Germ cell death in the testis and its relation to spermatogenesis in the wax moth, Galleria mellonella (Lepidoptera: Pyralidae), effects of facultative diapause

The dichotomous spermatogenesis of many Lepidopterans results in the production of two types of sperm: eupyrene sperm possessing a cell nucleus which participates in fertilisation, and apyrene ones, which lose their nuclei during development and whose function remains a mystery. The goal of our study was to analyse spermatogenesis at the end of the larval development of the wax moth, Galleria mellonella, at an optimal temperature of 30 degrees C as well as to describe how they are affected by diapause brought on by a reduction of temperature to 18 degrees C. Spermatogenesis in non-diapausing insects did not differ significantly from that described in other species of Lepidoptera, and any differences found were compared against available literature. Based on the results presented, it may be unequivocally stated that changes in spermatogenesis occur in diapause caused by a suboptimal temperature of 18 degrees C. The main effect of diapause observed in the testes is the degeneration of germ cells, immediately following their differentiation from bipotential spermatocytes. Eupyrene cells seem to reach a more advanced stage of development. Due to the absence of secondary eupyrene spermatocytes in the testis of diapausing insects, it may be surmised that the meiotic divisions, which lead to the formation of secondary spermatocytes and eventually spermatids, do not occur, or are somehow altered. Lastly, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) analyses we performed show that the degenerative changes of eupyrene cells are apoptotic in character.     

Repeated cycles of chilling and warming effectively terminate prolonged larval diapause in the chestnut weevil, Curculio sikkimensis

Curculio sikkimensis (Coleoptera: Curculionidae) requires one or more years to complete its life cycle, owing to prolonged larval diapause. To compare the effects of temperature cycles and total periods of chilling on the termination of prolonged diapause, larvae were subjected to different chilling (5 degrees C) and warming (20 degrees C) cycles ranging from 30 to 720 days, and all cycles were repeated until the sum of chilling and warming periods reached 720 days. The prolonged diapause of C. sikkimensis was more effectively terminated by repeated cycles of chilling and warming than by prolonging the continuous chilling period. However, extremely short temperature cycles were not highly effective in enhancing diapause termination, even when such cycles were repeated many times. To examine the role of warming periods on diapause termination, diapause larvae were subjected to a sequence of chilling (120 days at 5 degrees C) and warming (240 days at 20 degrees C) with a warming period (0-120 days at 20 degrees C) inserted in the chilling period. Diapause larvae that were not reactivated in the first chilling period required exposure to a certain period of warming before they were able to complete diapause development in the subsequent chilling. Thus, C. sikkimensis appears to spread its reactivation times over several years in response to seasonal temperature cycles.     

Movement of spermatozoa in the reproductive tract of adult male Spodoptera litura: daily rhythm of sperm descent and the effect of light regime on male reproduction

Sperm production and movement from the fused testes into the male reproductive tract of the common cutworm Spodoptera litura were studied in insects maintained in a 12 h:12 h light dark (LD) regime. Two types of sperm bundles, eupyrene (nucleated) and apyrene (anucleate) were present in the adult testes. Eupyrene bundles constituted about 25% of the total. Descent of spermatozoa from the testes into the upper vas deferens (UVD) first occurred about 24-30 h before adult eclosion. On entering the reproductive tract, eupyrene spermatozoa remained in bundles while apyrene bundles became dissociated before they reached the UVD. Downward movement of both eupyrene and apyrene spermatozoa within the male tract occurred in a daily rhythm. Sperm descent from the testes into the UVD occurred during the early scotophase, followed by their further descent into the seminal vesicle (SV) during the photophase. Spermatozoa remained in the SV for only a short duration, whence sperm quickly passed through the lower vas deferens into the duplex, which acted as the main sperm storage organ until mating was initiated. During mating 80% of sperm left the duplex, but mating did not influence the number of sperm bundles that subsequently descended into the duplex or the rate of their descent. There was no evidence of sperm reflux. Rearing in constant light (LL) and in constant dark (DD) reduced the number of eupyrene sperm present in the testes of adults that emerged in LL and DD compared to controls (LD), although there was no significant effect on the number of apyrene sperm in the testes. The rhythmic pattern of sperm descent was suppressed in both LL and DD regimes, and the number of sperm in the duplex was adversely affected, with a marked impact in LL reared insects. Male longevity, mating behaviour, oviposition and fertility were found to be more severely affected in LL than in DD.     

