Regulation of long-term oviposition in the house cricket Acheta domesticusRoles of prostaglandin and factors associated with sperm.код для вставкиСкачать
Archives of insect Biochemistry and Physiology 659-72 (1987) Regulation of Long-Term Oviposition in the House Cricket, Acheta domesticus: Roles of Prostaglandin and Factors Associated With Sperm Michael P. Murtaugh and David L. Denlinger Department of Entomology, Ohio State University, Columbus The transfer of spermatophore contents derived from testes during mating greatly stimulates ovipositional activity for long periods of time in the house cricket, Acheta domesticus (L.). Since prostaglandins appear t o play a role in reproduction i n several insect species, and since prostaglandin synthesis enzymes occur in cricket testes and spermatophores, we investigated the role of prostaglandins i n the regulation of long-term oviposition. Inactivation of prostaglandin synthesis enzymes in males or females using specific inhibitors failed to block mating-induced increases in egg laying. However, males lacking sperm because of X-irradiation were unable t o induce oviposition even though they mated, transferred spermatophores, and had high levels of prostaglandins in both testes and spermatophores. X-irradiation was also used t o generate males with nonfunctional sperm. Females mated t o these animals readily laid eggs, which failed t o develop. It appeared that sperm or a factor associated with sperm induced long-term oviposition in female house crickets. Prostaglandin synthesis enzymes transferred from the male t o females may have other roles in the female, for example, in sperm maintenance in the spermatheca. Previous observations strongly suggest that prostaglandins induce egg laying behavior and activity; they may be synthesized by female enzymes that are regulated by male-derived factors. Key words: X-irradiation, PGE, PGF2, INTRODUCTION In many insects, oviposition is induced or stimulated by male factors that are transferred during mating and act in females El]. Ovipositional stimuli Acknowledgments: We thank Laura Wiebers for preparing the manuscript. This research was supported by a Graduate Fellowship from the Ohio State University Graduate School to M.P.M., by a Sigma Xi Grant-In-Aid-of-Researchto M.P.M., and by grant 8600186 from the USDA Competitive Research Grants Office. Received Janary2,1987; accepted May 8,1987. Michael Murtaugh is now at the Department of Veterinary Pathobiology, University of Minnesota, 1971 Commonwealth Avenue, St. Paul, M N 55108. Address reprint requests to this address. 0 1987 Alan R. Liss, inc. 60 Murtaugh and Denlinger range from mechanical, the mating act itself, to chemical inducers secreted by various male reproductive organs [reviewed in 11. The transfer of malederived ovipositional stimuli with sperm at the time of mating serves to coordinate fertilization with oviposition and thus increases reproductive success. In the house cricket, Acketa domesticus (L.), mating is a powerful stimulus, which promotes oviposition at a high rate for many weeks 121. The malederived house cricket ovipositional stimulus is of some interest in that it appears to be both potent and stable. A single mating early in adult life stimulates the laying of fertilized eggs for 60 days, and the inducing activity is continuously present throughout this period . In addition, it can persist in recently emerged females, which mate readily but do not lay eggs for 1014 days. The biochemical nature of the male-derived ovipositional stimulus has not been determined, but enzymes and products of the prostaglandin (PG) synthesis system are present in male reproductive tissues and have been implicated in inducing oviposition [2-71. Prostaglandins are unsaturated, long-chain fatty acids that are found in all orders of animals and have diverse biological activities. PGs are widely distributed in insects in many tissues and organs. Their physiological function in insects has been studied most intensively with respect to reproduction, in which the E-type PGs appear to be involved in the regulation of the egg laying process. In crickets, the elevation of PGE in female reproductive tissues causes a short-lived ovipositional response [6,8,9]. However, whether prostaglandins also stimulate the long-term ovipositional activity of house cricket females has not been resolved. In this paper, we examine the role of PGs in the regulation of long-term oviposition in house crickets. The results obtained from these studies suggest that PGs do not mediate the long-term egg laying response in the house cricket. Rather, a factor associated with sperm appears to be the principal male-derived ovipositional stimulus in these insects. MATERIALS AND METHODS Insect Maintenance and Egg Collection A. domesticus (L.) adults and mid- to late-instar nymphs were maintained in continuous culture as described previously . Oviposition was monitored by holding mated females in 1pint containers with screen bottoms on moist perlite (Terra-lite, W.R. Grace, Cambridge, MA) as described previously . PG