lipids) activation energies (describing the frequency of water molecule collisions) are suppressed and lead to a lower water loss rate. 60

During diapause flesh fly pupae exhibit infradian cycles of oxygen consumption (Denlinger, et al. 1984), yet rates of water absorption and loss that we observed in this study occur at a fairly constant rate for many days. It is thus unlikely that the pupa's cycles of respiration and metabolism are affecting water flux. The water balance properties we have observed in S. crassipalpis are likely to be shared by other related species. Our results with S. bullata were quite similar to our observations with S. crassipalpis. Again, diapausing pupae absorb water at lower vapor activities and have higher CTT's than nondiapausing pupae. The rate of water loss in S. bullata is lower than in S. crassipalpis, and lower activation energies in S. bullata correspond to lower transpiration rates into dry air. This may indicate that S. bullata is better adapted to surviving in a dry environment, but insufficient distribution data are available to verify this prediction. 61

References

Adedokun, T. A. and Denlinger, D. L. (1985) Metabolic reserves associated with pupal diapause in the flesh fly S arcophaga crassipalpis. J. Insect Physiol. 31, 229-233.

Arlian, L. G. and Ekstrand, I. A. (1975) Water balance in Drosophila pseudoobscura, and its ecological implications. Ann. Ent. Soc. Am . 68, 827-832.

Arlian, L. G. and Veselica, M. M. (1979) Significance of passive sorption of atmospheric water vapor and feeding in water balance of the rice weevil, Sitophilus oryzae. Comp.Biochem. Physiol. 62 A, 725-733.

Arlian, L. G. and Wharton, G. W. (1974) Kinetics of active and passive water exchange between the air and a mite. J. Insect Physiol. 62 B, 725-733.

Bell, R. A., Nelson, D. R., Borg, T. G. and Caldwell, D. L. (1975) Wax secretion in nondiapausing and diapausing pupae of the tobacco hornworm, Manduca sexta. J. Insect Physiol. 21, 1 7 2 5 -1 7 2 9 .

Bursell, E. (1958) The water balance of tsetse pupae. Philos. Trans. B. 2 4 1 , 179-210.

Bursell, E. (1959) the water balance of tsetset. J. Insect Physiol. 62 A, 725-733.

Buxton, P. A. and Lewis, D. J. (1934) Climate and tsetse flies: laboratory studies upon Glossina submorsitans an d tachinoides. Philos. Trans. B. 224, 175-240. 62 Denlinger, D. L. (1972) Induction and termination of pupal diapause in Sarcophaga (Diptera: Sarcophagidae). Biol. Bull. mar. biol. Lab. Woods Hole. 142, 11-24.

Denlinger, D. L. (1986) Dormancy in tropical insects. Ann. Rev. Ent. 31, 239-264.

Denlinger, D. L., Campbell, J. J. and Bradfield, J. Y. (1980) Stimulatory effect of organic solvents on initiating development in diapausing pupae of the flesh fly,Sarcophaga crassipalpis, and the tobacco hornworm, Manduca sexta. Physiol. Ent. 5, 7-15.

Denlinger, D. L., Shukla, M. and Faustini, D. L. (1984) Juvenile hormone involvement in pupal diapause of the flesh fly Sarcophaga crassipalpis regulation : of infradian cycles of O 2 consumption. J. exp. Biol. 109, 191-199.

Devine, T. L. (1972) The kinetics of water sorption and its ecological implications for insect systems. J. Insect Physiol. 62 A, 725-733.

Devine, T. L. and Wharton, G. W. (1973) Kinetics of water exchange between a mite, Laelaps echidnina, and the surrounding air. J. Insect Physiol. 19, 243-254.

Edney, E. B. (1977) Water Balance in Land Arthropods. Springer- Verlag, New York.

Edney, E. B. (1978) The water balance of the flea beetle and its relationships with the surrounding air. J. Insect Physiol. 6 2 A, 725-733.

Gilbey, A. R. (1980) Transpiration, temperature and lipids in insect cuticle. Adv. Insect Physiol. 15, 1-33.

Glasstone, S. and Lewis, D. (1960) Elements of Physical Chemistry. D. Van Nostrand, New York. 63

Hegdekar, B. M. (1979) Epicuticular wax secretion in diapausing and nondiapausing pupae of the Bertha armyworm. Ann. Ent. Soc. Am. 72, 13-15.

Jack, R. W. (1939) Studies in the physiology and behaviour of Glossina morsitans Westsw. Mem. Dept. Agric. S. Rhod. 1,1- 203.

Johnson, C. G. (1940) The maintenance of high atmospheric humidities for entomological work with glycerol-water mixtures. Ann. appl. Biol. 27, 295-299.

Knulle, W. (1967). Physiological properties and biological implications of the water vapour sorption mechanisms, in larvae of the oriental rat flea Xenopsylla cheopis (Roths). J. Insect Physiol. 13, 333-357.

Lees, A. D. (1955) The Physiology of Diapause in Arthropods. University Press, Cambridge.

Lighton, J. R. and Feener Jr., D. H. (1989) Water-loss rates and cuticular permeability in foragers of the desert ant Pogonmyrmex rugosus. Physiol. Zool. 62, 1232-1256.

Machin, J. (1975) Water balance in Tenebrio molitar L. larvae; The effect of atmospheric water absorption. J. comp. Physiol. 101, 121-132.

Machin, J. (1980) Cuticle water relations: towards a new cuticle water proofing model. In: Insect Biology in the Future. Locke, M. and smith, D. S. (eds.) Academic Press, New York pp. 79- 103. Machin, J. (1984) The study of atmospheric water absorption. In: Measurements of Ion Transport and Metabolic Rate in Insects. Bradley, T. J. and Miller, T. A. (eds.) Springer-Verlag, New York. pp. 69-99.

Monteith, J. L. and Campbell, G. S. (1980) Diffusion of water vapor through integuments-potential confusion. J. therm. Biol. 5, 7 -9 . 64

Needham, G. R. and Teel, P. D. (1986) Water balance by ticks between blood meals. In: Morphology, Physiology, and Behavioral Biology of Ticks. Sauer, J. R. and Hair, J. A. (eds.) John Wiley and Sons, New York. pp. 100-151.

Noble-Nesbitt, J. (1970) Water uptake from subsaturated atmospheres: Its site in insects. N ature. 225, 753-754.

O'Donnell, M. J. (1977) Site of water vapor absorption in the desert cockroach, Arenivaga investigata. Proc. Nat. Acad. Sci., USA. 74, 1757-1760.

Rapoport, E. H. and Tschapek, M. (1967) Soil water and soil fauna. Rev. ecol. Biol. Soc. 4,1-58.

Rudolph, D. and Knulle, W. (1982) Novel uptake systems for atmospheric vapor among insects. J. exp. Zool. 222, 321- 334.

Seethaler, H. W., Knulle, W. and Devine, T. L. (1979) Water vapor intake and body water (3HOH) clearance in the housemite (Flycyphagus domesticus). Acarologia. 21, 440-450.

Sokal, R. R. and Rohlf, F. J. (1981) Biometry . W. H. Freeman Co., New York.

Tauber, M. J., Tauber, C. A. and Masaki, S. (1986) Seasonal Adaptations of Insects. Oxford: Oxford University Press.

Toolson, E. C. (1978) Diffusion of water through the arthropod cuticle: thermodynamic consideration of the transition phenomenon. J. therm. Biol. 3, 69-73.

Toolson, E. C. (1980) Thermodynamic and kineitc aspects of water flux through the arthropod cuticle. J. therm. Biol. 5, 1-6. 65

Wharton, G. W. (1985) Water balance of insects. In: Comprehensive Insect Physiology, Biochemistry, and Pharmacology, vol. 14 Kerkut, G. A. and Gilbert, L. I., Pergamon Press, Oxford, pp. 5 6 5 -6 0 1 .

Wharton, G. W. and Devine, T. L. (1968) Exchange of water between a mite, Laelaps echidnina, and the surrounding air under equilibrium conditions. J. Insect Physiol. 14, 1303-1318.

Williams, V. R. and Williams, H. B. (1967) Basic Physical Chemistry for the Life Sciences. W. H. Freeman Co., San Francisco. CHAPTER III

A COMPARISON OF THE WATER BALANCE CHARACTERISTICS OF TEMPERATE AND TROPICAL FLY PUPAE

A bstract Water balance characteristics of temperate zone fly pupae are compared with the characteristics of flies inhabiting the tropics. The flies, all of which were reared without diapause, had very similar equilibrium weights that were quite high (av 0.90 - 0.92), thus implying a limited capacity to absorb water from a subsaturated atmosphere. Likewise, the critical transition temperatures (CTT) were nearly the same for all the flies. Net transpiration rates at 20°C are a function of size, but the rate is less size dependent as temperature increases. When water loss is examined across a broad temperature range, as described by activation energies, it is apparent that the tropical flies lose water at a greater rate than their temperate zone counterparts. Activation energy may be a good parameter to use in evaluating habitat preference and suitability for a species because it describes water loss as a function of temperature, and thus is likely to be a

66 67 good indicator of the insect's response to the fluctuating temperatures that occur naturally.

Introduction A Central American tropical Tain forest is a vastly different environment from the temperate regions of North America, thus we might expect the water balance strategies for insects from these two regions to differ. In this paper we compare water balance in pupae of two tropical flesh fly species with three fly species inhabiting the temperate zone. The maintenance of water balance is especially important for pupae because they are incapable of imbibing free water. The temperate zone flesh flies overwinter in pupal diapause (review by Denlinger, 1981), and we previously demonstrated that during diapause, flesh fly pupae can absorb water vapour from subsaturated atmospheres, a mechanism that presumably enables the pupa to avoid desiccation during the nine to ten month period of dormancy (Yoder and Denlinger, 1990). The neotropical species we examine in this study do not have a diapause (Denlinger et al., 1988), thus this comparison with the temperate species examines only pupae that are not in diapause. For the flies in this study, we determine net transpiration rates at different temperatures and show their correlation with pupal size. The capacity to absorb atmospheric water vapour is evaluated and the equilibrium weight achieved after 24 h cannot be correlated with net transpiration rate. We also determine the 68

critical transition temperature (CTT) (Wharton, 1985), the temperature at which epicuticular lipids undergo a phase change resulting in dramatic water loss (Edney, 1977). Our experiments show that the activation energies (rate of water loss as a function of temperature (Toolson, 1978; Wharton, 1985)), from which the CTT is derived, are higher in tropical fly pupae, implying that they lose water more readily than flies from the temperate zone.

Materials and Methods Experimental Animals Tropical flesh flies (Sarcophagidae), Peckia abnormis (Enderlein) and Sarcodexia sternodontis Townsend, were collected from the lowland, moist forest of Barro Colorado Island, Panama (9°N, <137m) and maintained in colony along with temperate flesh flies, Sarcophaga crassipalpis Macquart from Urbana, Illinois (40°N) and Sarcophaga bullata Parker from Lexington, Massachusetts (42°N) as described (Denlinger, 1972). Small S. crassipalpis pupae were obtained by overcrowding the larvae. The house fly, Musca domestica (Muscidae), collected from Columbus, Ohio (40°N), was reared on artificial diet and maintained in the Ohio State University insectary. Tropical flies are incapable of diapause (Denlinger et al, 1988) and were reared at LD, 12:12 h (25 ±1°C) throughout development and temperate flies at LD, 15:9 h (25 ±1°C) to prevent diapause (Denlinger, 1981). To standardize developmental stage, all 69 experiments were initiated on the day of pupation (2 days after pupariation at 25°C). Experimental Conditions Individual pupae were weighed on an electrobalance (CAHN 25, Ventron Co.)- Experiments at 25°C utilized environmental rooms (±1°C), and experiments at other temperatures utilized environmental chambers ( ± 0.5°C). Water vapour activities (av) were maintained by glycerol-distilled water solutions (Johnson,

1940) in sealed desiccators; anhydrous CaSC>4 (Drierite) generated av 0.00. Within the desiccators pupae were placed on suspended steel mesh grids. Water balance characteristics, including body water pool (wet weight, % body water, water mass (difference between wet and dry weight)), net transpiration rate, and activation energies (Ea) used to derive the critical transition temperature (CTT), were determined as previously described (Wharton, 1985). Net transpiration (integumental and respiratory water loss) into dry air (av 0.00) is a first order kinetic relationship of exponential decay. The slope of a line on a semi-log plot of the water mass ratio at any time t to initial water mass (mt/mo) against time is the rate of water loss (%‘h'1) (Wharton, 1985).

The capacity to absorb water vapour from subsaturated air was evaluated by placing predesiccated pupae (av 0.52, 20°C for 24 h) in a series of av regimes. After a 24 h exposure to the experimental av, the percent change in weight was calculated 70

(difference between the weight at time t and initial wet weight divided by initial wet weight X 100). The av at which a 0% change in weight was observed indicates the equilibrium weight achieved after a 24 h av exposure and designates the activity above which a positive change in weight (water gain) occurs. The energy of activation (Ea) was derived from the slope of a line on an Arrhenius plot of water loss (In k) over a range of reciprocal absolute temperatures (T'1) (Toolson, 1980). A new temperature range designates a different Ea and was selected when the correlation coefficient between In k and T'1 was < 0.95. The CTT is the point of intersection of the two slopes defining the two activation energies (Wharton, 1985). These characteristics in the different experimental groups were compared using analysis of variance (ANOVA). Percentage body water data were arcsine transformed prior to statistical analysis. Parameters involving regression lines were compared according to Sokal & Rohlfs (1981) test for equality of several slopes.

