Sperm maturation screening and the effect of ecdysone on sperm development of silkworm Bombyx mori

There are two sperm morphs of silkworm, the nucleated spermatozoa (eupyrene) and anucleated spermatozoa (apyrene). Eupyrene sperm cannot complete fertilization successfully without the apyrene sperm. Here a modified rapid and efficient method for sperm identification was developed, after 10 s of fixation in paraformaldehyde and 30 s of 4′6-diamidino-2-phenylindole (DAPI) or propidium Iodide (PI) staining, the sperm bundles can be detected easily using a fluorescence microscope. Sperm maturation process of silkworm from the fifth instar larvae to the adult was described with the above method, the precise time of earliest elongate apyrene bundles was detected on day 2 of pre-pupation, with a ratio of 5% in total sperm bundles, after which the percentage of apyrene sperm bundles increased rapidly and attained a relatively stable ratio of 75% at the end of pupation and nearly 80% after eclosion. Delayed mating leads to apyrene sperm accumulation and damaged fertilization. Previous study showed that ecdysone can increase the frequency of apyrene sperm bundles in vitro. Here 20-hydroxyecdysone (20E) was injected into hemolymph of the 2-d-old fifth instar larvae, the worms entered into mounting period after three days injection, but no apyrene sperm bundles were induced unless day 2 of pre-pupation. Interestingly, maturation of eupyrene sperm bundles were accelerated, and the ratio of eupyrene sperm bundles increased and exhibited a dose-dependent effect after 20E injection, which indicated that the development of eupyrene sperm can be accelerated by ecdysone before pupation of silkworm in vivo. These results will provide new clues for lepidopteran pest control.     

Daily cycle of release of sperm from the testes of the Mediterranean flour moth

A daily rhythm of sperm release from the testes was found in young virgin male Mediterranean flour moths, Anagasta kühniella, maintained in a 12 hr light, 12 hr dark regimen. Passage of apyrene sperm from the testes began in the latter half of the light phase and that of eupyrene sperm somewhat later; an apparent peak of release occurred near the end of the light phase. A low level of sperm release continued through the night, but none occurred in the first half of the light phase. Maintenance of pupae and adults in continuous light or decapitation shortly after eclosiion greatly reduced the amount of eupyrene sperm released from the testes. Movement of sperm from the testes was not affected by continuous darkness or by severing the abdominal nerve cord.     

Effect of constant light on male sterility in the cotton leafworm Spodoptera littoralis

Females of the cotton leafworm (Spodoptera littoralis) reared in long day conditions (LD 16:8 h) and mated to males kept throughout the whole period of development in continuous light (LL) oviposit very small numbers of mostly sterile eggs. It was found that in control males reared from the first larval instar in long day conditions there was a large accumulation of euperene sperm bundles in their testes on day 1 after imaginal moult. On day 10 of adult life the number of the sperm bundles was very small. In males kept from the first instar in continuous light there was also high number of sperm bundles on day 1 after imaginal moult but it did not decrease significantly on day 10 as was observed in controls. Transfer of different developmental stages of S. littoralis from long day conditions to continuous light resulted in a big difference in the density of eupyrene sperm bundles in their testes. In control insects reared through the whole of their development in long day conditions there was a significant decrease in the density of eupyrene sperm bundles on day 10 of adult life. By contrast, in males in continuous light, regardless of their developmental stage when transferred, there was either no change in density of sperm bundles in day 10 adults or there was a significant increase in comparison with day 1 adults. The highest density of eupyrene sperm bundles was observed in day 10 adults when they were transferred to continuous light shortly before moulting to the last instar (as day 4 larvae in the last stadium or day 1 pupae). Generally, the density of eupyrene sperm bundles on day 10 of adult development was about 2–2.5 times higher in males in continuous light then those under long day conditions. The results presented here indicate that the last larval instar and the pupa are the stages most sensitive to constant light treatment, which greatly reduces the amount of eupyrene sperm bundles released from the testes.     

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.     

Motility and dynamics of eupyrene and apyrene sperm in the spermatheca of the monandrous swallowtail butterfly Byasa alcinous

Lepidopteran males produce two sperm types: nucleated eupyrene sperm and non‐nucleated apyrene sperm. Although apyrene sperm are infertile, both sperm types migrate from the spermatophore to the spermathecal after copulation. As a dominant adaptive explanation for migration of apyrene sperm in polyandrous species, the cheap filler hypothesis suggests that the presence of a large number of motile apyrene sperm in the spermatheca reduces female receptivity to re‐mating. However, apyrene sperm are also produced in males of the monandrous swallowtail butterfly Byasa alcinous Klug. To identify the role of apyrene sperm in these males, the present study examines the number of spermatozoa produced and transferred and the dynamics and motility of spermatozoa in the spermatheca for each type of sperm. Apyrene sperm represents approximatey 89% of the sperm produced and transferred, which is comparable to polyandrous species. Two‐day‐old males transfer approximately 17 000 eupyrene and 230 000 apyrene spermatozoa to a spermatophore; approximately 5000 eupyrene and 47 000 apyrene spermatozoa arrive at the spermatheca. Eight days after copulation, most eupyrene spermatozoa remain in the spermatheca and a quarter of them are still active. However, the number of apyrene spermatozoa decreases and those remaining lose their motility after the arriving at the spermatheca. Consequently, 8 days after copulation, no motile apyrene sperm are found. The high proportion of apyrene sperm in the spermatophore, as well as in sperm migration, suggests that the production and migration of apyrene sperm is not simply an evolutionary vestigial trait. The possible functions of apyrene sperm in monandrous species are discussed.     