Measurement PGE and PGF2, levels in cricket tissues were determined by radioimmunoassay as described elsewhere Data shown were adjusted for losses during purification . The extraction efficiency, which varied with the amount of starting tissue, averaged 35-40% [n. [n. Pharmacological Inhibition of PG Synthesis Aspirin, acetaminophen, and indomethacin from Sigma (St. Louis, MO) were administered by addition to the food in a concentration of 1%or, for Sperm Regulates Cricket long-Term Oviposition 61 aspirin, 5%. Acetaminophen was also provided as a saturated solution in water and was available ad libitum. X-Irradiation Adult males age 1-4 days were exposed to 250-8,000 rads of X-irradiation delivered from a Norelco MG X-ray tube operated at 250 KV and 10 mA. Cricket juveniles of mixed sex, and approximately at the stage when external genitalia appear, were given 1,200 rads from the X-ray tube operated 150 KV and 10 mA. RESULTS Inhibition of PG Synthesis in Males Previous studies showed that aspirin and acetaminophen, which were available ad libitum in the food and water, respectively, for a period of 10-20 days, decreased PGE levels in the testis by 91% and PGF2, levels by 64% [fl. Indomethacin at a concentration of 1%in food completely inhibited PG synthesis [7l. Inhibition of testicular PG synthesis also was reflected in reduced PG levels in spermatophores. The level of PGE (15 rt 4 pglspermatophore, n=25) was decreased 70% by aspirin and acetaminophen in these experiments. PGF2, was not detected in spermatophores from control or treated animals. Thus, continuous administration of PG synthesis inhibitors to adult males profoundly suppressed testicular PG levels. Inhibition of PG Synthesis in Males: Effect on Induction of Oviposition Figure 1 shows the ovipositional activity of females after mating with males that had been administered inhibitors of PG synthesis. The inhibition of enzymes involved in PG synthesis in male reproductive tissues and the reduction of PG levels in spermatophores failed to affect the pattern of egg laying by females mated to them. In females mated to both treated (Fig. 1, closed circles) and untreated (Fig. 1, open circles) males, oviposition was greatly stimulated within 4 days and continued at a high rate until the experiment was terminated after 12 days. The males referred to above were treated with aspirin and acetaminophen for 12-13 days, beginning on the day after adult ecdysis. However, in crickets, sperm development occurs prior to adulthood, so, at the onset of adulthood, the testis contains spermatozoa at all stages of differentiation [lo]. Thus, if PGs were required during sperm development or in the early stages of spermatogenesis, then inhibition of PG synthesis during adulthood would not affect the large number of spermatozoa that had already differentiated. Hence, a group of young nymphs that had not yet elaborated external genitalia were placed on the inhibitor diet. As adults, these males, which were 23-40 days old and had been on inhibitor diet as juveniles and adults for 61 days at the time of mating, induced oviposition just as proficiently as normal males (Fig. 1, open and closed squares). Furthermore, sperm viability as measured by egg hatchability indicated that sperm from males maintained on PG synthesis inhibitors was equivalent to sperm from untreated males. 62 Murtaugh and Denlinger Days a f t e r mating Fig. 1. Effect of PG synthesis inhibitors in the male on egg laying activity. One-day-old males were provided with untreated food and water (0)or food with 5% aspirin and water saturated with acetaminophen ( 0 ) After . 12-14 days, each male mated with one female. Eggs were collected for 12 days after mating. Data represent the mean number of eggs laid per day per female for five females mated to untreated males and for 13 females mated to treated males. In a second experiment, eggs were collected from females mated at 16-18 days of adult age t o individual males. Males either were untreated, 16-day-oldadults (El)or were 2340-day-old adults reared for a total of 61 days on food and water containing aspirin and acetaminophen (M)as described. Data represent the mean number of eggs laid per day by three females (0) or 18 females (M).Standard errors averaged 35% of the mean in the untreated group and 20% of the mean for the treated group. Eggs laid by females mated to untreated males hatched at a rate of &-82% (910 eggs examined) over the 10 day oviposition period. The fertility of eggs laid by females mated to treated males varied from 77% to 90% (5,904 eggs examined). The results of the preceding studies indicated that pharmacologic inhibition of PG synthesis enzymes in males before mating did not affect the pattern or the frequency of oviposition in mated females, nor did it affect embryogenesis and hatching in deposited eggs. Inhibition of PG Synthesis in Females PG levels are increased greatly in the female spermatheca after mating [6,7,11]. For example, PGE, which was not detected in virgin spermathecae, averaged 215 pglgland in mated animals [ 7 ] . The administration