Results Body Water Mass Wet weights for all pupae studied (range of 14.11 - 136.95 mg) are shown in Table 1. Body water mass was a positive correlate of dry weight (R>0.89), and slopes of regression lines describing this relationship were all significantly different from 7 1 zero (F>325.07, df=399, p<0.0001). Despite variation in size (wet weight), percent water content (ratio of water mass to wet weight X 100) for the six flies (range of 65.1 - 67.0%) was not significantly different. All flies successfully eclosed in dry air (av 0.00) at 25°C. Net Transpiration Rates Total water loss at 20°C into dry air (av 0.00) for the fly pupae is shown in Table 1. These net transpiration rates, range of

0.018%‘IT1 (S. bullata) to 0.069%‘ h '1 (5. sternodontis), are a function of pupal size (R=0.92, Fig. 6A), and within a single species, S. crassipalpis, water is lost from small pupae (wet weight 33.09 mg) at a significantly greater rate (0.045%*h '1) than from large pupae (128.79 mg) (0.023%* h’1) (F=5.027, df=89, p<0.01). Net transpiration rates at 20°C for large-sized pupae, S. crassipalpis (temperate zone) and P. abnormis (tropical) did not differ significantly; small-sized pupae, M. domestica (temperate zone) and S. sternodontis (tropical) also lost water at the same rate. Thus, at 20°C there appears to be no relationship between net transpiration and locality. However, the strength of the correlation between pupa size and net transpiration rate becomes less as temperature increases (at 25°C, y=0.1847-0.0289x, R=0.77 and at 30°C, y=1.5636-0.2602x, R=0.38), suggesting that factors other than a large surface area to volume ratio also contribute to water loss when a range of temperatures is considered. 72

Equilibrium Weight The equilibrium weights (av at which a 0% change in weight was observed after a 24 h av exposure) at 20° C for the temperate and tropical flies (predesiccated av 0.52, 20°C for 24 h), all of which were reared under non-diapausing conditions, are shown in Table 1. Among these pupae, equilibrium weight estimates (range av 0.90 - 0.92) did not significantly differ, and the equilibrium weight values did not correlate with net transpiration rate at 20°C (Fig. 6B, R=0.32) Activation Energies and Critical Transition Temperature The activation energies (Ea) were significantly different in the temperate and tropical fly pupae that were pre-desiccated at av 0.52, 20°C for 24 h (Table 1). The activation energies for temperate fly pupae (range 0.14 - 0.38 kcaHmol'1 at low temperatures (below the CTT), and 3.55 - 4.95 kcaFmoT1 at high temperatures (above the CTT)) are consistently lower than the activation energies of Panamanian flies at both low and high temperatures. However, critical transition temperatures (CTT), determined from the intercept of the slopes defining the two activation energies, did not differ significantly for the fly pupae studied. As these data demonstrate, different species can have the same CTT even though their activation energies are quite different. The most accurate comparisons of activation energies can be made between pupae of the temperate fly M. domestica and the tropical fly S. sternodontis 73 because their water balance parameters are very similar. Both share the same % body water content, transpiration rate (at 20°C), 24 h equilibrium weight and CTT. Yet, the two species differ greatly with respect to the activation energies describing water loss in both the low and high temperature ranges, thus implying that water is lost more readily from the tropical fly species across a broad range of temperatures. Table 1. A comparison of wet weight, % body water (arcsine transformed), net transpiration rate (-kt, 20°C), 24 h equilibrium weight (20°C), CTT, and activation energies (Ea) below (<) and above (>) the CTT for temperate zone and tropical fly pupae. Mean values + S. D. (each n=45) followed by the same letter within a column are not significantly different (ANOVA, p<0.05).

WET EQUILIBRIUM CTT Ea < CTT Ea > CTT BODY - k t 2 0°C SPECIES WEIGHT WATER WEIGHT (°C ) (kcalmol"^) ( k c a lm o r * ) (m g) {%) (fth '1) (av)

Temperate species

S. crassipalpis 128.79 + 2.8a 66.41 + Z la 0.023a 0.92 + .02a 30.2 + 2.1a 0.25 + .14a 4.95 + ,lla

S. crassipalpis (small) 33.09 + 2.3b 65.40 + 2.3a 0.045b 0.91 ± .04a —— ...

S. bullata 126.43 + 3.2a 65.13 + 1.9a 0.018c 0.91 + .03a 30.4 + 1.6a 0.14 + .06b 3.55 + .12b

M. domestica 14.11 + 2.1c 66.53 + 2.4a 0.066d 0.90 + ,03a 30.1 + 2.4a 0.38 + ,04c 4.95 + ,09a

Tropical Species

P. abnormis 136.95 + 2.2d 67.00 + 1.6a 0.024a 0.90 + ,04a 31.1 + 1.4a 0.41 + .09c 13.08 + ,08c

S. sternodontis 31.87 + 1.8b 65.14 + Z0a 0.069d 0.91 ± .03a 30.4 + 2.7a 0.60 + ,08d 79.47 + ,13d Fig. 6. The relationship between net transpiration rate (-kt, 20°C) in six species or sizes of fly pupae and (A) In wet weight (Wo), R=0.92, and (B) 24 h equilibrium weight (av at 20°C), R=0.32. B y = 0.1281 - 0.0216X R = 0.92 y = 0.9065 • 0.9529X R = 0.32

S. sternodontis 0 .0 7 - □ S. sternodontis □ M. dom estica M. dom estica

JC 0.05- crassipalpis (small) □ S. crassipalpis (small)

l 0 .03- ’. abnorm is P. abnormis Q □ S. crassipalpis crassipalpis □ bullata S. bullata 0.01 ------* i 1 1 1 1 2 . 5 3 . 5 4 . 5 5 . 5 0.89 0.90 0.91 0.92 0.93 In W0 Equilibrium Weight (av)

Fig. 6

- j CT\ 77

Discussion Many water balance characteristics of the six types of fly pupae examined in this study are quite similar, in spite of differences in species, locality of origin, and body size. Water content was relatively constant (65.1 - 67.0%) and is also similar to the water content previously reported for pupae of another cyclorraphous Diptera, tsetse (71% reported by Bursell, 1958), and for many other insect species (Edney, 1977). The equilibrium weights for all pupae in this study are quite high (av 0.90 - 0.92) and show very little variation among the species. Thus, all of these non-diapausing pupae have very limited capacity to absorb water vapour from the air, and the water gain that does occur at vapour activities near saturation may be due to the hygroscopic properties of the puparium, as suggested by Jack (1939) and Bursell (1958). The equilibrium weights for the five fly species do not correlate with transpiration rate at 20°C. Transpiration rates for several species of Ixodid ticks do not correlate with their critical equilibrium activities (CEA) (Needham and Teel, 1986) and these investigators concluded that the CEA, and as shown by these data, equilibrium weight cannot be used to infer an organism's habitat preference in terms of their humidity requirements. Critical transition temperature, the temperature at which the transpiration rate dramatically increases, is also very similar for the five species tested. Though the significance of the CTT is controversial (Edney, 1977; Toolson, 1978; Gilbey, 1980; Machin, 1980), the similarity of 78

the critical transition temperatures of all the pupae in this study suggests there are no substantial quantitative or qualitative differences in the properties of the epicuticle. Net transpiration rates differ among the flies in this study. The rate of total water loss at 20°C increases with a decrease in wet weight, hence at that temperature net transpiration is a function of size, as demonstrated in other insects (Schmidt-Nielsen, 1984), though the degree of correlation decreases with increasing temperature, implying that water loss at some temperatures is independent of size. Though diapausing flesh flies can tolerate a 24.5% loss in body water (Yoder and Denlinger, 1990) and tsetse pupae a 28% loss (Bursell, 1958), the small pupae would appear quite vulnerable to desiccation. The danger of desiccation in the small tropical flesh fly S. sternodontis is perhaps partially reduced by its rapid developmental time; while the larger tropical fly (P. abnormis) and the temperate zone flies (S. bullata and S. crassipalpis) complete a generation in 30 - 32 days at 25°C, S. sternodontis requires only 20 days (Denlinger et al., 1988). The one feature that distinguishes temperate and tropical flies is activation energy (Ea). Activation energies are consistently higher in the tropical flies, implying that they lose water faster than flies from the temperate zone. Though transpiration rates at 20°C correlate well with body size when a range of temperatures is considered, as in calculation of the Ea, transpiration rates are independent of pupal size (low correlations at 25°C and 30°C) and 79

lead to dramatically different water loss rates at given temperatures and thus yield different activation energies. A large Ea corresponds to low collision frequencies of water molecules, thus more water is partitioned into the gaseous phase and water escape is facilitated. Activation energies describe transpiration as a two component curve with different water loss rates dependent on temperature and, as shown by these data, independent of size. Because activation energies describe transpiration rate over a broad temperature range, they are likely to be a useful water balance parameter for predicting habitat suitability for a species. All comparisons in this study were based on non-diapausing individuals. The neotropical flies lack the genetic capacity for diapause (Denlinger et al., 1988) and the temperate species were reared under environmental conditions (long day length) that prevent the expression of pupal diapause (Denlinger, 1981). This study and the work on tsetse (Bursell, 1958) thus suggest that most water balance parameters of non-diapausing fly pupae are quite similar. Diapause, however, invokes a distinctly different syndrome of water balance characteristics in flesh flies (Yoder and Denlinger, 1990). Unlike non-diapausing pupae, pupae in diapause have reduced net transpiration (-kt=0.008%•h"1), can absorb water vapour from the atmosphere even at low humidities (ca. av 0.64, 24 h equilibrium weight for pupae predesiccated at av 0.52, 20°C for 24 h), and pupae in diapause have a much higher CTT (39°C) than their non-diapausing counterparts (30°C), which implies that the epicuticular lipids of the two types of pupae are likely to differ. The relatively brief duration of the pupal stadium in non- diapausing flies apparently has not necessitated evolution of the water conservation and absorption properties associated with diapause. 81

References

Bursell, E. (1958) The water balance of tsetse pupae. Philosophical Transactions. 241 B:179-210.

Denlinger, D. L. (1972) Induction and termination of pupal diapause in Sarcophaga (Diptera: Sarcophagidae). Biological Bulletin. 142: 11-24.

Denlinger, D. L. (1981) The physiology of pupal diapause in flesh flies. In: Current Topics in Insect Endocrinology and Nutrition. Bhaskaran, G., Friedman, S. and Rodriguez, J. G. (eds.) Plenum, New York. pp. 131-160.

Denlinger, D. L., Chen, C-P & Tanaka, S. (1988) The impact of diapause on the evolution of other life history traits in flesh flies. Oecologia. 77:350-356.

Edney, E. B. (1977) Water Balance in Land Arthropods. Springer- Verlag, New York.

Gilbey, A. R. (1980) Transpiration, temperature and lipids in insect cuticle. Advances in Insect Physiology. 15:1-33.

Jack, R. W. (1939) Studies in the physiology and behaviour of Glossina morsitans Westw. Memoirs of the Department of Agriculture of Southern Rhodesia. 1:1-203.

Johnson, C. G. (1940) The maintenance of high atmospheric humidities for entomological work with glycerol-water mixtures. Annals of Applied Biology. 27:295-299. 82 Needham, G. R. and Teel, P. D. (1986) Water balance by ticks between blood meals. In: Morphology, Physiology and behavioral biology of Ticks. Sauer, J. R. and Hair, J. A. (eds.), John Wiley and Sons, New York. pp. 100-151.

Machin, J. (1980) Cuticle water relations: towards a new cuticle waterproofing model. In: Insect Biology in the Future. Locke, M. and Smith, D. S. (eds.) Academic Press, New York. pp. 79- 103.

Schmidt-Nielsen, K. (1984) Scaling: Why Is Animal Size So Im portant? Cambridge University Press, New York.

Sokal R. R. & Rohlf, F. J. (1981) Biometry. W. H. Freeman and Co., New York.

Toolson, E. C. (1978) Diffusion of water through the arthropod cuticle: thermodynamic consideration of the transition phenomenon. Journal of Thermal Biology. 3:69-73.

Wharton, G. W. (1985) Water balance of insects. In: Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 14, Kerkut, G. A. and Gilbert, L. I. (eds.) Pergamon Press, Oxford, pp. 565-601.

Yoder, J. A. and Denlinger, D. L. (1991) Water balance in flesh fly pupae and water vapor absorption associated with diapause. Journal of Experimental Biology (in press). Chapter IV

WATER VAPOR UPTAKE BY DIAPAUSING EGGS OF A TROPICAL WALKING STICK

A bstract

This study is the first to demonstrate the capacity of an arthropod egg, a tropical walking stick Extatosoma tiaratum (Mac Ieuy), to absorb water vapor from the air. This species diapauses both as an early embryo and then again as a pharate first instar larva, and both stages are capable of absorbing water vapor. Water vapor absorption occurs at lower humidities and at a lower rate for an egg in early embryonic diapause (ca. av 0.30, 0.516 m g^h^'Aa/ *) than in the diapausing pharate first instar (ca. av 0.60, 0.725 m g'h'^A ay'1) at 25°C. In addition to having the capacity to gain water at very low vapor activities, water is efficiently conserved as indicated by the low rate of water loss (0.015 %*h_1 in the early embryo and 0.046 %*h_1 in the pharate larva at 25°C). Eggs that have been killed lose water when held at a hydrating vapor activity, thus implying that active uptake contributes to net absorption. Wax block experiments suggest that water is absorbed over the entire chorionic surface rather than at a specific site. Eggs

83 84

of five other insect species that were examined [Lymantria dispar (L.), Bombyx mori (L.), Antheraea polyphemus (Cram.), Oncopeltus fasciatus (Dallas) and Diaferomera femorata (Say)] lacked the ability to absorb atmospheric water.