Abdominally entrained periodicities of testis and vas deferens activity in the Mediterranean flour moth

In a light-dark (LD) regimen, sperm, first apyrene and then eupyrene, start moving out of the fused testes of the Mediterranean flour moth, Anagasta kuehniella, toward the beginning of the scotophase. At 27° ± 2°C, the sperm mass remains in the proximal part of the vasa deferentia for 10 to 12 hr and then passes rapidly into the seminal vesicles, remains in these organs for about 5 hr, and is then transported to the ductus ejaculatoris duplex where it becomes available for ejaculation. The phases of sperm movement appear to be closely related to sperm development, and the reproductive activity of the moths. In isolated abdomens there is a significant reduction in the amount of sperm released from the testes, but normal periodicity of sperm release and movement continues in either LD or continuous dark (DD) regimens, and rapid phase shifting occurs when a LD regimen is reversed. All stages of sperm movement are disrupted in continuous light (LL), but normal periodicity is usually resumed when isolated abdomens of the LL moths are placed in LD or DD regimens. Normal periodicity also occurs in moths paralyzed with tetrodotoxin or procaine. Removal of any one of the four abdominal ganglia from LL moths does not prevent increased sperm release when the moths are placed in LD, though with each ganglion there is some disruption of the normal pattern of movement down the vasa deferentia. It is thought that the testes and vasa deferentia down to at least the seminal vesicles represent a semiautonomous complex in which periodicity is maintained by endogenous circadian activity in cells of the testes (and possibly the vasa deferentia) or more probably in a peripheral control center.     

BmPMFBP1 regulates the development of eupyrene sperm in the silkworm,Bombyx mori

Sperm deliver the male complement of DNA to the ovum, and thus play a key role in sexual reproduction. Accordingly, spermatogenesis has outstanding significance in fields as disparate as infertility treatments and pest-control, making it a broadly interesting and important focus for molecular genetics research in a wide range of species. Here we investigate spermatogenesis in the model lepidopteran insect Bombyx mori (silkworm moth), with particular focus on the gene PMFBP1 (polyamine modulated factor 1 binding protein 1). In humans and mouse, PMFBP1 is essential for spermatogenesis, and mutations of this gene are associated with acephalic spermatozoa, which cause infertility. We identified a B. mori gene labeled as “PMFBP1” in GenBank’s RefSeq database and sought to assess its role in spermatogenesis. Like in mammals, the silkworm version of this gene (BmPMFBP1) is specifically expressed in testes. We subsequently generated BmPMFBP1 mutants using a transgenic CRISPR/Cas9 system. Mutant males were sterile while the fertility of mutant females was comparable to wildtype females. In B. mori, spermatogenesis yields two types of sperm, the nucleated fertile eupyrene sperm, and anucleated unfertile apyrene sperm. Mutant males produced abnormal eupyrene sperm bundles but normal apyrene sperm bundles. For eupyrene sperm, nuclei were mislocated and disordered inside the bundles. We also found the BmPMFBP1 deficiency blocked the release of eupyrene sperm bundles from testes to ejaculatory seminalis. We found no obvious abnormalities in the production of apyrene sperm in mutant males, and double-matings with apyrene-deficient sex-lethal mutants rescued the ΔBmPMFBP1 infertility phenotype. These results indicate BmPMFBP1 functions only in eupyrene spermatogenesis, and highlight that distinct genes underlie the development of the two sperm morphs commonly found in Lepidoptera. Bioinformatic analyses suggest PMFBP1 may have evolved independently in lepidoptera and mammals, and that despite the shared name, are likely not homologous genes.     

The evolution of sperm length in moths

Sperm form and function remain poorly understood despite being of fundamental biological importance. An instructive approach has been to examine evolutionary associations across comparable taxa between sperm characters and other, potentially selective reproductive traits. We adopt this approach here in a comparative study examining how sperm lengths are associated with male and female reproductive characters across moths. Primary data have revealed Lepidoptera to be an ideal order for examination: there is profound variation in the dimensions (but not organization) of the reproductive traits between closely related species which all share a monophyletic ancestry, for example, eupyrene sperm length varies from 110 to 12,675 microm. Eupyrene (normal fertilizing) and apyrene (anucleate and non-fertile) sperm lengths are positively correlated across taxa and both sperm types show positive associations with mating pattern (as measured by the residual testis size). At fertilization, eupyrene sperm must migrate down the often elongated female spermathecal duct from storage to unite with the ovum. Across taxa, the elongation of this duct is associated with increased eupyrene sperm length, suggesting a positive female influence on sperm size since longer, more powerful sperm may be selected to migrate and/or compete successfully down greater ductal lengths. Apyrene sperm length is not associated with female reproductive tract dimensions. However, we found a positive relationship between the residual testis volume and spermathecal volume, suggesting coevolution between male investment in spermatogenesis and the extent of the female sperm storage capacity. Within males, there is a positive association between the two organs which form the ejaculate-containing spermatophore: the testes and the accessory gland. The 'trade-up' in investment to these components is discussed in relation to paternal investment and mating patterns.     