of aspirin and acetaminophen to females completely blocked the mating-induced increase in PG levels . An equally effective inhibition of PG synthesis in the spermatheca after mating was also observed when the inhibitors were fed to males [ 7 ] . The effect on egg deposition of PG inhibitor administration to females is shown in Figure 2. Untreated females displayed a burst of ovipositional activity immediately after mating followed by a lower but sustained level of activity for 8 days (Fig. 2, closed circles). Females fed aspirin and acetamino- Sperm Regulates Cricket Long-Term Oviposition 63 Days a f t e r m a t i n g Fig. 2. Effect of administering PG synthesis inhibitors to females on subsequent egg laying. Females were placed on diets containing aspirin and acetaminophen from the day of eclosion until they were mated 16-17 days later to untreated males. Eggs were collected at 2 day intervals. Data shown are the means and standard errors from 18 untreated females ( 0 )and 12 treated females (0). phen for 16 days before mating showed a low rate of egg deposition which increased to the control rate in 7-8 days (Fig. 2, open circles). A similar stimulation of long-term egg laying activity without an initial burst was observed in two additional experiments (not shown). The lack of a large burst of egg laying activity immediately after mating in these experiments was attributed to nonspecific effects of aspirin and acetaminophen that were unrelated to PG synthesis in reproductive tissues, since, as indicated previously, postmating increases in spermathecal PG levels were blocked totally by treating males with PG inhibitors a procedure that had no effect on ovipositional activity (see Fig. 1). PG synthesis inhibitors in females have no apparent effect on the survivability of deposited eggs. The frequency of hatching in three experiments in which females received maximally effective levels of aspirin and acetaminophen throughout the period of oogenesis varied from 76% to 80% in a total of 1,361 eggs examined. [n, PGs and Egg Deposition in Young Females The preceding experiments attempted to evaluate the role of PGs in stimulating oviposition by observing the effects of PG synthesis inhibitors in mature females that were competent to lay eggs as determined previously . The results did not indicate that PGs mediate or constitute the matinginduced stimulation of egg laying. However, another feature of house cricket reproductive physiology is that young adult females mate readily but do not Murtaugh and Denlinger 64 lay eggs until approximately days 12-14 of adulthood . To determine if this delay in oviposition was due to the failure of mating to increase spermathecal PG levels, PG levels were measured in the spermatheca of young mated females. Four- to six-day-old females were mated, and spermathecae were removed 24 h later for determination of PGE and PGF2, levels. These individuals would not be expected to commence oviposition until about 7 days later. Nevertheless, high levels of spermathecal PGE were detected (Table 1). Furthermore, these values were substantially higher than in mature females treated with PG synthesis inhibitors , which were able to lay eggs (see Fig. 2). We also attempted to overcome the ovipositional delay in young females with exogenous PG. Four- to five-day old females were injected with PGE2 or were mated. Comparison of the egg laying behaviors of injected and mated females is shown in Figure 3. A pharmacological dose (100 pg) of PGE2 was completely ineffective, failing to elicit any egg laying in young females (Fig. 3, closed circles). Females from the same group that were allowed to mate commenced prolonged ovipositional activity 5 days later (Fig. 3, open circles). Injection of 4-5 day-old-females with PGF2, (100 pg) likewise failed to elicit ovipositional activity (data not shown). Injection of PGE2 into 20-day-old adult females induced a 1 day burst of egg laying similar to the short-term effects of PGE2 injection reported previously [6,8,9]. One hundred micrograms of PGE2 administered intraabdominally induced females to lay an average of l2+1 eggs in the following 24 h as compared to lrt0.2 eggs per female in buffer-injected controls (sample size = 15-8 crickets per group). PGF2, at 100 &female was not significantly different from controls. In the following six days, all groups laid less than one egg per female per day. Identical results were obtained irrespective of incubation temperatures from 25 to 31°C. Effect of X-Irradiation on Male Stimulation of Egg Laying From the previous experiments, it appeared that PGs or active PG synthesis enzymes from the male were not required to stimulate long-term egg laying in females. Furthermore, the accumulation of PGs in female reproductive tissue was not sufficient to induce ovipositional activity. Therefore, we evaluated other components of the spermatophore for egg laying stimulatory activity. A likely candidate was sperm, since the use of sperm to induce egg TABLE 1. Effect of Prostaglandin Synthesis Inhibitors on Prostaglandin Levels in the