Overwintering success in adults of the Japanese common grass yellow Eurema mandarina

The evolution of reproductive diapause is controversial in males as compared to females that must overwinter to leave offspring, because late‐autumn males can obtain offspring by pre‐overwintering copulation. The Japanese common grass yellow Eurema mandarina is suitable to examine the evolution of male reproductive diapause, because direct comparisons are possible between males that do and do not exhibit reproductive diapause. Approximately one‐half of males are insensitive to diapause‐inducing conditions, and emerge as non‐diapause summer‐form. Most autumn‐form females mate with summer‐form males in late autumn. Females that have overwintered re‐mate with autumn‐form males before the onset of oviposition. Because last‐male‐precedence is general in sperm competition in Lepidoptera, it is unclear why half of males emerge as summer‐form in late autumn. A potential adaptive benefit for emerging as summer‐form is increased sperm overwintering success, if autumn‐form females have a higher overwintering success than autumn‐form males. In the present study, overwintering success was estimated for both sexes of autumn‐form adults by rearing under seminatural conditions and a mark–release–recapture technique. Both approaches estimated an overwintering success of approximately 5% for both sexes. The absence of difference in overwintering success between the sexes suggests that pre‐overwintering copulation does not increase sperm overwintering success. However, a considerably low overwintering success may explain, at least partly, the presence of summer‐form males in late autumn. The degree of overwintering success might be more important than the sexual differences of overwintering success in the evolution of male reproductive diapause.     

Effects of temperature during chilling and pre-chilling periods on diapause and post-diapause development in a katydid, Eobiana engelhardti subtropica

In Eobiana engelhardti subtropica, early laid eggs reach the diapause stage in early autumn. For long periods before winter, the eggs are exposed to temperatures higher than their theoretical lower threshold for development. In contrast, late-laid eggs cannot reach their diapause stage before winter. Our study showed that E. e. subtropica copes with these difficulties via the thermal response involving embryonic diapause. In this katydid, the almost fully developed embryo undergoes an obligatory diapause. When diapause eggs were maintained at a temperature of 20 degrees C or higher, diapause persisted for a long time. Diapause was effectively terminated by temperatures ranging from 1 to 11 degrees C, and hatching occurred successfully at temperatures from 11 to 15 degrees C. In addition to the chilling temperature, pre-chilling temperature modified diapause intensity and hatching time. Diapause eggs hatched earlier after chilling when the pre-chilling temperature was lower, within a range of 14.5-25 degrees C. Thus, the low-temperature requirement for diapause termination prevents early laid eggs from untimely hatching in autumn, and low temperatures before and during winter decrease diapause intensity and shorten the hatching time in the following spring. When eggs were chilled before diapause, they tolerated chilling and averted diapause. Thus, even if eggs encounter low temperatures before diapause, they can hatch in the following spring.     

Sperm reflux and its role in multiple mating in males of a butterfly Polygonia c-aureum Linnaeus (Lepidoptera: Nymphalidae)

The relationship between sperm quantity in the duplex and that in the vasa deferentia was examined in the Asian comma butterfly, Polygonia c-aureum. In virgin males, the number of eupyrene sperm bundles in the duplex increased linearly with age, whereas that in the vasa deferentia was consistently small. However, numerous sperm were found in the vasa deferentia of males immediately after mating. The number of eupyrene sperm bundles in the vasa deferentia after mating significantly increased with age and with increasing the time interval between matings. From these and other results, it was suggested that some sperm in the duplex were moved back to the vasa deferentia during mating, and that such sperm reflux provides a means to save sperm for multiple mating.     