Introduction

Few experiments have examined water balance strategies of arthropod eggs, and those species that have been investigated belong to only two orders, Hemiptera and Orthoptera (Edney, 1977). Maintenance of water balance is especially important for eggs because they are incapable of drinking free water, they are frequently deposited in an environment with a low water content (Edney, 1977), and their large surface area to volume ratio makes them especially vulnerable to water loss (Schmidt-Nielsen, 1984). Under these circumstances, eggs must regulate water loss and/or gain to remain viable. This challenge is especially formidable among species that may spend many months in an egg diapause. Eggs of several species, when in contact with free water, are capable of absorbing water, and this absorption may initiate development and synchronize hatching with the new growth of host plants (Edney, 1977). In this study we examine water balance characteristics of several species of insect eggs and focus more detailed experiments on the diapausing eggs of a tropical walking stick, Extatosoma tiaratum. Egg diapause for this species, like other 85

tropical phasmids, is obligatory and occurs in two stages, first as an early embryo and then as a pharate first instar larva (Bedford, 1970) In this paper we report that eggs ofE. tiaratum have the capacity to absorb atmospheric water at very low vapor activities. This is the first demonstration that an insect egg is capable of water vapor uptake from the atmosphere. In previous reports of water uptake by arthropod eggs, the eggs were in contact with free water (Edney, 1977; Hinton, 1981). For E. tiaratum, we show that the capacity to absorb water from subsaturated atmospheres can be retained for many days. We determine the rates of water vapor uptake and seek to identify the site of absorption.

Materials and Methods Insects and Experimental Conditions Eggs examined in this study (Table 2) represent three insect orders, Lepidoptera, Hemiptera, and Phasmida. All are temperate zone species except the phasmid,E. tiaratum, which originated from Queensland, Australia. All species are in culture at the Ohio State University insectary, except L. dispar which was received from the USDA Forest Service Laboratory in Hamden, Connecticut. Diapausing eggs from temperate species were held at 4°C for > 80 days before experimentation and the diapausing walking stick eggs were held at 25 + 1°C. Insects that do not have an egg diapause (A. polyphemus and O. fasciatus) were held at 25°C and utilized for 86

experiments 2-4 days after oviposition. All eggs were synchronized physiologically by predesiccation at av (R.H./100) 0.00 (anhydrous

CaSC>4 ), 20 ± 0.5°C for 24 h, unless otherwise noted, prior to experimentation so that weight changes reflect water flux (Wharton, 1985). Each egg was monitored individually, and data sets from eggs that failed to eclose were not included. Water vapor activities were maintained in sealed desiccators using glycerol- distilled water solutions (Johnson, 1940). Body Water Pool and Net Transpiration Rate Individual eggs were weighed on an electrobalance (CAHN 25, Ventron Co.) to determine initial wet weight and subsequent weights during the experiments. Dry weight was determined after

drying the eggs over anhydrous CaSC>4 at 50°C until constant mass. Total percent body water was determined by dividing the water mass (difference between wet and dry weight) by the initial wet weight X 100 (Wharton, 1985). The percent change in weight in egg water content was calculated as in equation 1, where mt = the mass at any time t and mo = the initial mass:

(1) (mt - m0) mo'1 (100) = % change in water

Net transpiration rates for the eggs were calculated by determining their wet weight on day 0, placing the eggs in dry air (av 0.00) and reweighing them at 24 h intervals. The ratio of the water mass at time t (mt) to initial mass (mo) describes the amount of water lost over time relative to initial water mass. The kinetics of water loss at av 0.00 is a function of exponential decay in which water content at any time t (mt) is equal to the water mass (m) at time 0 (mo) X e'kt, where k is percent water lost over time t between mo and mt (Wharton, 1985) (equation 2):

(2) mt = mo e"kt

The negative slope of an exponential regression through a semi-log plot of In mt/mo against time is a first order rate function and is expressed in %-h'1 (Wharton & Devine, 1968; Devine & Wharton, 1973). Water Vapor Absorption and Uptake Kinetics The change in water weight (m) results from the difference between net transpiration from insect to air, mx, and net water gain (active and passive uptake) from air to insect, ms- (Wharton, 1985) (equation 3):

(3) m = ms - rriT

In dry air ms is zero, thus mx=m, and transpiration proceeds so that a constant percentage of the water mass (m) is lost in unit time and rate of loss is equal to kt (eqn. 2). Water balance is achieved when mx= ms, thus in addition to net transpiration (mx) its competing 88 flow (ms), rate of uptake, must also be determined (Wharton, 1985). To test for water vapor absorption, eggs of all six species were cycled at least once between av's 1.00 and 0.20 for 12h at each humidity before transferring them to test humidities. By synchronizing the body water av (>0.99) with ambient air (av 1.00) prior to experimental treatment it is possible to determine whether water uptake is wholly passive (net loss to av's

R esu lts Weight Changes at Saturation for Eggs of Six Species The six species examined in this study ranged widely in size (range 0.26 to 44.05 mg) and in the percent change in weight at saturation (av 1.00) (range -3.92 to +20.71%, 20°C) as shown in Table 2. The diapausing eggs of the tropical walking stick, E. tiaratum, were the only eggs that gained weight from saturated air. E. tiaratum enters diapause as an early embryo and then again as a pharate first instar larva. In both stages of diapause, eggs of E. tiaratum gained weight at saturation. Eggs of other species, regardless of their developmental status (diapause or nondiapause), lost weight at 20°C, suggesting that in most species no water can be absorbed as vapor at vapor activities greater than the activity of the body water (aw). 90

For eggs diapausing as early embryos, the percent increase in weight of E. tiaratum at saturation was not significantly different at 20°C (20.71 + .19%) and 25°C (21.09 + .23%), but at 25°C pharate first instar larvae gained more (25.30 + .23%) than eggs in early embryonic diapause (F=3.217, df=89, P<0.05). Eggs in early embryonic diapause had a significantly greater wet weight (F=5.409, df=89, P<0.01) and lower percent body water content (52 + 3 % versus 67 ± 4 %, F=5.431, df=89, P<0.01) than diapausing pharate first instar larvae. The dry weights of early embryos and pharate larvae of E. tiaratum are positive correlates of their respective body water masses (r2>0.91) and the regression describing this relationship is significantly different from zero (F>602.09, df=399, P<0.0001). Because eggs of E. tiaratum were the only ones capable of absorbing water, all further experiments focused on this species. Net Transpiration Rate In dry air, depletion of the egg's water mass proceeds as a first order kinetic relationship of exponential decay (eqn. 2). Eggs of E. tiaratum in early embryonic diapause held at av 0.00 lost water at a significantly slower rate at 20°C (0.006 %-h'1) than at 25°C (0.015 %-h'1) (F=5.497, df=89, P<0.01, Fig. 7A, 7B). Diapausing pharate first instar larvae at 25°C lost water at a significantly faster rate (0.046 %‘h '1) (Fig. 7C) than early embryos at the same temperature (Fig. 7B) (F=5.395, df=89, PcO.Ol). 91

Water Retention Capacity at Hydrating Vapor Activities Percent change in weight was documented for five days at 20°C for diapausing early embryonic eggs of E. tiaratum that were exposed to av 1.00 for 12h and then held at experimental humidities (Fig. 8). At av 0.33, the eggs gained 0.18 ± .07 % water by the second day, and that rate was maintained for the remaining five days of the experiment. Rehydrated eggs gained weight from subsaturated air, thus implying an active process, but the percent increase in weight was progressively higher at av 0.50 and av 0.60 (ANOVA, P<0.05). A subgroup of eggs that were killed by freezing at -70 0 C (N=45) and held at a hydrating atmosphere (av 0.60) lost water (-3.24 ± .20 %) over the five day period, thus implying that the physical attributes of the egg are not responsible for the absorption of water vapor. These results suggest an active process. Site of Uptake Eggs in early embryonic diapause were blocked with wax in select regions (entire egg, anterior half, posterior half, micropylar plate,operculum-capitulum, entire egg excluding micropylar plate, and entire egg excluding operculum-capitulum, each N=45), and then held at av 0.60 (hydrating av) at 20°C for 24 h after which percent change in weight was calculated (eqn. 1). Blocking of select regions did not affect water uptake; percent gain for each group was within the range (± .06 %) of control eggs (gain of 0.39 ± .04 %). Water uptake was only inhibited when the entire egg was covered with wax (net gain 0.007 + .003 %) (F=5.441, df=89, P<0.01). This 92 suggests that water vapor is absorbed over the entire egg surface and is not restricted to a specific region, but the actual points of entry have not been identified. Uptake Kinetics Water vapor uptake rates were derived from the slope of a regression of water vapor exchange on av (Machin, 1984) following a 24 h exposure to the experimental av. At 20°C, eggs ofE. tiaratum in early embryonic diapause absorbed water at a rate of 0.499 mg'h'^Aav'^Fig. 9A). This rate did not differ significantly from the rate of uptake at 25°C (0.516 mg-h'^Aay"1) (Fig. 9B). The fact that the rate of uptake for the early embryos were not affected by temperature indicates that water loss, although greater at 25°C (Fig. 9B), does not affect vapor absorption kinetics. The rate of uptake at 25 °C for diapausing pharate first instar larvae is significantly greater (0.725 m g'h'^A ay'1) (Fig. 9C) than the rate for the early embryos at the same temperature (Fig. 9B) (F=5.283, df=89, P<0.01). A perpendicular line drawn to the x axis from the intercept of the regression line describing the rate of vapor uptake and zero water vapor exchange defines the av at which the rate of water loss equals gain under these conditions. Early embryonic eggs are capable of absorbing water vapor from lower vapor activities (ca. av 0.30, Fig. 9B) than pharate first instar larvae (ca. av 0.60, Fig. 9C) at 25°C. Increasing temperature decreases the absorptive affinity of early embyronic eggs (av0.23 ± .04 at 20°C compared to av 0.30 ± .03 at 25°C). The values (mg*h_1) used to determine rate of uptake were 93 from data collected for eggs predesiccated at av 0.00. This pretreatment differs from that used for the percent change in weight data plotted in Fig. 8 (cyclic pretreatment, av's 1.00 and 0.20), thus the values in the two figures cannot be interconverted. Table 2. A comparison of wet weight (mg) and weight change (%, cqn.l) at saturation (av 1.00) for six species of eggs exhibiting different developmental patterns (ND=nondiapause, D=diapause).

Developmental Wet Weight Weight Change Species Status (mean + S.D. mg) (a v 1.00, 24 h) temperature 5L Lepidoptera

Bombyx mori (L.) D 0.50 + 0.64 20°C -3.26

Lymantria dispar (L.) D 0.63 + 0.10 20 -1.92

Aniheraea polyphemus (C ra m .) ND 3.74 + 0.73 20 -3 .9 2

Hemiptera

Oncopeltus fasciatus (D alla s) ND 0.26 + 0.04 20 -1.76

Phasmida

Diaferomera fem orata (S a y ) D 3.34 + 4.46 20 -1.03

Extatosoma tiaratum (Mac Ieuy) D (early embryo) 42.26 + 5.42 20 + 20.71

" ” D (early embryo) 44.05 + 4.87 25 + 21.09

” “ D (pharate larva) 28.74 + 4.33 25 + 25.30

4^ 95

Fig. 7. Net transpirational water loss from eggs of E. tiaratum (predesiccated av 0.00, 20°C, 24 h) as early embryos at 20°C (A) and at 25°C (B) and as pharate first instar larvae at 25°C (C). The negative slope (mg^d'1) on a semi-log plot of the water mass ratio (mt/mo; mass at time t, mt to original mass, mo) against time describes transpiration rate (-kt) as a function of exponential decay (eqn. 2). Common regressions (r2>0.98) fit to the means of 45 eggs for each group. 96

o.oo- a □ □ a A - 0.01 -

- 0. 02 - B

-0 .0 3 -

-0 .0 4 -

-0 .0 5 -

-0 .0 6 H------1------1------1------1------1 0 1 2 3 4 5

Time (days)

Fig. 7 97

Fig. 8. Percent change in weight in response to long term exposure to three hydrating atmospheres at 20°C for early embryonic E. tiaratum eggs (cycled for 12 h each at av 1.00 and 0.20, 20°C prior to transfer to experimental conditions). Weight change was also monitored in a group of eggs killed at -70°C and held at av 0.60. Each point is the mean of 45 eggs. Vertical error bars lie within the confines of the symbol used on the graph (S.D. <0.23). Change in Weight (%) -4.0 - -3.0- - 2 0.0 0.3- 0 0.4- 1 . . . 0 0 2 - - - ie (days) Time i. 8 Fig. 98 99

Fig. 9. Rates of water vapor exchange for eggs of E. tiaratum (predesiccated av 0.00, 20°C, 24 h) as early embryos at 20°C (A) and at 25°C (B) and as pharate first instar larvae at 25°C (C). The slope of the regression line at hydrating vapor activities indicates the mean rate of uptake (mg'h'^Aav’1). Each point is the mean of 45 eggs; vertical error bars signify 95% confidence limits. 0.4 •