Spermathecae of Young Females* Experiment No. of individuals 1 2 3 12 13 13 PGE (pg) per Individual mg Protein 90 90 54 550 464 320 PGF2, (pg) per Individual mg Protein 18 46 36 107 239 214 *Four- to six-day-old females were mated to untreated males. After mating, spermathecae were removed for PG determinations. Protein determinations were carried out according to Bradford  after chloroform extraction of the samples to remove lipids. Sperm Regulates Cricket long-Term Oviposition 65 Days after t r e a t ment Fig. 3. Effect of PGE2 or mating on house cricket oviposition. Four- to five-day old adult females were mated (0,n = 18) or were injected with 100 p g of PGE2in 5 pI of 20 mM sodium phosphate, pH 7.4, through an intersegmental membrane of the ventral abdomen ( 0 ,n = 20). Data represent the mean and standard error. Results over the first four days were the same for both groups. laying would be the simplest method to link oviposition with fertilization. To examine the role of sperm or factors associated with sperm in the matinginduced stimulation of egg laying, X-irradiation was used to deplete males of sperm. X-irradiation damages chromosomal DNA and interferes with meiosis and mitosis. Thus developing sperm fail to differentiate, and mature spermatozoa are incapable of fertilization. In these experiments, it was important that treatment with X-irradiation specifically block spermatogenesis without affecting PG biosynthesis. To determine whether X-irradiation affected PG synthesis in reproductive tissues, males were irradiated as nymphs and PG levels were determined in the testes and spermatophores of adults at various intervals. The intensity of irradiation, 1,200 rads, was sufficient to block sperm development completely, as shown below but development of somatic tissue was unaffected. Treated animals molted successfully and survived as adults at the same rate as untreated siblings. The levels of PGE and PGF2, in testes of irradiated males were striking (Fig. 4).At the earliest time tested, PGE and PGF2, levels were greater than those in untreated controls. At later times, levels of both PGs were even higher. The data shown in Figure 4 are expresssed on a per individual basis. Treated testes lack sperm and were significantly smaller, as indicated by protein determinations (Fig. 4, inset). Thus the PG concentrations were even greater on a mass basis. PGE levels in spermatophores of irradiated males were about the same (10-30 pglspermatophore) as in unirradiated males. PGF2,, which was not detected in spermatophores of normal males, was abundant in irradiated males (10-100 pglspermatophore). The high concentration of PGs observed in both testes and spermatophores of 66 . Murtaugh and Denlinger 2400- br 0 2000 ((I 3 'P 1600> 10 I4 C (3 a. 21 18 16 adult age '0 1200- a 800 - 400 - 0- - 10 0 Y 14 18 22 26 Adult age at analysis Fig. 4. Effect of X-irradiation on PG levels in testes. Males were irradiated as nymphs 30-41 days before PG measurement. Each data point i s based on an assay of eight to 18 individuals. PGE (M) and PGF, (0) levels in testes of untreated 23-day-old males are shown for comparison. Inset: protein content of the testes in irradiated ( @ ) and untreated (0)males. The samples used for protein determinations were taken from the pooled samples used for PC measurements. irradiated males indicated that the enzymes involved in PC biosynthesis were present and were active. Disruption of normal chromosome function and sperm inactivation was assessed by the inability of sperm from irradiated adult males to fertilize eggs. The data in Figure 5 show that increasing doses of X-rays up to 4,000 rads caused infertility in males. Figure 5 also shows that the process of irradiation itself had no effect on the ability of males to induce oviposition (closed circles). Infertility was not due to the absence of sperm; inspection of spermathecae from females at the end of the experiment showed that in every case sperm were present. The tissue damage induced by X-irradiation was specific for the reproductive system in males and females. Treatment of juveniles with 1,200 rads Xirradiation completely blocked reproductive development, but treated animals continued to develop and molt. They reached adulthood as rapidly as untreated siblings, and their adult longevity was uninpaired. Yet, if irradiated 10 days before adult eclosion, the development of reproductive tissue was severely retarded. In irradiated females, 12-13 days after the final molt, the median ovarian wet weight was 4.5 mg (n=10) and no eggs were found. In untreated siblings, median weight was 156 mg (n=10), and ovaries were filled with eggs. In males, irradiation of juveniles caused lack of sperm production as adults and testes were reduced in size. However, as adults, Sperm Regulates Cricket long-Term Oviposition 67 I Y v) x m -0 u) (J, (J, W Fig. 5. Effect of X-irradiation of males on oviposition and fertility in females to which they were mated. Males were irradiated as described in Materials and Methods at 1-4 days of adult age. After 7 days, males were