Spermatogenesis in the testes of diapause and non-diapause pupae of the sweet potato hornworm, Agrius convolvuli (L.) (Lepidoptera: Sphingidae)

Dichotomous spermatogenesis was examined in relation to diapause in the sweet potato hornworm, Agrius convolvuli. In non-diapause individuals, eupyrene metaphase began during the fifth larval instar and eupyrene spermatids appeared in wandering larvae. Bundles of mature sperm were found after pupation. Apyrene spermatocytes also appeared during the fifth larval instar, but meiotic divisions occurred irregularly and their nuclei were discarded from the cells during spermiogenesis. Morphometric analyses of flagellar axonemes showed a variable sperm number in apyrene bundles. The variation ranging from 125 to 256 sperm per bundle indicated abnormal divisions or the elimination of apyrene spermatocytes. In diapause-induced hornworms, spermatogenesis progressed similarly during the larval stages. The cessation of spermatogenesis during diapause is characterized by 1) secondary spermatocytes and sperm bundles degenerating gradually as the diapause period lengthens, and 2) spermatogonia or primary spermatocytes appearing throughout diapause. A TUNEL (TdT-mediated dUTP-biotin nick end-labeling) assay revealed that DNA fragmentation occurred in the nuclei of secondary spermatocytes and early spermatids. Aggregates of heterochromatin along the nuclear membrane indicated the onset of apoptosis, and condensed chromatin was confirmed by electron microscopy to be the apoptotic body. These results show that the degenerative changes in spermatogenic cells during pupal diapause were controlled by apoptosis.     

Temporal shift between daily sperm movement and mating (sperm reflux) in the Asian comma butterfly,Polygonia c-aureum

Temporal changes in mating behavior and daily sperm movement were examined in adult males of the butterfly Polygonia c-aureum L. (Lepidoptera: Nymphalidae), reared under L15:D9 photoperiod at 21 °C. In this butterfly, when extra sperm is present in the duplex (storage organ) at mating, sperm reflux occurs: extra sperm are moved from the duplex to the lower and middle portions of the vasa deferentia by peristaltic movement of the lower portion of the vasa deferentia. It is known in several moth species that daily sperm movement from the testis to the upper portion of the vasa deferentia and from the upper vasa deferentia to the duplex occurs in a circadian manner. If sperm reflux and daily sperm movement occur at the same time, it could create confusion, because these sperm movements are directed oppositely. In this study, we examined the temporal changes of sperm movement and mating behavior in P. c-aureum in order to clarify whether the two sperm movements are separated in time. Our results showed that most matings occurred during the second half of the photophase, whereas daily eupyrene (nucleate) sperm movement from the upper to the middle portion of the vasa deferentia occurred immediately after light on (early photophase), indicating the presence of a time lag between mating and daily sperm movement. As sperm reflux occurs during mating, these two oppositely directed sperm movements could be performed without conflict.     

Effect of anoxia on diapause termination in eggs of the false melon beetle, Atrachya menetriesi

The effect of anoxia on diapause development in the leaf beetle Atrachya menetriesi was investigated to elucidate the role of oxygen in regulation of egg diapause. While anoxia alone had no effect on diapause termination, it decreased diapause intensity before chilling. Such an effect reached a maximum level when anoxia lasted for about 10 days. Anoxia applied during the pre-diapause stage also reduced diapause intensity. On the other hand, anoxia terminated diapause when the diapause intensity had been lowered by sufficient duration of chilling (50 days at 7.5 degrees C). The effect of anoxia was temperature dependent; the larger effect was elicited when anoxia was combined with a higher temperature. A 50-day chilling caused more than 20% of eggs to terminate diapause upon transfer to warm conditions. However, when this chilling period was interrupted on the 20th day by a 5-day exposure to a high temperature of 20, 25 or 30 degrees C, the effect of the former chilling was cancelled partially or completely, suggesting that warming reversed diapause development. This reversing effect of a high temperature, however, was not manifested when the warming was combined with anoxia. The results suggest that anoxia inhibits diapause reversal and facilitates a certain process of diapause development. The sequence of exposure to anoxia and chilling is not important.     