0 .3 - y=-0.109+0.499X r*=0.9^j

0.2 •

0.0

- 0.1

0.4

y =- 0.154 + 0.5 16x r2=0.96 O)

a . £> 0.0

01 - 0.1 £

0.3 i

y =- 0.442+0.725x r =1.00 0.2 -

0.1

0.0

- 0.1 0 0.2 0.4 0.6 0.8 1.0 av

Fig. 9 101

D isc u ssio n Absorption of liquid water by passive diffusion has been well documented for a few arthropod eggs (Lees, 1955; Edney, 1977; Hinton, 1981), and for some insects it is essential for development (Matthee, 1951; Edney, 1977). But, eggs of relatively few species actually require contact with moisture, and most can develop in unsaturated air (Edney, 1977). All previous work has indicated that arthropod eggs are incapable of absorbing water as vapor from atmospheres as high as av 0.98 (Shulov, 1952; Edney, 1977; Hinton, 1981). The fact that diapausing eggs of E. tiaratum gain weight at saturation is extraordinary because most other insect eggs not in contact with liquid water lose water under these conditions (Hinton, 1981), and this was again verified by the other eggs examined in this study. Eggs of some Saturniid moths do gain a slight amount of water in saturated air (Ludwig, 1942, 1943; Ludwig & Anderson, 1942), and Hinton (1981) attributes this to either the hygroscopic nature of the chorion or the embryo itself. This study on diapausing walking stick eggs is the first demonstration of an arthropod egg being able to absorb water vapor from subsaturated air. Both stages of egg diapause in E. tiaratum, the early embryo and the pharate first instar larva, have the capacity to absorb water vapor from subsaturated air, but the capacity of the early embryo is especially impressive. For these early embryos, the vapor activity at which water vapor can be utilized for rehydration is 102 incredibly low (ca. av 0.23 at 20°C). For the second diapausing stage of E. tiaratum, the pharate first instar larvae, the vapor activity at which absorption occurs is considerably higher (ca. av 0.60 at 25°C) and approximates the critical equilibrium activity (CEA) for larvae of the oriental rat flea Xenopsylla cheopis (av 0.65) (Knulle, 1967) and prepupae ofX. brasiliensis (av 0.50) (Edney, 1977). Increasing the rate of water loss from early embryonic eggs by increasing temperature elevates the equilibrium weight. This indicates that the rate of water loss exceeds net gain until higher vapor activities are reached, and no gain can occur below av0.30 due to the high rate of water loss. The increase in temperature may enhance evaporation of adsorbed surface water such that the egg does not re-equilibrate until a higher av. The hygroscopic nature of the chorion may be responsible for some water gain by passive chemisorption and physical adsorption of water vapor. Several features suggest this possibility, the plateau reached by day 2 increases with increase in av, and a new equilibrium water content is reached after 1-2 days. It is also possible that 12 h of rehydration at av 1.00 may not have been sufficient to replenish the total water pool after 12 h of dehydration, thus accounting for passive water gain by av 1.00 synchronized eggs. But, the vapor activity at which the egg of E. tiaratum in both stages of diapause absorbs water lies hundreds of atmospheres below saturation and presents a dramatic case of water movement against a large atmospheric gradient. If water 103 vapor uptake is an active process, uptake would cease at death (Edney, 1977). Killed eggs of E. tiaratum in early embryonic diapause lost water at a vapor activity that hydrated viable eggs. We observed no first-day gain in killed eggs that could account for passive absorption alone, and no significant (passive) absorption was observed in eggs after transfer from dry air to humidities below 'hydrating' humidities. Thus, the total water recruited from subsaturated atmospheres cannot be attributed solely to passive absorption, suggesting that active water vapor uptake contributes to net absorption. Hydrated early embryonic eggs of E. tiaratum absorb water at av 0.33, an incredibly dehydrating atmosphere equivalent to tens of osmoles of osmotic gradient. How does an egg achieve such a feat? Several arthropod species secrete hyperosmostic fluids that capture atmospheric water vapor from subsaturated air (O'Donnell & Machin, 1988). Whether a solute-driven mechanism of water vapor absorption is utilized by the walking stick eggs is unknown. One possible scenario is that the yolk of this species is highly hygroscopic (generated by solute or some other molecule), and the low av at which rehydration begins and low rates of net water flux could be the consequence of a large pool of inert, hygroscopic yolk. But, water vapor can be absorbed by not only the early embryo but also the pharate first instar larva, and at that stage the yolk has been depleted. Clearly, in that case, the diapausing pharate larva 104

must be responsible for the uptake, but how this is achieved remains puzzling. Previous work on the site of liquid water uptake in insects eggs has not revealed a specific universal structure, but the hydropyle has this capacity in some species (Lees, 1955; Hinton, 1981). The site of water absorption in many arthropods has been identified by blocking specific structures or regions with wax (Noble-Nesbitt, 1970). If the hydropyle in eggs of the orthopteran Melanoplus sp. is sealed, no water is absorbed (Slifer, 1938; Salt, 1952). In Schistocerca gregaria, most water uptake occurs at the posterior tip of the egg (Mathur, 1944), but when this region is covered with wax some water continues to be absorbed through uncovered areas (Moloo, 1971). Water is collected over the whole egg surface of Chorthippus brunneus, and the hydrofuge properties of the chorion conduct the water to a ring of micropyles at the posterior pole where water enters the egg (Hartley, 1961; Moriarty, 1969). Edney (1977) suggests that many egg chorions have air cavities (aeropyles) through which water might diffuse. Our wax block experiments with eggs of E. tiaratum suggest that water is absorbed across the entire surface of the chorion. Though uptake may normally occur over the whole egg surface, our experiments show that the egg can become hydrated by any contact with moist air, thus implying that uptake is not limited by the contact area between the chorion and air. The actual site of water entry 105

(perhaps via aeropyles or hydrofuge conduction to micropyles) remains unknown. In addition to the capacity to absorb water vapor, the walking stick eggs also effectively conserve water. We observed low rates of water loss in E. tiaratum during both stages of diapause. This is a characteristic feature of arthropod eggs (Edney, 1977; Hinton, 1981), and indeed the permeabilities of some egg membranes (Rhodnius prolixus, reported by Beament 1946 and Lucilia sericata, reported by Davies, 1948) are the lowest recorded for any animal membrane (Edney, 1977). The two stages of egg diapause seen in E. tiaratum appear to be a characteristic shared by other tropical phasmids, e.g., Didymuria violescens (Readshaw, 1965) and Clonopsis gallica (Voy, 1954a, b). The environmental factors regulating these two stages of diapause in E. tiaratum have not yet been identified, but under our laboratory rearing conditions (25°C, variable photoperiod) the diapause periods encompass about 6-7 months (unpublished results). Our preliminary experiments indicate that termination of the second diapause and eclosion of the first instar larva can be initiated by immersion in water, thus suggesting that rainfall serves as an environmental cue used by the phasmid to coordinate its development with fresh growth of its plant host. From this study it is apparent that the egg's survival in a dry environment during these long months of diapause is greatly facilitated by water conservation and its unique ability to absorb atmospheric water vapor. 107

References

Beament, J. W. L. (1946) The formation and structure of the chorion of the egg in an hemipteran, Rhodnius prolixus. Quarterly Journal of Microscopic Science, 87, 393-439.

Bedford, G. O. (1970) The development of the egg of Didymuria violescens and determination of the stage at which first diapause occurs. Australian Journal of Zoology, 18, 155-169.

Davies, L. (1948) Laboratory studies on the egg of the blowfly, Lucilia sericata (Mg.). Journal of Experimental Biology, 25, 7 1 -8 5 .

Devine, T. L. and Wharton, G. W. (1973) Kinetics of water exchange between a mite, Laefaps echidnina, and the surrounding air. Journal of Insect Physiology, 19, 243-254.

Edney, E. B. (1977) Water Balance in Land Arthropods. Springer- Verlag, New York.

Hartley, J. C. (1961) The shell of acridid eggs. Quarterly Journal of Microscopic Science, 102, 249-255.

Hinton, H. E. (1981) Biology of Insect Eggs. Pergamon Press, New York.

Knulle, W. (1967) Physiological properties and biological implication of the water vapour sorption mechanims in larvae of the oriental rat flea, Xenopsylla cheopis (Roths). Journal of Insect Physiology, 13, 330-357.

Lees, A. D. (1955) The Physiology of Diapause in Arthropods. Cambridge University Press, New York. 108

Ludwig, D. (1942) The effect of different relative humidities, during the pupal stage, on the reproductive capacity of the luna moth Tropaea luna L. Physiological Zoology, 15, 48-60.

Ludwig, D. and Anderson, J. M. (1942) Effects of different humidities, at various temperatures, on the early development of four saturniid moths (Platysamia cecropia Linnaeus, Telea polyphemus Cramer, Samia walkeri Felder and Felder, and Callosamia promethea (Drury) and on the weights and water contents of their larvae. Ecology, 23, 259-274.

Ludwig, D. (1943) The effect of different relative humidities, during the pupal stage, on the reproductive capacity of the Cynthia moth, Samia walkeri Felder and Felder. Physiological Zoology, 16, 381-388.

Machin, J. (1984) The study of atmospheric water absorption. In: Measurements of Ion Transport and Metabolic Rate in Insects,T. A. Miller, T. A. (eds.), pp. 69-99. Springer-Verlag, New York.

Machin, J. (1985) Evidence for an active water uptake mechanism in the rectal complex of the midge. Journal of Insect Physiology, 19, 243-254.

Mathur, C. B. (1944) The site of the absorption of water by the egg of Schistocerca gregaria. Indian Journal of Entomology, 5, 3 5 -4 0 .

Matthee, J. J. (1951) The structure and physiology of the egg of Locustana pardalina (Walk). Union of Southern Africa Department of Agriculture Bulletin, 316, 3-83.

Moloo, S. K. (1971) Some aspects of water absorption by the developing egg of Schistocerca gregaria. Journal of Insect Physiology, 17, 1489-1495. 109

Moriarty, F. (1969) Water uptake and embryonic development in eggs of Chorthippus brunneus Thunberg (Saltatoria:Acrididae). Journal of Experimental Biology, 50, 327-333.

Noble-Nesbitt, J. (1970) Water uptake from subsaturated atmospheres: Its site in insects. N atu re, 225, 753-754.

O'Donnell, M. J. and Machin, J. 1988. Water vapor absorption by terrestrial organisms. Advances in Comparative Environmental Physiology, 2, 47-90.

Readshaw, J. L. (1965) A theory of phasmatid outbreak release. Australian Journal of Zoology, 13, 475-490.

Salt, R. W. (1952) Some aspects of moisture absorption and loss in eggs of Melanoplus bivittatus (Say). Canadian Journal of Zoology, 30, 55-82.

Schmidt-Nielsen, K. (1984) Scaling: Why is Animal Size So Important? Cambridge University Press, New York.

Shulov, A. (1952) The development of eggs of Schistocerca gregaria (Forskal) in relation to water. Bulletin of Entomological R esearch, 43, 469-476.

Slifer, E. H. (1938) The formation and structure of a special water absorbing area in the membranes covering the grasshopper egg. Quarterly Journal of Microscopic Science, 80, 437-457.

Sokal R. R. and Rohlf, F. J. (1981) Biometry. W. H. Freeman and Co., New York.

Voy, A. (1954a) Sur I'existence de deux categories d'oeufs dans la ponte globale du phasme (Clonopsis gallica Charp.). Comptes Rendus de I’Academie des Sciences a Paris,238, 625-627.

Voy, A. (1954b) Biologie et croissance chez le phasme femelle (Clonopsis gallica Charp.). Bulletin Biologique de la France et de la Belgique, 88, 101-129. 110

Wharton, G. W. (1985) Water balance of insects. In: Comprehensive Insect Physiology, Biochemistry, and Pharmacology, Kerkut, G. A. and Gilbert, L. I. (eds.), pp. 565-601. Pergamon, Oxford.

Wharton, G. W. and Devine, T. L. (1968) Exchange of water between a mite, Laelaps echidnina, and the surrounding air under equilibrium conditions. Journal of Insect Physiology, 14, 1 3 0 3 -1 3 1 8 .

Wharton, G. W. and Devine, T. L. (1969) Water vapor exchange kinetics of terrestrial arthropods. Journal of Insect Physiology, 19, 243-254. CHAPTER V

PUPARIA OF DIAPAUSING FLESH FLIES ARE ENHANCED WITH ADDITIONAL HYDROCARBONS

A b stra ct

Puparia of diapausing flesh flies, Sarcophaga crassipalpis, are lined with twice as much hydrocarbon as their nondiapausing counterparts. Most of the hydrocarbons, alkanes in the range of

C 2 6-C 3 0 . are present in both types of puparia, but a straight-chain

C 29 alkane is abundant only on the puparia of diapausing pupae. The quantity of epicuticular hydrocarbon increases throughout the life of the fly, but it is only in the pupal stage that a distinction can be made between diapausing and nondiapausing cohorts. Though some of the additional hydrocarbon is deposited on the exterior surface of the puparium, the bulk of the additional hydrocarbon is on the interior surface of the puparium. The few flies that fail to diapause when reared under short day conditions also produce puparia enhanced with an abundance of hydrocarbon, thus implying that the increase in production of hydrocarbons is not invariably linked to the expression of diapause. Elevation of temperature can increase the quantity of hydrocarbon produced in

111 112 puparia from nondiapausing flies, but this effect is modest in comparison to the effect of short-day (diapause) programming. The hydrophobic barrier created by the additional hydrocarbons reduces water loss, enhances atmospheric water absorptivity and may also prevent drowning in a water-saturated environment. In addition we demonstrate that microsomal preparations from the flesh fly convert fatty acid to alkane with CoA, ATP and NADH as required cofactors. Conversion of an aldehyde to an alkane and CO by the microsomes does not require any cofactors. Thus, we provide the first evidence that alkane synthesis in insects involves decarbonylation.

INTRODUCTION

The challenge of maintaining water balance is particularly acute during diapause. If it is a diapause occurring in the egg or pupal stage, the insect for months has no opportunity to replenish its water supply by feeding or imbibing free water. In the flesh fly, Sarcophaga crassipalpis, we have observed several adaptations of diapausing pupae that favor conservation of water: the net transpiration rate is reduced, water vapor can be absorbed at low humidities, and the critical transition temperature is high (Yoder and Denlinger, 1991). But, what is the basis for this difference? One possibility is a difference in epicuticular hydrocarbons, the class of lipids well known to protect insects from desiccation, 113

abrasion and microbial penetration (Blomquist and Dillwith,1985; Lockey, 1985; Wharton, 1985). Some insects that diapause as pupae secrete additional cuticular hydrocarbon [e.g. Manduca sexta (Bell et al., 1975) and Mamestra configurata (Hegdekar, 1979)], which presumably reduces water loss. In this study, we compare the epicuticular lipid chemistry of nondiapausing and diapausing groups of S. crassipalpis throughout ontogeny and show significant quantitative and qualitative differences in hydrocarbons associated with the puparium, the tanned third instar larval cuticle which encases the pupa. We demonstrate that empty puparia are hygroscopic and define the relative humidities at which the two types of puparia equilibrate (equilibrium weight). The major site of hydrocarbon deposition within the puparium is identified, and we evaluate the effects of temperature and humidity on the quantities of hydrocarbon synthesized. We also provide the first evidence for insects that the mechanism used to synthesize long chain n-alkanes is decarbonylation.