allowed to mate with IS-23-day-old females. Each point represents t h e mean number of eggs laid per female in 8 days ( 0 )or percent egg fertility (0) for 6-8 females. these animals manufactured spermatophores and transferred them successfully to females. Irradiation at earlier development times, before spermatozoa were present in the testes, blocked sperm development. Thus irradiation effectively froze sperm development so that no further differentiation would take place. Males irradiated 11-12 days before eclosion with 1,200 rads produced spermatophores with sperm as 10-11-day-old adults, but testicular growth was greatly retarded: Median wet weight of these testes was 18.5 mg (n=lO), whereas that in a group of siblings reared under identical conditions was 43.5 mg (n=10). Increasing the dosage of X-rays administered to penultimateinstar nymphs increased the percentage of mature males lacking sperm. When these males mated with females, the effects on oviposition depended dramatically on whether sperm were present (Fig. 6). At 400 rads, 72% of surviving males transferred sperm to females. In each case, long-term oviposition was stimulated. At 800 rads, only 18% of males transferred sperm, but again in these cases oviposition was stimulated. At 1,600 rads, no males transferred sperm. At every dosage level, males that failed to transfer sperm failed to elicit an ovipositional response even though spermatophores were attached, as determined in all cases by visual observation. Treating juvenile crickets at midinstar ages with X-irradiation produced mature males whose spermatophores were usually devoid of sperm. Following mating with these males, females failed to oviposit (Fig. 7, cross-hatched bars). Occasionally, sperm were transferred (determined by examination of the spermathecal contents at the end of the experiment), and in these cases 68 Murtaugh and Denlinger sperm t r m r f e r r e d El sperm not transferred n:5 L 400 800 ii 1600 X-ray dose to male (rads) Fig. 6 . Effect of various doses of X-rays on sperm transfer and oviposition in the house cricket. Last-instar male nymphs were irradiated, and 41 days later (as 30-38-day-old adults) surviving males were each given two to five females. After mating, eggs were collected for 7 days. Data represent the mean number of eggs laid per day per female. The numbers displayed above each bar are the number of mated females that did (stippled bars) or did not (open bars) receive sperm. No males given 1,600 rads of X-rays transferred sperm at mating. egg laying was induced (Fig. 7, open bars). However, in these animals, the rate of oviposition declined steadily during the 10 day period, in contrast to that of females mated to untreated males (Fig. 7, solid bars). It appeared from these results that an intact PG synthesis system was not a sufficient stimulus to provoke oviposition in mated females but that the presence of sperm, even nonviable sperm, was required to induce oviposition. The last experiment was to determine if the presence of sperm was necessary to stimulate the delayed oviposition response of young females. Therefore, 2-4-day-old females were mated to X-irradiated or untreated males and provided with an oviposition substrate for 20 days. Females that received sperm from normal or irradiated males (Fig. 8) exhibited an elevated rate of egg deposition 8 days after mating, which persisted to the end of the experiment. The data for those mating to untreated or irradiated males was combined since the numbers were indistinguishable. Mated females that failed to receive sperm as determined by examination of spermathecal contents laid eggs at the same rate as unrnated control crickets (Fig. 8, open circles and open squares). DISCUSSION In crickets, especially A. domesticus and Teleogryllus cornrnodus, female egg laying is greatly stimulated by spermatophore contents derived from the Sperm Regulates Cricket long-Term Oviposition 69 100- 2 80m -0 0 2 . 5 . c 60 a, m 40 *- v) u, u, w 20 2 4 8 6 Days after 10 mating Fig. 7. Effect of X-irradiation on sperm transfer and oviposition in house crickets. Males 1316 days old received 1,200 rads of X-irradiation 48 days before mating to individual females. Of 25 females that accepted spermatophores, four had sperm in the spermatheca (open bars) and 21 did not (cross-hatched bars). Five females mated to untreated siblings had sperm (solid bars). Data represent means and standard errors. 4 8 1: Days after mating Fig. 8. Oviposition by female house crickets after mating at an early age with X-irradiated males. Two- to 4-day-old females were mated to X-irradiated or untreated males 10-13 days old or were not mated. Closed circles represent the means of seven mated females that received sperm from five normal and from two X-irradiated males. Open circles represent means of 22 mated females that did not receive sperm. Open squares represent means of seven unmated females. 