Effect of mating on survival at low temperature in females of the Japanese common grass yellow,Eurema mandarina

1. In some insects that overwinter as adults, mating occurs both before and after overwintering. Two hypotheses have been proposed to explain the adaptive significance of pre‐overwintering copulation of females. One is the bet‐hedging hypothesis, which explains pre‐overwintering copulation as a preparation for less chance of mating in the following spring. The other is the nuptial gift hypothesis, which states that secretions derived from males increase overwintering success of females. 2. In Eurema mandarina, both diapause autumn‐ and non‐diapause summer‐form male adults emerge with autumn‐form female adults in the last generation in a year. Most autumn‐form females mate with summer‐form males before winter, and re‐mate with autumn‐form males in the following spring. Because autumn‐form females have sufficient chances for mating after overwintering, the nuptial gift hypothesis has been regarded as the more probable hypothesis. 3. To test the nuptial gift hypothesis, the survival period was compared under short‐day conditions at 10 °C between mated and unmated females that had been reared on sucrose solution at 25 °C for 15–21 days. The mated females had significantly greater longevity than the unmated females, supporting the nuptial gift hypothesis. Body size also affected the survival period. 4. The results suggest that the nuptial gift is an important factor for the evolution of pre‐overwintering copulation in species in which females mate both before and after overwintering.     

Effects of 20-hydroxyecdysone on sperm release from the testes of the Mediterranean flour moth,Anagasta kuehniella (Zeller)

Abdominal injection of 1 μg aqueous 20-hydroxyecdysone into Anagasta kuehniella, anytime prior to the initiation of sperm release from the testes, prevents the impending release of eupyrene sperm bundles. Apyrene sperm release is not prevented and there is complete recovery of eupyrene release by the following cycle 24 hr later. If 20-hydroxyecdysone is administered on consecutive days, no eupyrene bundles are released and although apryene sperm release continues, it diminishes with time. The effect of 20-hydroxyecdysone in preventing eupyrene release is dose dependent. Administration of decreasing 20-hydroxyecdysone dosages results in increasing numbers of eupyrene bundles released. When a single injection of 20-hydroxyecdysone is administered to isolated abdomens, recovery time of eupyrene sperm release is slower than in whole moths and total recovery is not seen even by 5 days after administration. Apyrene sperm release is also affected to a greater extent than in whole moths, and in some cases, no apyrene release was detected at all. Treatment with 20-hydroxyecdysone prevents cupyrene bundles from passing through the testicular basilar membrane into the vasa efferentia, thus causing a build up of bundles near the basilar membrane but no disintegration of these eupyrene sperm bundles.     

Effects of photoperiod and temperature on seasonal morph development and diapause egg oviposition in a bivoltine race (Daizo) of the silkmoth,Bombyx mori L

A bivoltine race (Daizo) of the silkmoth, Bombyx mori, has two types of adults which are classified on the basis of the number of dark-brown and brown scales distributed along the outer lines of the anterior wings. Development of these two types of adults is determined by photoperiod and temperature experienced during the embryonic and larval stages. The two types of adults are thought to be seasonal (summer and autumn) morphs. All adults developed under short days at low temperature (15 degrees C and 23 degrees ) were classified as summer morphs and oviposited only nondiapause eggs (ND-eggs), and those that developed under long days at high temperature (28 degrees C and 25 degrees C) were autumn morphs ovipositing only diapause eggs (D-eggs), only ND-eggs or a mixture of D- and ND-eggs. Larval exposure to long days at 25 degrees C shifted the wing pattern towards the autumn morphs and decreased the incidence of female adults ovipositing only D-eggs. Larval exposure to long days elicits an opposite effect on D-egg oviposition to that observed when long days are received only during the embryonic stage.     

Demarcation of diapause development by cold and its relation to time-interval activation of TIME-ATPase in eggs of the silkworm, Bombyx mori

We investigated the mode of action of winter cold in the termination of diapause by investigating Time-Interval-Measuring Enzyme (TIME). First, we determined the period of cold required for the completion of diapause development. Synchronously developing egg batches of a pure strain (C108 Bombyx mori silkworm) were used to minimize variations in hatching time. Hatching occurred with only 18 days chilling at 5 degrees C when the incubation at 25 degrees C after the chilling was elongated. The 18-day period was much shorter than we expected; diapause in B. mori is known to terminate completely with about 100 days of chilling. Even in such a short period of chilling, no sporadic hatching occurred. Moreover, we determined that a temperature-insensitive stage, which we called "Neboke", followed the short cold-requiring stage. Thus, the stage of diapause development was demarcated from other stages of diapause. While the length of diapause development was elongated when chilling was delayed after oviposition, the Neboke stage length was invariant. Cold evidently exerts its effect only on diapause development. When TIME was purified from eggs and chilled in test tubes, a transitory burst of its ATPase activity occurred at a time equivalent to shortly before the completion of diapause development; this was an interval-timer activation. The mechanism by which cold activates TIME to measure the time interval may help explain in biochemical terms the insect's adaptation to its seasonal environments.     