MATERIALS AND METHODS Fly Rearing A colony of the flesh fly, Sarcophaga crassipalpis Macquart, was maintained in the laboratory as described (Denlinger, 1972). Parental adults were reared at 25°C under a diapause-inducing photoperiod (LD, 12:12 h) or a nondiapausing photoperiod (LD,15:9 114 h). Larvae and pupae were kept at 20°C under the maternal photoperiod. Short day conditions at 20°C produced a high incidence (>95%) of pupal diapause, while no diapause was observed under long day conditions. Extraction and Quantification of Hydrocarbon and Other Lipids Puparial hydrocarbons were quantified as described (Jackson et a/., 1974). Briefly, groups of 100 empty puparia were digested in 20% KOH for 24 h at 70°C and extracted twice with chloroform:methanol (2:1 v/v) for 5 min. The extract was then passed through a silica gel column (Millipore, Waters Associates); hydrocarbons were eluted with hexane and other lipids with chloroform. The eluant was N 2 -dried onto predesiccated (0% R.H., 25°C, 24 h), preweighed aluminum pans and stored at 0% R.H. under

N 2 and then reweighed after 24 h. Weighing was done on an electrobalance (Cahn G-2, Ventron Co.). Third instar larvae, unopened puparia and adult females were extracted twice with chloroform:methanol (2:1 v/v) for 5 min, and extracts were treated as above. Wandering third instar larvae were extracted after they completed the purging of their gut, puparia were extracted three days after pupariation (phanerocephalic pupa within the puparium), and adult female flies were extracted 4 h after ecdysis. 115

Analysis of Puparial Hydrocarbons and Other Lipids Empty puparia from nondiapausing and diapausing pupae were extracted twice with chloroform:methanol (2:lv/v) for 5 min,

extracts were dried with N2 to a 50 pi volume and spotted onto N2 -

dried, predeveloped (chloroform:methanol, 2 : 1 v/v) thin layer chromatography (TLC) silica gel plates (DC-Fertigplatten, silica gel

60 F 2 5 4 , 0.25 mm, EM Reagents). Hydrocarbons were separated from other lipids by developing the plate in HPLC grade hexane. The solvent front, containing hydrocarbons, was scraped from the plate and eluted with anhydrous diethyl ether. The eluant was

dried with N2 and stored at 4°C. Each dried extract was reconstituted with 20 pi hexane and injected into a GC-MS fit with a

SP-5 nonpolar column. Carrier gas (N 2) pressure was maintained at 18 psi over a temperature range of 150 - 300°C (10°C‘min’1). Correlation of log elution time with carbon number (y=13.18+1.29x, R=1.00) was obtained by injection of known hydrocarbon standards (Sigma Co., St. Louis, Mo.), and hydrocarbons were verified by their mass spectra. Neutral lipids were determined by comigration of lipid standards (Sigma Co., St, Louis, Mo.) on silica gel TLC plates developed in hexane:ether:acetic acid (70:30:lv/v/v, Davis, 1974) and detected by charring with H 2 SC>4 /ethanol (3:lv/v). Phospholipids, separated in chloroform:methanol:water:acetic acid (65:25:4:lv/v/v, Davis, 1974), were detected by charring with molybdenum blue. 116

Calculation of Equilbrium Weight The hygroscopic nature of empty puparia from nondiapausing and' diapausing pupae was examined by predesiccating the puparia (0% R.H, 20°C for 24 h) so that weight changes represent water flux (Wharton, 1985) and placing them in a series of humidity regimes. After a 24 h exposure to the experimental humidity at 20°C, the percent change in weight (on the electrobalance) was calculated

(equation 1 ),

(1) Wt-Wo-Wo' 1 (100) where Wt is the weight at any time t and Wo the original wet weight. A positive percent change in weight indicates net water gain whereas a negative percent indicates net water loss. The point of intersection of a regression line through a plot of percent change in weight (y) against relative humidity (x) and 0% change in weight defines the equilibrium weight achieved after 24 h on the x axis (R.H.) and identifies the humidity at which the puparium re­ equilibrates under these conditions [adapted from critical equilibrium activity (CEA) determination by Wharton, 1963]. Net Transpiration Rates Total water loss rates (integumental and respiratory water loss) were determined for untreated diapausing pupae and diapausing pupae that had been stripped of epicuticular lipid as third instar wandering larvae. Any difference in net transpiration 117

rate should indicate an alteration of respiration or cuticular permeability. Diapause-destined third instar larvae (<24 h before pupariation) were immersed in hexane for 2 min to extract epicuticular lipids and then held with untreated controls at 93% R.H., LD 12:12 h, 20°C until pupation (2 days after pupariation). Newly pupated flies were transferred to dry air (0% R.H.), LD 12:12 h, 20° C, weighed on an electrobalance after 24 h and at daily intervals for 1 week. Dry weight was determined by drying the fly over anhydrous CaS 0 4 , 50°C until constant weight. Loss of the pupa's water mass, m, (difference between wet and dry weight) proceeds as a first order kinetic function of exponential decay (equation 1) (Wharton, 1985).

( 1 ) mt=m 0 e"kt

The slope of an exponential regression through a semi-log plot of In mt/mo (water mass at any time t, mt, to original water mass, mo) against time is the rate of water loss and is expressed as %-h' 1 (Wharton and Devine, 1968; Devine and Wharton, 1973). Enzyme Preparation 100 Nondiapausing pupae were removed from their puparia and homogenized in 2 vol (w/v) 0.1 M potassium phosphate, pH 7.0, containing 0.3 M sucrose for 2 min using a microhomogenizer. The homogenate was centrifuged 1 min at 2,940 x g in an Eppendorf microfuge. The supernatant was centrifuged 1.5 h at 105,000 x g. 118

The supernatant from this 105,000 x g centrifugation was assayed directly, and the microsomal pellet from this centrifugation was resuspended in an equal volume of reaction buffer [0.1 M potassium phosphate, pH 7.0, 5 pM P-mercaptoethanol and 0.1% Triton X-100 (v/v)] and assayed after a 30 min incubation. Protein was determined by the method of Bradford (1976). Aldehyde and Fatty Acid Substrates [1-14C], [9,10-3H]octadecanal and [1-14C], [9,10- 3H]octadecanoic acid were synthesized and purified by thin layer chromatography on Whatman silica gel 60A as described (Cheesbrough and Kolattukudy, 1988). Enzyme Assays Alkane synthesis was assayed as described (Cheesbrough and Kolattukudy, 1988; Dennis and Kolattukudy, 1991). In brief, assays were run anaerobically in 16 x 100 mm test tubes sealed with serum stoppers containing two polypropylene cups. One cup contained RhCl[(C6H 6)3 P]3 to trap the enzymatically released CO.

After flushing the reaction tube 2 min with N 2 , a freshly prepared solution of 12.5% pyrogallol in 20% KOH was injected into the second cup to remove any remaining O2 . Each reaction mixture contained 200 pM [1-14C], [9,10-3H]octadecanal or when fatty acid was used as a substrate 200 pM [1-14C], [9,10-3H]octadecanoic acid and the following cofactors: 6.0 mM ATP, 0.2 mM CoA and 0.2 mM NADH. The reaction was started by the addition of enzyme and stopped after 45 min at 25°C with the addition of 200 pi 2N HC1. 119

After photolysis the CO trap was removed and assayed for 14C (Cheesbrough and Kolattukudy, 1984). Lipids extracted from the mixture were separated by TLC and the isolated alkane fraction was assayed for radioactivity. Chain length of the alkane product was determined by radio-gas chromatography.

RESULTS Equilibrium Weights of Empty Puparia We previously demonstrated that diapausing pupae are capable of absorbing water vapor from atmospheric air at lower humidities than nondiapausing pupae (Yoder and Denlinger, 1991), and in this experiment we test whether this difference can be at least partially attributed to the puparium. Empty puparia of nondiapausing flies attained equilibrium weight (after 24 h at the experimental humidity) at a significantly higher relative humidity (18% R.H.) than empty puparia from flies that completed a diapause of >40 days (7% R.H.) (F=3.457, df=89, P<0.05). The lower equilibrium weight of diapausing puparia thus indicates that they are better suited to passively absorb atmospheric water than their nondiapausing counterparts. Percent weight change was significantly greater in empty nondiapausing puparia than in empty diapausing puparia at 0% R.H. (-5.5 ± 0.2% for nondiapausing puparia versus -1.1 + 0.3% for diapausing puparia, each N=45, Student's t test, P<0.05) and at a hydrating humidity (33% R.H.) 120

(+5.1 ± 0.3% for nondiapausing puparia versus +4.0 + 0.3% for diapausing puparia, each N=45, Student's t test, P<0.05). These data indicate that the rate reduction in both net transpiration (water loss, % change in weight data at 0% R.H.) and water vapor absorption (water gain, % change in weight data at 33% R.H.) we observed in diapausing pupae of S. crassipalpis (Yoder and Denlinger, 1991) can be, at least partially, attributed to the properties of the puparium. Developmental Changes in Quantity of Cuticular Hydrocarbons and Other Lipids The quantity of epicuticular hydrocarbon changes throughout the lifecycle of S. crassipalpis, and the most striking increase (ca. 10-17 fold) was observed during the pupal-adult transformation (Table 3). Slight, but significant, quantitative differences in epicuticular hydrocarbon were detected between intact puparia from nondiapausing and diapausing pupae (each N=100, Student's I test, P<0.05): 2.29 pg versus 3.89 pg respectively. But, no differences were observed between diapause- and nondiapause- destined larvae nor between adults that emerged from diapausing and nondiapausing pupae. The quantitative difference between nondiapause and diapause puparia was much more pronounced when empty puparia were compared. The use of empty puparia permitted extraction from both the exterior and interior surface of the puparium, as well as from the pupal exuvium that remains inside the puparium after 121 adult eclosion. Much more hydrocarbon was extracted, and the amount of extractable hydrocarbon was nearly twice as great in puparia from diapausing pupae as in puparia from nondiapausing individuals (15.61 versus 7.92 pg/individual, Table 3). A subset of empty puparia were also digested in 20% KOH, 70°C for 24 h (Jackson et al., 1974), extracted with chloroform:methanol (2:lv/v) and hydrocarbons quantified. With this alternative extraction technique, puparia from the diapausing group again were shown to contain nearly twice as much hydrocarbon as puparia of nondiapausing flies (15.51 ± 0.18 versus 7.94 + 0.22 pg/empty puparium, each N=100, Student's t test P<0.05). Results obtained from these two techniques (base digest and surface extraction) did not vary significantly, thus indicating that extractions with chloroform:methanol (2 :1 v/v) without base digestion are reliable in removing total surface wax. In contrast to hydrocarbons, the total quantity of other lipids remained approximately the same (ca. 1 0 pg/individual) in all developmental stages (Table 3). Analysis of empty puparia from diapausing and nondiapausing pupae showed no evidence for qualitative differences among the neutral lipids (cholesterol, free fatty acids, wax esters), and no phospholipids were detected (data not shown). 122

Localization of Hydrocarbons within the Puparium The above experiments showing that more hydrocarbons were extracted from empty puparia than from the surface of intact puparia suggested that the hydrocarbons were most abundant on the interior surface of the puparium or in the pupal exuvium. To separate these two possible sites, pupal exuvia were removed and extracted separately from empty puparia. Each was digested in base and hydrocarbons quantified. The amounts of hydrocarbon extracted from nondiapausing and diapausing pupal exuvia were nearly the same (4.6 ± 0.33 pg versus 4.8 ± 0.29 pg/exuvium, mean + S.D., each N=100). Empty puparia, excluding the pupal exuvium, from 100 nondiapausing pupae contained 2.56 ± 0.21 pg hydrocarbon/puparium. But, puparia from diapausing pupae reared at the same temperature, 20°C, contained 11.42 ± 0.31 pg hydrocarbon/puparium, a 4.5-fold increase over the amount extracted from the nondiapausing puparia (N=100, Student's t test, P<0.05). Thus, the abundance of hydrocarbons associated with diapause are primarily deposited on the interior surface of the puparium . If the hydrocarbons that are important in maintaining water balance are located on the interior surface of the puparium we would anticipate little effect from removal of hydrocarbons from the exterior of the puparium. Net transpiration rates for diapausing pupae are much lower than in nondiapausing pupae (Yoder and Denlinger, 1991), yet removal of the epicuticular lipids from larvae 123

shortly before pupariation did not affect the net transpiration rate of diapausing pupae (0.009 %-h'1 for hexane-washed individuals

versus 0.010 %*h -1 for untreated controls). This again suggests that the properties of the exterior surface of the puparium make little contribution to the difference observed in diapausing and nondiapausing pupae. Linkage with Diapause The above experiments demonstrate a consistent elevation of hydrocarbons in puparia of diapausing pupae. Pupal diapause inS. crassipalpis is programmed by short daylength during late embryonic development and early larval life, in concert with low temperature (Denlinger, 1971). To test whether synthesis of extra hydrocarbons is consistently linked to the expression of diapause we also examined puparia from the few flies reared under short day conditions at 20°C that failed to enter diapause (approximately 5% of the laboratory population). Empty puparia from those flies yielded the same quantity of extractable hydrocarbon (15.27 ± 0.31 pg/empty puparium) as those that actually entered diapause (15.32 + 0.24 pg/empty puparium, each N=100, Student's t test P<0.05). Thus, synthesis of extra hydrocarbons is not invariably linked to the expression of diapause but is dependent upon the environmental signals that program diapause. Normally, diapause and accumulation of extra hydrocarbons would coincide, but this experiment suggests that these two developmental characteristics can be separated. 124

Effects of Temperature and Humidity Larvae destined for continuous development were reared at 20°, 25°, or 30°C and maintained there until adult eclosion. Empty puparia from flies reared at 25°C contained about 25% more hydrocarbon (9.63 ± 0.23 pg/empty puparium) than puparia held at 20°C (7.87 ± 0.21 pg/empty puparium) (each N=100, Student'st test, P<0.05), but quantities extracted at 25°C and 30°C (9.79 ± 0.21 pg/empty puparium) were not significantly different. The amount of hydrocarbon extracted from nondiapausing puparia over this temperature range never approached the quantity of hydrocarbon extracted from diapausing puparia at 20°C (15.61 + 0.16 pg/empty puparium, each N=100, Student's t test, P<0.05). A parallel experiment could not be done with diapausing pupae because temperatures higher than 20°C avert diapause. Third instar larvae were also reared at 20°C under 0% R.H.