70 Murtaugh and Denlinger male testis [12,13]. This observation, combined with the finding of PGs and PG synthesis enzymes in testes and spermatophores [4,5,7,11], has led to the suggestion that PGE2, or the enzymes responsible for its synthesis, are the male-derived oviposition stimulus. Additional experiments, including the inhibition of egg deposition in crickets by inhibitors of PG synthesis , and the induction of egg laying in crickets by the injection of PGE2 ,supported this hypothesis. However, the episodes of egg laying involved in these experiments were of short duration, typically lasting about 24 h. We were interested in determining if the same stimulus was responsible for inducing the prolonged period of egg deposition lasting for a period of weeks that commences after A. domesticus females mate . PG synthesis inhibitors were used to evaluate the role of PGE2 and PGF2, in the induction of long-term oviposition. Aspirin and acetaminophen irreversibly inhibit PG endoperoxide synthase [14-17'J and prevent large postmating increases in PGE and PGF2, levels in the spermatheca [q. Administration of these inhibitors to males blocked PG synthesis in the testis  and, presumably, results in the packaging of inactive synthetase in spermatophores. Nevertheless, these inhibitors, when given to males, do not block mating-induced oviposition. We were unable to detect any effect of inhibitors on oviposition when they were administered to males. Both young females and older, ovipositionally competent, females displayed normal egg laying patterns after mating with inhibitor-treated males. Thus a contribution of PGs or active PG synthesis enzymes from the male was not required to stimulate oviposition. In females, PG synthesis inhibitors acted in a more complex manner. Egg laying by mated females initially was suppressed in animals given diet containing inhibitors, but, after several days without inhibitor egg laying activity began. From these results, it appears that PG synthesis in the female is important in the ovipositional process. Combined with the results from inhibiting PG synthesis in the male, these observations indicate that femalederived PGs play a physiological role in the egg laying process. If PGs are required for the ovipositional event itself, then these experiments cannot address questions about earlier events associated with induction of long-term ovipositional responses. If PG synthesis in the female reproductive tract is required for induction of the long-term response, then these experiments indicate that the male-derived factor functions by activating or inducing female PG synthesis enzymes. These data are interesting in light of previous observations that an intact spermatheca with intact neural connections is required for egg laying .Thus oviposition at some point involves interaction with the nervous system. It is also clear from the experiments with young females that the induction of egg laying involves much more than increasing PG levels in female reproductive tissues. These animals have mature eggs 131, are mated, and have high levels of PG (Table 1)yet will not lay eggs for another 5-7 days [ 3 ] . In a variety of insect species, sperm have been implicated as an inducer of egg laying [18-221. Our studies indicate that sperm or factors associated with sperm induce egg laying in the house cricket. In contrast to the lack of effect of PG synthesis inhibitors on male-induced egg laying, elimination of sperm Sperm Regulates Cricket Long-Term Oviposition 71 from testes and spermatophores rendered males incapable of stimulating oviposition. Removal of sperm from the insemination mixture was accomplished by irradiating adult or immature males with X-rays. The effect of Xirradiation to abolish the egg laying stimulus was due specifically to the removal of sperm, since the application of even very high doses of X-rays to males did not interfere with the induction of oviposition if sperm were transferred during mating (Fig. 5). Furthermore, treatment of males with Xrays did not impair PG synthesis. In fact, it appeared to enhance PG synthesis; irradiated males had extremely high levels of both PGE and PGF2, in testicular tissue and in spermatophores. Ionizing radiation has been associated with increased PG synthesis , an effect that may be due to the inhibition of thromboxane A2 biosynthesis with resultant overproduction of other PGs 1241. Thus, in irradiated males, enzyme complexes involved in PG biosynthesis were active. Previous studies showed that mating provided a continuous stimulus for oviposition which could be titered out by removing the spermatophore at short time periods after transfer . The data in Figure 6, showing that egg laying activity and sperm production diminished as X-ray dosage increased, also indicate that the presence of sperm is required for egg laying activity. Likewise, the transfer of small numbers of sperm would be expected to stimulate a burst of ovipositional activity that would decline as sperm were depleted, as is shown in Figure 7. The results of these experiments strongly suggest that sperm contain the information that induces prolonged ovipositional activity in mated females. 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