In vitro cultivation of spermatocysts to matured sperm in the silkworm Bombyx mori

Bombyx spermatogonia are bipotential, producing nucleate eupyrene sperm and anucleate apyrene sperm. An in vitro cultivation of spermatocysts of Bombyx mori from spermatocytes to matured sperm was established. The present experiment made clear that: (i) spermatocysts must be isolated; (ii) constant shaking at 45 r.p.m. was necessary; and (iii) the addition of Bombyx hemolymph (BH) was indispensable for successful cultivation. In the absence of BH, spermatogenesis proceeded normally for 2 or 3 days and, thereafter, spermatocytes and sperm bundles began to degenerate. The best results for normal eupyrene spermatogenesis were obtained when culture medium containing BH of the corresponding stage was used in every exchange of the medium at 72 h intervals. None or only a small number of apyrene sperm bundles was produced by this culture system when spermatocysts from larval testes were used, although eupyrene spermatogenesis proceeded normally to form matured, or squeezed, sperm bundles.     

Low temperature storage of larvae and synchronization of adult emergence in the predatory midge Aphidoletes aphidimyza.

Diapause larvae of Aphidoletes aphidimyza were stored at a temperature of 3 degrees C under continuous darkness for up to 7 months with survival rates above 50%; after storage for 1 year the survival rate dropped to 12%. Diapause was terminated in the majority of individuals within 120 days of chilling under storage conditions. Brief exposure (10-60 s) to the vapor of n-hexane appeared to be a useful alternative to chilling for the termination of diapause. The larvae with terminated diapause required, on average, an additional 31 days at 22 degrees C and long-day conditions in order to reach the adult stage. The 10-90% adult emergence spanned a period of 21.1 days. When the larvae with terminated diapause were exposed to 30 degrees C for 1 week after the end of low temperature storage, the survival rate was not affected, the average "time-to-adult" shortened moderately to 28 days, and the synchrony of adult emergence improved considerably to 10 days. Low temperature storage of nondiapause larvae resulted in a decrease in survival from 98 to 31% during the first 60 days of storage. Nondiapause larvae did not enter diapause during low temperature storage and, as a consequence, the adults emerged relatively rapidly (after 14-15 days) and synchronously (within 2-3 days) after the end of storage. Directions for future research, which might bring further improvement in low temperature storability and synchrony of adult emergence in A. aphidimyza, are proposed.     

Mating prior to overwintering in the imported willow leaf beetle, Plagiodera versicolora (Coleoptera: Chrysomelidae)

Abstract. 1. We report on the mating status of overwintering adults of the imported leaf beetle, Plagiodera versicolora Laicharting. This species overwinters as adults, under the bark of willow trees.
2. Individuals were collected both in the autumn, just after initiation of diapause, and in the spring, just before natural termination of diapause. From the autumn sample, we discovered that diapause can be terminated by husbanding the adults at 4°C for 4 weeks.
3. Most females lay fertile eggs upon the termination of diapause; however, a significant number of females lay non-fertile eggs after overwintering in the field.
4. Many females mate with more than one male before entering diapause. Progeny analysis using allozyme genetic markers shows that, on average, females who are fertile upon termination of diapause utilize sperm from 1.2 males to fertilize their eggs.     

Morphological changes in eupyrene and apyrene spermatozoa in the reproductive tract of the male butterfly Atrophaneura alcinous Klug