(anhydrous CaSC>4 ) and 93% R.H. (saturated KNO3 solution, Winston and Bates, 1960) to determine the effects of humidity on hydrocarbon deposition within the puparium. The quantities of hydrocarbon obtained by base digestion of empty puparia from both nondiapause and diapause groups in dry air (7.71 ± 0.25 pg and 15.43 ± 0.20 pg/puparium, respectively) and near saturation (7.84 ± 0.19 pg and 15.29 ± 0.21 pg/puparium, respectively) were not significantly different. Thus, humidity does not appear to affect the amount of hydrocarbon synthesized. 125

Qualitative Analysis of Puparial Hydrocarbons Analysis of the hydrocarbons by capillary gas liquid chromatography and mass spectrometry show that hydrocarbons of empty puparia from nondiapausing flies also differ qualitatively from puparia of flies that entered diapause (Fig. 10). A straight- chain C29 hydrocarbon, comprising about 2 0 % of the total hydrocarbon content of puparia in diapausing pupae, was only present in puparia of nondiapausing flies in trace amounts. Identification of this hydrocarbon was made by comparing elution time (Fig. 10B, 12.08 min) to that of a known C 29 standard (Sigma, Co., St. Louis, Mo.) (12.14 min) and their mass spectra (Fig. 11). Other differences between the puparia of nondiapausing and diapausing flies (Fig. 10, peaks a-f) were strictly quantitative. Consistently, each hydrocarbon was more abundant in puparia from diapausing flies. Comparison of elution times and mass spectra of components a, b and c (Fig. 1 ) indicate that b is straight-chain C 27 whereas a and c are methyl-branched C 27 and C2 8, respectively. Peaks d, e and f (Fig. 10) did not yield readily interpretable mass spectra, but similar elution times of known hydrocarbon standards suggest they are C 30 molecules. Biosynthesis of Hydrocarbon by Decarbonylation Changes in the levels of hydrocarbons observed in this study indicate that the flesh flies synthesize hydrocarbons. However, hydrocarbon synthesis has not been directly demonstrated in this insect and the mechanism by which a fatty acid is converted into hydrocarbons in insects is not known. To test whether the insect can generate hydrocarbon by decarbonylation of an aldehyde as previously observed with other animal and plant systems (Cheesbrough and Kolattukudy, 1984; Cheesbrough and Kolattukudy, 1988; Dennis and Kolattukudy, 1991) [1-14C], [9,10- 3H]octadecanal was incubated with microsomal and supernatant preparations from the fly pupae. Both the supernatant and the microsomes were able to catalyze the decarbonylation of aldehyde into CO and alkane, with more than 70% of the activity in the microsomes (Fig. 12). Boiling the microsomal preparation for 15 min prior to the addition of the substrate aldehyde destroyed more than 92% of the decarbonylase activity. The ratio of alkane to CO produced was 1.0:0.6, somewhat less than the predicted 1.0:1.0 stoichiometry of the reaction. When the labeled alkane generated from [1-14C], [9,10-3H]octadecanal was subjected to radio-gas chromatography, the radioactivity was found exclusively in [3H]heptadecane (Fig. 13). The microsomal preparation generated alkane from fatty acid at only 8% of the rate observed with aldehyde as the substrate (Fig. 14). When ATP, CoA and NADH were added to the microsomal fraction, alkane formation from the fatty acid increased to 60% of the rate obtained with aldehyde. Alkane synthesis from aldehyde did not require the addition of cofactors. When the above cofactors were added, alkane synthesis from aldehyde was only 58% the rate 127 obtained without cofactors (Fig. 14), due to the conversion of the aldehyde into alcohol and/or acid. 128 Table 3. Quantities of epicuticular hydrocarbon and other lipids extracted from the surface of S. crassipalpis at different developmental stages. All flies were reared at 20°C, either at a long daylength to avert diapause or at a short daylength that programmed the pupae for diapause. Extraction of 100 individuals/instar with chloroform:methanol (2:lv/v). Numbers (mean + S.D.) followed by the same letter within a column are not significantly different (Student's t test, P>0.05).

Developmental stage Hydrocarbons Other lipids (pg/individual) (pg/indi vidual)

Third instar larva

Diapause destined 1.53 + ,20a 9.83 ± .25a Not destined for diapause 1.27 + .22a 10.02 ± .21a

Puparium, external surface

Diapausing pupa 3.89 + .19b 9.87 ± .24a Nondiapausing pupa 2.29 + .23° 9.92 + .30a

Empty puparium

Diapausing pupa 15.61 + .16d Nondiapausing pupa 7.92 + ,30e

Adult female

Emerged from pupal diapause 38.51 ± .22f 9.90 + .20a Emerged from nondiapausing pupa 38.35 ± .20f 9.76 + .25a 129

Fig. 10. Total ion count (TlC)-gas chromatograms (abundance versus carbon number) of hydrocarbons from empty puparia (each N=300 empty puparia) of nondiapausing (A) and diapausing (B)S. crassipalpis. Both groups were reared at 20°C. indicates the C29 alkane unique to diapause. Abundance X 10 15- 10 i 4 3-i - 62 29 28 26 627 26 27 i. 10 Fig. abn No. Carbon c uv 28 29 130 131

Fig. 11. Mass spectrum (abundance versus mass/charge ratio) of

C 29 indicates a straight-chain alkane. CO o 4: X 57 CD O c 2- 1 13 407 CO / TJ 155 225 308 c 1^ / r» X) j LI I I L : — — 1—- 1 —I" 1 < 100 200 300 400

Mass/charge

Fig. 11 u> to 133

Fig. 12. Formation of CO and alkane from [1-14C], [9,10- 3H]octadecanal by the 105,000 x g supernatant and resuspended microsomal pellet. "Boil" indicates a 15 min boiling of the same microsomal preparation prior to aldehyde substrate formation. Activity (pmoles/min/mg) 50 uentn Mcooe Microsomes Microsomes Supernatant i. 12 Fig. Alkane ■ m co 134 135

Fig. 13. Radio-gas chromatography of the alkane generated from [1-14C], [9,10-3H]octadecanal by the microsomal preparation. A 2m x 1/8" OD column packed with 3% OV-101 on Chromosorb W-HP 80-100 mesh was used with a temperature program from 120°C to 300°C at a rate of 10°C*min'1 with an argon flow of 70cm3•min'1. The numbers above the flame ionization detector response indicate the chain length of the co-injected authentic n-alkanes. 136

. Radioactivity

5 TO Time (min)

Fig. 13 Fig. 14. Effects of the cofactors ATP, CoA and NADH on the formation of alkane from octadecanal and octadecanoic acid catalyzed by the microsomes of flesh fly pupae. Activity (pmoles alkane/min/mg) 300

Cofactors

Cofactors

Cofactors

Cofuctors 139

DISCUSSION

Systematic changes in epicuticular hydrocarbons and other lipids occur throughout development in S. crassipalpis, but the major difference between diapause and nondiapause focuses on the hydrocarbons of the puparium. Puparia from diapausing flies contain nearly twice as much hydrocarbon as puparia from nondiapausing flies and a straight-chain C29 hydrocarbon is unique to diapause. The amount of hydrocarbon coating the cuticular surface of S. crassipalpis increases during ontogeny, as it does in S. bullata (Armold and Regnier, 1975), in correlation with an increased need for water conservation. Flesh fly larvae feed on decaying carrion, an environment in which water is readily available. The pupal habitat under the soil surface may have a high moisture content, but the pupa cannot imbibe free water, and if the pupa enters diapause, it must conserve its limited water supply for many months. The free-flying, active adult flesh fly has the highest quantity of epicuticular hydrocarbon, suggesting that the adult environment is especially stressful for maintaining water balance. In spite of having a higher quantity of epicuticular hydrocarbon, adult flies still lose water faster than either larvae or pupae (Yoder 140 and Denlinger, 1991), presumably due to a high rate of respiratory loss. The deposition of extra cuticular hydrocarbon that we observe in puparia of diapausing flesh flies has also been noted in association with diapause in other insects: pupae of Manduca sexta (Bell et al., 1975), Mamestra configurata (Hegdekar, 1979) and Pieris rapae crucivora (Kono, 1973), and eggs of Melanoplus differentialis (Slifer, 1946) and Petrobia latens (Lees, 1955). Few experiments have integrated epicuticular lipid analysis with water loss rates, but those that have show a consistent reliance on high quantities of saturated hydrocarbons for reduction of net transpiration [in tiger beetles, Cicindela sp., (Hadley and Schultz, 1987) and in the scorpion, Paruroctonus mesaensis, (Hadley and Jackson, 1977)]. In S. crassipalpis, net transpiration rate is 0.023 % 'h'1 for nondiapausing pupae, but in the diapausing pupae that have twice as much hydrocarbon, the rate is only 0.008 %-h'1. A correlation between increased production of hydrocarbon in response to increased water stress can also be seen in our temperature experiments. Flesh flies reared at higher temperatures produced more hydrocarbons, a correlation also noted in M. configurata (Hegdekar, 1979) and several other species (Downer, 1985). Hydrocarbons extracted from puparia of S. crassipalpis are predominately C2 6-C 30 alkanes, and C 29 is much more abundant in puparia from diapausing flies. A similar composition of long, 141 straight-chain alkanes is common to the epicuticle of many insect pupae (Goodrich, 1970; Armold and Regnier, 1975; Lockey, 1976;

Cbudron and Nelson, 1978). C 2 9, the straight-chain hydrocarbon unique to puparia of diapausing flies, makes up 20% of the total hydrocarbon content and is a dominant hydrocarbon in the waxes of many insects (Jackson and Blomquist, 1976; Lockey, 1985) and plants (Kolattukudy, 1970; Tulloch, 1976). The scorpion, Paruroctonus mesaensis, which has one of the lowest rates of water loss known (Hadley, 1974), also contains high quantities of C 29

(Hadley and Jackson, 1977). C29 comprises 10% of the cuticular hydrocarbon in both nondiapausing and diapausing pupae of Heliothis virescens (Coudron and Nelson, 1978), 16% in nondiapausing pupae of S. bullata (Armold and Regnier, 1975) and 37% of the hydrocarbon content of empty puparia in nondiapausing L. cuprina (Goodrich, 1970). It not clear whether the presence of

C 29 contributes any unique features to the puparium of diapausing pupae or if the enhanced water balance properties are simply due to the overall increase in hydrocarbon production. The bulk of hydrocarbon on the flesh fly puparium adheres to the interior surface of the puparium. Exactly how it gets there is unknown. Wax is usually deposited on the exterior surface of the procuticle at each moult, but in this case the wax is present on the interior surface of the old larval exocuticle (puparium). Possibly the wax is synthesized at or shortly before pupation, transported to the surface of the pupal cuticle, and from there transferred to the 142

inner surface of the puparium. If so, transfer to the inner surface of the puparium is nearly complete because very little of the hydrocarbon content remains on the surface of the pupa. After the pupal cuticle has physically separated from the puparium, at the time of the pupal moult, there would appear to be no conduit for transfer of hydrocarbon to the puparium. Thus, deposition must occur before the separation is complete. The extra hydrocarbon associated with the diapause of Sarcophaga affects the properties of the puparium. Empty puparia of diapausing pupae re-equilibrate at lower humidities than nondiapausing puparia, thus the absorptive capacity of diapausing pupae is at least partially due to the puparium. In diapausing pupae of Sarcophaga, reduced rates of both net transpiration (0.008 % 'h'1 compared to 0.023 %*h_1 in nondiapausing pupae) and vapor absorption (1.76 mg'h^AR.H."1 versus 3.49 mg'h^AR.H."1 for nondiapausing pupae) at 20°C (Yoder and Denlinger, 1991) are likely consequences of a more impervious puparium. A greater amount of cuticular hydrocarbon requires a higher temperature to induce a phase change and this is reflected in the higher critical transition temperature observed in diapausing pupae (39° versus 30°C in nondiapausing pupae) (Yoder and Denlinger, 1991). The permeability barrier generated by additional hydrocarbon on the puparium could not only protect the pupa from desiccation but may serve an equally important role as a hydrophobic barrier that prevents drowning. Remaining dry may be especially important to 143 an overwintering flesh fly pupa that is vulnerable to innoculative freezing. Though our experiments show that puparia from diapausing pupae consistently contain high amounts of hydrocarbon, expression of diapause and synthesis of additional hydrocarbons are not invariably linked. A few individuals reared under strong diapause-inducing conditions fail to enter diapause, yet they produce puparia with an abundance of hydrocarbons. Several environmental stresses can act on the young pupa to reverse the decision to enter diapause (Denlinger et al., 1988), and possibly the developmental decision was reversed after the commitment to produce hydrocarbons was already made. However, the possibility that the synthesis of abundant hydrocarbons may not actually be a component of the diapause syndrome cannot be ruled out. Both may simply be programmed independently by environmental signals that are nearly, but not entirely, identical. Regardless of the linkage between these two events, expression of diapause normally coincides with increased hydrocarbon synthesis. Temperature alone has a modest effect on the quantity of hydrocarbons synthesized (more abundance at higher temperature), but this cannot account for the dramatic difference we observe at 20°C between puparia of diapausing and nondiapausing pupae. Our results with flesh flies provide the first evidence that alkane synthesis in insects proceeds via decarbonylation of an aldehyde to yield CO and an alkane of one carbon less than the 144

precursor aldehyde. Though not previously reported in insects, such a novel decarbonylation reaction has been observed in peas (Cheesbrough and Kolattukudy, 1984), birds (Cheesbrough and Kolattukudy, 1988) and green algae (Dennis and Kolattukudy, 1991). When [1-14C], [9,10-3H]octadecanal was incubated with the microsomal preparation from flesh fly pupae, labeled heptadecane was the only alkane generated. The observed ratio of alkane to CO of 1.0:0.6 was less than the predicted ratio of 1.0:1.0, probably caused by some components of the enzyme preparation binding a

portion of the generated CO. The decarbonylase did not require O 2 or the addition of any cofactors, indicating that the reaction did not involve a net oxidation or reduction. That the decarbonylase activity could be severely inhibited (>92%) by boiling the pupal microsomal preparation prior to aldehyde substrate addition suggests the enzymatic nature of the reaction. The microsomal localization of decarbonylase is consistent with previous observations showing that alkane formation from fatty acids is catalyzed by microsomal preparations in insect (Chu and Blomquist, 1980; Vaz et al., 1988) and non-insect systems (Cassagne et al., 1977; Cheesbrough and Kolattukudy, 1988; Dennis and Kolattukudy, 1991). The previous step, fatty acyl-CoA reduction, is also known to be catalyzed by microsomes (Kolattukudy, 1976). From our experiments it is clear that fatty acid conversion to alkane requires the cofactors CoA, ATP and NADH, but the cofactors are not needed for the final step, conversion of aldehyde to alkane by decarbonylation. Whether the change in hydrocarbon level in diapausing flesh flies involves increased levels of the acyl-CoA reductase and decarbonylase remains to be elucidated. 146

REFERENCES

Armold M. T. and Regnier F. E. (1975) A developmental study of cuticular hydrocarbons of Sarcophaga bullata. J. Insect Physiol. 21,1827-1833.

Bell R. A., Nelson P. R., Borg T. K. and Caldwell D. L (1975) Wax secretion in non-diapausing and diapausing pupae of the tobacco hornworm, Manduca sexta. J. Insect Physiol. 21,1725-1799.

Blomquist G. J. and Dillwith J. W. (1985) Cuticular lipids. In Comprehensive Insect Physiology, Biochemistry, and Pharmacology vol 4 (Edited by Kerkut G. A. and Gilbert L. I.), pp. 117-154. Pergamon Press, Oxford.

Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.

Cassagne C., Darriet D., and Bourre J. M. (1977) Evidence of alkane synthesis by the sciatic nerve of rabbit. FEBS Lett. 82, 51- 54.

Coudron T. A. and Nelson D. R. (1978) Hydrocarbons in the surface lipids of pupal tobacco budworms, Heliothis virescens. Insect Biochem. 8, 59-66.

Cheesbrough T. M. and Kolattukudy P E. (1984) Alkane biosynthesis by decarbonylation of aldehydes catalyzed by a particulate preparation from Pisum sativum. Proc. Natl. Acad. Sci. USA. 81, 6613-6617. 147

Cheesbrough T. M. and Kolattukudy P. E. (1988) Microsomal preparation from an animal tissue catalyzes release of carbon monoxide from a fatty aldehyde to generate an alkane. J. Biol. Chem. 263, 2738-2743.

Chu A. J. and Blomquist G. J. (1980) Decarboxylation of tetracosanoic acid to n-tricosane in the termite, Zootermopsis angusticollis. Comp. Biochem. Physiol. 6 6 B, 3 1 3 -3 1 7 .

Davis M-T. B. (1974) Critical temperature and changes in cuticular lipids in the rabbit tick, Haemaphysalis leporispalustris. J. Insect Physiol. 20, 1087-1100.

Denlinger D. L. (1971) Embryonic determination of pupal diapause in the flesh fly Sarcophaga crassipalpis. J. Insect Physiol. 17, 1 8 1 5 -1 8 2 2 .

Denlinger D. L. (1972) Induction and termination of pupal diapause in Sarcophaga (Diptera:Sarcophagidae). Biol Bull. 142, 11-24.

Denlinger D.L., Giebultowicz J. and Adedokun T. (1988) Insect diapause: Dynamics of hormone sensitivity and vulnerability to environmental stress. In Endocrinological Frontiers in Physiological Insect Ecology (Edited by Sehnal F., Zabza A. and Denlinger D. L.), pp.309-324. Wroclaw Technical University Press, Wroclaw, Poland.

Dennis M. W. and Kolattukudy P. E. (1991) Alkane biosynthesis by decarbonylation of aldehyde catalyzed by a microsomal preparation from Botryococcus braunii. Arch. Biochem. Biophys. In press.

Devine T. L. and Wharton G. W. (1973) Kinetics of water exchange between a mite, Laelaps echidnina, and the surrounding air. J. Insect Physiol. 19, 243-254. 148

Downer R. G. H. (1985) Lipid metabolism. In Comprehensive Insect Physiology, Biochemistry, and Pharmacology vol. 3 (Edited by Kerkut G. A. and Gilbert L. I.), pp. 77-113. Pergamon Press, Oxford.

Goodrich B. S. (1970) Cuticular lipids of adults and puparia of the Australian sheep blowfly Lucilia cuprina (Wied). J. Lipid Res. 1 1 , 1- 6 .

Hadley N. F. (1974) Adaptational biology of desert scorpions. J. Arachnol. 2, 11-23.

Hadley N. F. and Jackson L. L. (1979) Chemical composition of the epicuticular lipids of the scorpion, Paruroctonus mesaenis. Insect Biochem. 7, 85-99.

Hadley N. F. and Schultz T. D. (1987) W ater loss in three species of tiger beetles ( Cicindela): correlations with epicuticular hydrocarbons. J. Insect Physiol. 33, 677-682.

Hegdekar B. M. (1979) Epicuticular wax secretion in diapause and non-diapause pupae of the Bertha armyworm. Ann. Ent. Soc. Am . 72, 13-15.

Jackson L. L., Armold M. T. and Regnier F. E. (1974) Cuticular lipids of adult flesh flies Sarcophaga bullata. Insect Biochem. 4, 3 6 9 -3 7 9 .

Jackson L. L. and Blomquist G. L. (1976) Insect waxes. In The Chemistry and Biochemistry of Natural Waxes (Edited by Kolattukudy P. E.), pp.201-233. Elsevier, Amsterdam.

Kolattukudy P. E. (1970) Plant waxes. Lipids 5, 259-275.

Kolattukudy P. E. (1976) Chemistry and Biochemistry of Natural Waxes, Elsevier, Amsterdam. 149

Kono Y. (1973) Differences of cuticular surface between diapause and non-diapause pupae of Pieris rapae crucivora (Lepidoptera:Pieridae). Appl. Entomol. Zool. 8, 50-52.

Lees A. D. (1955) The Physiology of Diapause in Arthropods. Cambridge University Press.

Lockey K. H. (1976) Cuticular hydrocarbons of Locusta, Schistocerca, and Periplaneta, and their role in waterproofing. Insect Biochem. 6, 457-472.

Lockey K. H. (1985) Insect cuticular lipids. Comp. Biochem Physiol. 81B, 263-273.

Slifer E. H. (1946) The effects of xylol and other solvents on diapause in the the grasshopper egg; together with a possible explanation for the action these agents. J. exp. Zool. 102, 3 3 3 -3 5 6 .

Tulloch A. P. (1976) Chemistry of waxes of higher plants. In The Chemistry and Biochemistry of Natural Waxes (Edited by Kolattukudy P. E.), pp. 235-287. Elsevier, Amsterdam.

Vaz A. H., Jurenda R. A., Blomquist G. J. and Reitz R. C. (1988) tissue and chain length specificity of the fatty acyl-CoA elongation system in the American cockroach. Arch. Biochem. Biophys. 267, 551-557.

Wharton G. W. (1963) Equilibrium humidity. Adv. Acarology. 1, 201- 208.

Wharton G. W. (1985) Water balance of insects. In Comprehensive Insect Physiology, Biochemistry, and Pharmacology vol 14 (Edited by Kerkut G. A. and Gilbert L. I.), pp. 565-601. Pergamon Press, Oxford.

Wharton G. W. and Devine T. L. (1968) Exchange of water between a mite, Laelaps echidnina, and the surrounding air under equilibrium conditions. J. Insect Physiol. 14, 1303-1318. 150

Winston P. W. and Bates D. H. (1960) Saturated solutions for the control of humidity in biological research. E cology 41, 232- 237.

Yoder J. A. and Denlinger D. L. (1991) Water balance in flesh fly pupae and water vapor absorption associated with diapause. J. exp. Biol. In press. CHAPTER VI

EVIDENCE FOR A BRAIN FACTOR THAT STIMULATES SYNTHESIS OF PUPARIAL HYDROCARBONS IN DIAPAUSING FLESH FLIES

Puparia from diapausing pupae of the flesh fly,Sarcophaga crassipalpis, are lined with twice as much hydrocarbon as puparia from nondiapausing pupae (Yoder, Denlinger, Dennis and Kolattukudy, unpublished). The additional hydrocarbon favors water conservation during diapause by reducing water loss, enhancing water vapor absorptivity and elevating the critical transition temperature (Yoder and Denlinger, 1991). In this study we seek to identify the source of a hormonal modulator that would account for this difference in hydrocarbons. Our results suggest that a factor unique to the brains of diapause-programmed larvae is responsible for increasing the quantity of hydrocarbon deposited on the puparium. cAMP elicits the same effect, thus suggesting that the factor is likely a neuropeptide. A colony of the flesh fly Sarcophaga crassipalpis Macquart was maintained in the laboratory as previously described (Denlinger, 1972). Parental adults were reared under nondiapausing (LD, 15:9h, 25°C) or diapausing (LD, 12:12h, 25°C)

151 152

conditions. All larvae and pupae were maintained at LD, 12:12h and 20°C. Under these conditions, a high incidence of pupal diapause (>95%) was observed in the progeny of short-day mothers, while none of the progeny from long-day mothers entered diapause. The photosensitive stage is actually the embryo which develops within the uterus of the female (Denlinger, 1971). Thus, diapausing and nondiapausing groups in this experiment differed only in maternal photoperiod. Brain extracts, hemolymph, and known hormonal agents were tested for their efficacy in stimulating hydrocarbon synthesis. Methoprene (Zoecon Corp., Palo Alto, CA.), a juvenile hormone (JH) analog was diluted in acetone and applied topically to larvae. 20-hydroxyecdysone (Sigma, St. Louis, MO.) was diluted in 10% ethanol and injected using a finely drawn glass capillary, and the dibutyryl derivative of cyclic AMP (N6,0 2- dibutryladenosine-3':5'-cyclic monophosphoric acid [Na+], Sigma) was coinjected with the phosphodiesterase inhibitor, aminophylline (Sigma). To collect larval hemolymph third instar larvae were punctured and hemolymph was collected on chilled parafilm. Hemolymph was withdrawn from pupal heads after first centrifuging pupae in a heads-up position. Within a few minutes of collection the hemolymph was injected into recipient larvae. Brain extracts were prepared from larvae that were within 12 h of pupariation. Brains dissected from chilled larvae were placed in 153 acetone, homogenized and centrifuged; the supernatant was dried under nitrogen and reconstituted in distilled water for injection. Unless otherwise noted, all recipients were nondiapause- destined larvae that were within 24 h of pupariation. Recipient larvae were immobilized on ice before injection and were returned to ice for a few minutes of recovery after injection. All recipients were returned to 20°C (LD, 12:12h) until pupation (3 days after pupariation). At pupation, a subset of pupae (N=45) from each experimental group was transferred to 0% R.H. to determine net transpiration rates; the others were permitted to complete adult eclosion and their empty puparia were then analyzed for hydrocarbon content. To determine net transpiration rates (integumental and respiratory water loss), pupae were held at 0% R.H. (generated by anhydrous CaSCU) in sealed glass desiccators. Pupae were first predesiccated [0% R.H., 20°C for 24 h, so that weight change reflects water flux (Wharton, 1985)], and weighed on an electrobalance (Cahn 25, Ventron Co.). After predesiccation, pupae were maintained at 0% R.H. and reweighed every 24 h for 5 days. Pupal dry weight was determined after drying the individual over an.