Summary

Eupyrene and apyrene spermatozoa are contained in separate cysts in the testis of the butterfly Atrophaneura alcinous. Spermatozoa of both types from various parts of the male reproductive tract were examined with particular reference to their morphological characteristics. All spermatozoa collected from the vas deferens and the vesicula seminalis were found to be immotile under a dissecting microscope. No spermatozoa of either type were recognized in any part of the ejaculatory duct. Within the testis, eupyrene spermatozoa are present in bundles and each spermatozoon has a slender nucleus with an acrosome and a long flagellum containing mitochondrial derivatives. Two kinds of appendages, lacinate and reticular, are present on the surface of the sperm membrane. They are replaced with an extracellular sheath during passage through the vas deferens. In contrast, apyrene spermatozoa have neither nucleus nor acrosome, whereas a cup-shaped structure was found at the sperm tip instead of the acrosome. Unlike eupyrene spermatozoa, they are surrounded by a concentric sheath outside the sperm membrane in the vas deferens. Individual apyrene spermatozoa and coiled bundles of eupyrene spermatozoa were both found to accumulate in the vesicula seminalis before mating. These morphological changes during passage through the male reproductive tract suggests the occurrence of a kind of maturation and capacitation process reminiscent of mammalian spermatozoa.     

The control of ovarian development during adult diapause in the cabbage whitefly, Aleyrodes proletella

ABSTRACT. Field collections of overwintering cabbage whitefly, Aleyrodes proletella L. (Homoptera: Aleyrodidae) taken during the autumn and winter of 1982/83 and 1983/84 have shown that ovarian development proceeds at a low, but near uniform rate throughout diapause. Extensive fat body is recorded during the autumn and early winter of both periods, but this gradually diminishes during February and March. There does not appear to be a true refractory phase during the dormancy. A period of chilling primes the females for accelerated ovarian development in favourable conditions. When field collected females were transferred to light: dark 16:8 h (15°C) or L:D 12:12 h (15°C) in the laboratory, the subsequent rates of ovarian development were not significantly different, suggesting that temperature is the major regulator of the activation phase, although there was some evidence to suggest that an increase in photoperiod also has a role.     

Overwintering strategy in Pyrrhocoris apterus (Heteroptera): the relations between life-cycle, chill tolerance and physiological adjustments

Seasonal dynamics of ecophysiological parameters are described which are relevant to overwintering in field-collected adults of a Czech population of the red firebug, Pyrrhocoris apterus. Five life-cycle phases were distinguished using the duration of pre-oviposition period as a criterion: reproductive activity (spring-early summer), intensification of reproductive diapause (RD) (peak of summer), maintenance of RD (late summer-early autumn), termination of RD (late autumn-early winter), and low temperature quiescence (LTQ) (winter). The supercooling capacity and chill tolerance (c.t.) increased simultaneously with the termination of RD and all three processes were triggered/conditioned by autumnal decrease in ambient temperatures. Maximum supercooling capacity and c.t. 'outlived' the end of diapause and persisted throughout the LTQ state. The limits of c.t. were estimated as -15 degrees C/1-2 weeks for 50% survival. Ribitol, sorbitol, arabinitol, and mannitol were accumulated in the winter-sampled insects. Relatively low concentrations of polyols (dominating ribitol reached ca. 1% FW) indicate that they do not function as colligative cryoprotectants. However, because their seasonal occurrence coincided with the highest c.t., their non-colligative cryoprotectant effects would merit further study. Although the overwintering microhabitat of P. apterus is buffered, the temperatures may fall to -13 degrees C during exceptionally cold winters and thus, the parameters of c.t. seem to be just appropriately tuned to the local overwintering conditions.     

Effect of temperature on the termination of prolonged larval diapause in the chestnut weevil Curculio sikkimensis (Coleoptera: Curculionidae)

Adults of the chestnut weevil Curculio sikkimensis emerged over a 3-year period under laboratory and quasi-field conditions due to a prolonged diapause that occurred at the mature larval stage. Variable proportions of the larvae remained in diapause after a single cold (5 degrees C) treatment of 120 days. Extension of the chilling period to as long as 540 days did not increase the percentage of diapause termination, and excessively long chilling actually reduced the percentage. Chilling was not indispensable to the termination of larval diapause. Diapause intensity was very high and variable, and more than 1000 days at 20 degrees C was necessary to reactivate all diapause larvae. When the diapause larvae were exposed to cycles of low (5 degrees C for 120 days) and high (20 degrees C for 240 days) temperatures, the percentage of diapause termination reached 100% after two or three such cycles. Thus, the prolonged diapause of C. sikkimensis has characteristics similar to the common short winter diapause in other insects, but has unique characteristics that ensure polymodal reactivation over several years.