CaSC>4 , at 50°C for 7 days. Percent body water content was determined by dividing the water mass (m, the difference between wet and dry weight) by initial wet weight X 100% (Wharton, 1985). In dry air, the pupa's water mass depleted exponentially. The slope of an exponential regression through a plot of In mt/mo 154

[water masses at time t (mt) and initial mass (mo)] versus time is the rate of net water loss (-kt) and is expressed as %-h'1 (Wharton, 1985). Slopes of regression lines were compared according to Sokal and Rohlfs (1984) test for the equality of several slopes. To quantify the hydrocarbons, empty puparia were extracted with chloroform:methanol (2:lv/v) for 10 min, dried (N2 ) to a 50 |il vol and passed through a silica gel column (Waters Associates, Milford, MA). Hydrocarbons were eluted with HPLC grade hexane, dried under nitrogen onto predesiccated (0% R.H., 25°C, 24 h), preweighed aluminum pans and held at 0% R.H., 25°C for >24h. Total hydrocarbon quantity was calculated as described by Bligh and Dyer (1959). Pupal wet weight and percentage body water were not significantly different among the pupae tested (data not shown). In all cases, body water mass was a positive correlate of dry weight (R>0.89) and slopes of regression lines describing this relationship were all significantly different from zero (F>524.27, df=399, P<0.001). Percentage body water data were arcsine transformed prior to statistical analysis. Puparia from diapausing pupae have twice as much hydrocarbon as their nondiapausing counterparts and the greater amount of hydrocarbon correlates with lower rates of net transpiration for diapausing pupae (Yoder, Denlinger, Dennis and Kolattukudy, unpublished; Yoder and Denlinger, 1991; Table 4). The concentration ranges of juvenile hormone analog and 20- 155

hydroxyecdysone that were tested did not affect the quantity of puparial hydrocarbon synthesized and water loss from these pupae was similar to that of the controls (Table 4). Armold and Regnier (1975) reported ecdysteroid stimulation of hydrocarbon biosynthesis at pupariation in a closely related species, Sarcophaga bullata. But, ecdysteroid titres at the time of pupariation are similar for both nondiapause and diapause-destined larvae of S. crassipalpis (Denlinger, 1985), thus it seems unlikely that ecdysteroids could be responsible for doubling the quantity of puparial hydrocarbon associated with diapause. Unique cycles of JH activity are associated with pupal diapause in flesh flies (Denlinger et al., 1984), and this could potentially be a stimulant of hydrocarbon synthesis, but our results do not suggest that JH is involved. Dibutyryl cAMP, however, when injected with aminophylline was effective in increasing hydrocarbon levels of the puparium and such pupae lost water at a significantly slower rate than control pupae (Table 4). The accumulation of lipid stores is a feature of the diapause syndrome in Sarcophaga (Adedokun and Denlinger, 1985), but dibutyryl cAMP did not increase overall levels of lipid (control=0.073 ± .012 mg/pupa, db cAMP=0.076 ± .014 mg/pupa), suggesting that cAMP is selective in stimulating hydrocarbon production. Though dibutyryl cAMP promoted the hydrocarbon synthesis characteristic of diapause, the pupae themselves did not enter diapause. 156

Recipients of hemolymph from diapause-destined larvae, young (2 day old) and older (>20 day) diapausing pupae lost water at the same rate as nondiapausing pupal controls (Table 4), thus suggesting that hydrocarbon levels were not affected by hemolymph transfusions. Similar transfusions into nondiapausing larvae at the time of pupariation also showed no effect. Recipients of brain extracts from diapause-programmed larvae (7 brain eq/larva) had a reduced rate of pupal net transpiration, and this was associated with a higher quantity of puparial hydrocarbon (Table 4). Injection of 3 brain equivalents/larva had no effect. As with cAMP, the brain extract did not alter the pupa's developmental (nondiapause) status. Injection of brain extracts from nondiapause-programmed larvae neither enhanced quantities of puparial hydrocarbon nor lowered net transpiration rate (Table 4). Thus, a factor unique to a diapause-programmed larval brain, and not simply the presence of additional brain material, was responsible for stimulating hydrocarbon synthesis. While hydrocarbon quantities in normal diapausing puparia and in hydrocarbon-enhanced nondiapausing puparia were nearly the same, transpiration rates were lower in diapausing pupae. This difference is most certainly due to the fact that diapause greatly suppresses the metabolic rate, a feature that also contributes to a low transpiration rate. The difference we note in transpiration rates between normal nondiapausing pupae and 157 those with enhanced levels of puparial hydrocarbon can likely be attributed directly to the amount of hydrocarbon in the two types of puparia. These results suggest that the extra puparial hydrocarbon associated with the diapause of Sarcophaga is linked to a factor present in the brain of diapause-programmed flies, and the fact that cAMP, a well-known secondary messenger for peptide hormones, mimics this effect indicates that this factor is most likely a neuropeptide. I 158 Tabic 4 . A comparison of net transpiration rate (-kt) at 20°C and quantities of hydrocarbon (pg/empty puparium) for pupae of S. crassipalpis. All treated flics were programmed for nondiapausc development. Numbers (mean + S. 3.) followed by the same letter within a column arc not significantly different (ANOVA, P>0.05). Mean wet weights for experimental pupae ranged from 120.51-123.98 mg, and body water ranged from 66.28-67.31%; differences between groups were not significant (ANOVA, P>0.05).

Experimental Treatments -kl(20°C ) Hydrocarbon (%h‘*) (pg/cm pty puparium)

Untreated controls

nondiapausing pupae 0.024* 8.07 + .18* diapausing pupae 0.009b 15.12 + .22b

Hormonal agents

Juvenile hormone analog acetone (5pl) 0.025* 8.08 ± .26* O.OOlpg 0.024* 8.02 + .21* 0.01 0.026* 7.96 ± .24* 0.1 0.024* 8.06 + .25* 1.0 0.025* 8.04 ± .23*

20-Hydroxyccdysone 10% ethanol (5pi) 0.023* 7.99 + .26* O.OOlpg 0.026* 8.09 + .25* 0.01 0.025* 7.91 + .23* 0.1 0.024* 8.11 +.21* 1.0 0.023* 8.07 ± .19*

cyclic AMP (dibutyryl derivative) water (5pl) 0.023* 8.10 + .21* aminophyllinc (lOpg) 0.026* 8.13 + .18* db cAMP (lOOpg) + aminophyllinc (lOpg) 0.018® 13.64 + .25®

Hemolymph transfusions (50pl)

from diapause-destined larvae 0.024* from 2 d old diapausing pupae 0.026* from >20 d old diapausing pupae 0.023*

Brain extracts

from nondiapause-dcstincd larvae 7 brain equivalents 0.025* 7.95 + .26*

from diapause-destined larvae 3 brain equivalents 0.025* 7.92 + .27* 7 brain equivalents 0.016® 12.84 + .24® 159

R e f e r e n c e s

Adedokun, T. A. and Denlinger, D. L. (1985). Metabolic reserves associated with pupal diapause in the flesh fly S arcophaga crassipalpis. J. Insect Physiol. 31, 229-233.

Armold, M. T. and Regnier, F. E. (1975). Stimulation of hydrocarbon biosynthesis by ecdysone in the flesh fly, Sarcophaga bullata. J. Insect Physiol. 21, 1581-1586.

Denlinger, D. L. (1971). Embryonic determination of pupal diapause in the flesh fly Sarcophaga crassipalpis. J. Insect Physiol. 17, 1815-1822.

Denlinger, D. L. (1972). Induction and termination of pupal diapause in Sarcophaga (Diptera:Sarcophagidae).Biol. Bull, mar. biol. Lab. Woods Hole. 142, 11-24.

Denlinger, D. L., Shukla, M. and Faustini, D. L. (1984). Juvenile hormone involvement in pupal diapause of the flesh fly Sarcophaga crassipalpis: regulation of infradian cycles of O 2 consumption. J. exp. Biol. 109, 191-199.

Denlinger, D. L. (1985). Hormonal control of diapause. In Comprehensive Insect Physiology, Biochemistry, and Pharmacology, vol. 11 (ed. G. A. Kerkut and L. I. Gilbert), pp. 353-412. Oxford: Pergamon Press.

Hadley, N. F. and Jackson, L. L. (1979). Chemical composition of the epicuticular lipids of the scorpion, Paruroctonus mesaenis. Insect Biochem. 7, 85-89.

Hadley, N. F. and Schultz, T. D. (1987). Water loss in three species of tiger beetles (Cicindela): correlations with epicuticular hydrocarbons. J. Insect Physiol. 33, 677-682. 160 Sokal, R. R. and Rohlf, F. J. (1984). Biometry. San Francisco: W. H. Freeman Co.

Wharton, G. W. (1985). Water balance of insects. In Comprehensive Insect Physiology, Biochemistry, and Pharmacology, vol. 14 (ed G. A. Kerkut and L. I. Gilbert), pp. 565-601. Oxford: Pergamon Press.

Yoder, J. A. and Denlinger, D. L. (1991). Water balance in flesh fly pupae and water vapor absorption associated with diapause. (J. exp. Biol) In press. SUMMARY

I. Water balance in flesh fly pupae and water vapor absorption associated with diapause:

1. The body water pools are the same for both nondiapausing and diapausing pupae of Sarcophaga crassipalpis and dry weight is a positive correlate of water mass. Diapausing pupae can only tolerate a loss of ca. 24% of their total percentage body water (ca. 67%). 2. Diapausing pupae of S. crassipalpis lose and gain water at slower rates than nondiapausing pupae, and diapausing pupae absorb water at lower humidities than nondiapausing pupae. Patterns of water loss and gain do not cycle in phase with the cyclic patterns of O2 consumption typical of diapausing pupae. At hydrating humidities, no metabolic water is produced that would account for the observed gain. 3. The puparium, the inert third instar larval cuticle that encases the pupa, is sufficiently porous to permit water entry but is restrictive to water loss. Wax block experiments indicate that the entire puparium is porous to water entry but the actual site of water entry into the pupa has not been determined.

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4. The critical transition temperature (CTT) for diapausing pupae is higher than nondiapausing pupae, thus implying an epicuticular layer that is unique to diapause. Activation energies (Ea) are greater for nondiapausing pupae than those in diapause. Thus, water is lost more readily from nondiapausing pupae than diapausing pupae over a range of temperatures. Rate of uptake increases with temperature for both types of pupae, but once within the range of the CTT, the rate drastically declines. 5. The water balance characteristics are similar for a more northern, closely related species, Sarcophaga bullata, but water loss rates are lower, and hence Ea is higher, in S. crassipalpis.

II. A comparison of the water balance characteristics of temperate and tropical fly pupae:

1. Pupal net transpiration rates at 20°C are a function of size, but the strength of the correlation declines as temperature increases. Thus factors other than a large surface area:volume ratio contribute to total water loss. 2. All pupae examined have the same percentage body water (67%). Dry weight correlates positively with water mass. 3. The pupae have a limited capacity to absorb atmospheric water and equilibrate at the same humidity (ca. av 0.90-0.92). The 24h equilibrium weight does not correlate with pupal net transpiration rates. 163

4. All pupae have similar CTT's but values of Ea in both temperature ranges were greater for tropical fly pupae than those occupying the temperate zone, implying that pupae from the tropics are more prone to water loss over a range of temperatures. 5. The pupae of all temperate species (M. domestica, S. crassipalpis and S. bullata) were photoperiodically programmed (LD, 15:9h) to avert diapause. Tropical fly pupae (P. abnormis and S. sternodontis) lack the genetic capacity for diapause. Both groups of pupae have the capacity to gain water and the values of CTT indicate that nondiapausing pupae, regardless of their origin, have similar patterns of metabolism and respiration.

III. Water vapor uptake by diapausing eggs of a tropical walking stick:

1. Eggs of Extatosoma tiaratum are capable of water vapor absorption, but the eggs of 5 other species (L. dispar, B. mori,A. polyphemus, O. fasciatus, and D. femorata) lack this ability. Both diapausing stages of E. tiaratum (early embryo and pharate first larva) have the capacity to absorb water from subsaturated air. 2. Water vapor absorption occurs at lower humidities and at a lower rate for early embryonic eggs than diapausing pharate first instar larvae. Temperature has no affect on uptake rate by early embryonic eggs, but temperature increase decreased the capacity to absorb atmospheric water, presumably due to greater evaporation 164

of adsorbed surface water. The greater rate of water loss may exceed rate gain, and the egg cannot equilibrate until a higher humidity is encountered. 3. Eggs in early embryonic diapause lose water at a slower rate than pharate first instar larvae. The low rate of net transpiration and gain for early embryos may be a consequence of a large pool of inert hygroscopic yolk combined with low rates of respiratory water loss. 4. Water is absorbed over the entire surface of the egg and can be maintained for many days. Killed eggs fail to gain water when placed at a humidity that hydrated viable eggs.

IV. Puparia of diapausing flesh flies are enhanced with additional hydrocarbons:

1. Empty puparia of diapausing flesh flies equilibrate at a much lower humidity (7%) than puparia from nondiapausing flies (18%). 2. Empty puparia of diapausing pupae are lined with twice as much hydrocarbon than puparia from nondiapausing flies. Quantities of other lipids (cholesterol, free fatty acids, triglycerides and wax esters) are approximately the same in the two kinds of pupae. No phospholipids were detected. 3. Temperature affects the quantity of hydrocarbon synthesized in puparia of nondiapausing flies, but the increase is 165 modest when compared to the two-fold increase associated with diapause programming (12:12, 20°C). Humidity does not affect the quantity of hydrocarbon synthesized. 4. For diapausing pupae, the bulk of the hydrocarbon is found on the inner wall of the puparium. 5. The hydrocarbons of empty puparia from both nondiapause and diapause groups are predominantly straight chained C2 7, methyl-branched C 27 and C28 and C30 alkanes. The quantity of puparial hydrocarbon is consistently higher in the diapause group.

6 . A straight-chain C 2 9 alkane is more abundant on empty puparia from diapausing flies than puparia from flies not programmed for diapause. The additional C 2 9 may enhance adherence to the inner puparial wall after larval-pupal ecdysis and is not found in puparia from nondiapausing flies. 7. Hydrocarbon synthesis in the fly proceeds via decarbonylation of precursor fatty acids, a reaction not previously described in insects.

V. Evidence for a brain factor that stimulates synthesis of puparial hydrocarbons in diapausing flesh flies:

1. Brains from diapause-destined third instar larvae injected into nondiapausing larvae induce extra puparial hydrocarbon. Consequently, total water loss is reduced in these experimental 166 nondiapausing pupae. Nondiapausing larval recipients of diapause- programmed larval brains fail to respond developmentally as diapausing pupae. Thus, patterns of respiration (respiratory water loss) and metabolism are not interrupted. Therefore, the extra hydrocarbon reduces only the transcuticular component of net water loss. 2. cAMP injected into nondiapausing larvae produce more puparial hydrocarbon than controls. The additional hydrocarbon also reduces water loss. cAMP possibly serves as a second messenger for the brain factor. 3. 20-hydroxyecdysone, juvenile hormone and hemolymph from diapause-programmed fly instars fail to stimulate additional hydrocarbon in puparia of nondiapausing flies. Consequently, pupal net transpiration rates are unaffected. 4. Nondiapause-destined larval brains injected into nondiapause-destined larvae fail to elevate puparial hydrocarbons. Thus it is the presence of a brain factor, most likely a neuropeptide, unique to a diapause-destined larval brain and not additional brain material that is responsible for elevating hydrocarbon biosynthesis.