The circadian rhythm of sperm release in the codling moth, Cydia pomonella

The release of sperm bundles from testes to the vas deferens is controlled by a circadian clock in several moth species. We investigated the pattern of sperm release in the codling moth, Cydia pomonnella L. (Lepidoptera: Tortricidae). Sperm release in the codling moth follows a two-step rhythm in light-dark cycles: sperm is released from the testis before lights-off, remains in the vas deferens during the dark phase and is transferred to the seminal vesicles after lights-on. This rhythm continues in constant darkness indicating that it has a circadian nature. The release of sperm is asynchronous in moths held in constant light. In contrast to previously investigated moths, constant light has no adverse effects on the male reproductive capacity in the codling moth.     

Effects of Environmental Conditions and Aging on Eupyrene Sperm Movement in Male Adults of Polygonia c-aureum (Lepidoptera: Nymphalidae)

The effects of temperature, photoperiod and aging on eupyrene sperm movement from the testis to the duplex in Polygonia c-aureum male adults were examined in relation to seasonal form and imaginal diapause. In males of both summer and autumn forms obtained under long-day and short-day conditions, respectively, the number of eupyrene sperm bundles in the duplex increased linearly with age during the early stage of adult life at 21 degrees C, and no marked difference was observed between the two seasonal forms. Photoperiod during the adult stage did not influence the rate of sperm movement in autumn forms with diapause. The lower the temperature, the smaller the number of sperm bundles moved from the testis to the duplex. Sperm movement did not appear to occur during a period of chilling at 5 degrees C. Males of the autumn form which were pre-incubated for 30 days and chilled for 60 days failed to resume rapid sperm movement at the final incubation temperature of 21 degrees C. Those which were pre-incubated only for 15 days at 21 degrees C had much fewer sperm bundles in the duplex after chilling than those pre-incubated for 45 days. These results suggest the possibilities that eupyrene sperm movement is suppressed strongly during and after overwintering in the field, and that the eupyrene sperm to be used for reproduction in spring are transferred from the testis to the duplex before overwintering.     

Sperm transfer during mating, movement of sperm in the female reproductive tract, and sperm precedence in the common cutworm Spodoptera litura

Abstract. Mating behaviour, sperm transfer and sperm precedence were studied in the moth Spodoptera litura (Fabr.) (Lepidoptera: Noctuidae). There existed a rhythmic, diel pattern of mating behaviour of this moth during the scotophase, presumably set with respect to an endogenous activity rhythm. Approximately 30 min after copulation had started, the formation of the corpus of the spermatophore began in the bursa copulatrix of the female moth, but full inflation of the corpus was not completed until 45–60 min after mating had started. The mature spermatophore contained about 350 eupyrene sperm bundles and a large number of individual (loose) apyrene spermatozoa. The mating status and the age of the male insect influenced the number of sperm transferred to the female within the spermatophore, and also affected the consequent fertility. There was no evidence of sperm reflux within the male tract. Within the female, dissociation of eupyrene sperm bundles was evident within the spermatophore less than 15 min after the completion of mating. Spermatozoa began to move from the bursa (in which the spermatophore is lodged) into the spermatheca 30–45 min after the end of the copulation, and the quantity of sperm in the spermatheca reached a plateau at 90 min after mating. Apyrene sperm reached the spermatheca first, followed by eupyrene sperm. Examination of total (apyrene plus eupyrene) sperm in the female tract showed that 86% of mated females received an apparently normal amount of total sperm from the male. Examination of eupyrene sperm alone showed that 81% of matings resulted in an apparently normal transfer of eupyrene sperm. A small proportion (approximately 8%) of the matings, however, were identified as transferring a clearly subnormal quantity of eupyrene sperm to the spermatheca. The eggs produced as a result of such pairings displayed much reduced fertility (about 43%) compared to those from matings confirmed to have transferred normal quantities of sperm, which showed about 92% fertility. This shows that the availability of eupyrene sperm in the spermatheca may be an important constraint on fertility in normal populations of insects. In the laboratory, S. litura females exhibited multiple matings. Of the females, 93% mated, and the mean frequency of mating was 1.69. Mating with a fertile male led to the oviposition of an increased number of eggs. This effect continued even when the female subsequently mated with an infertile male. Displacement of sperm from previous matings is known to be an important factor in the evolution of multiple mating strategies. Our results on sperm utilization by S. litura indicated that after a second mating, the sperm utilized for subsequent fertilization were almost exclusively from the last mating with little mixing. The proportion of eggs fertilized by sperm from the second mating (P₂) was calculated as 0.95, indicating almost complete sperm precedence from the last mating.     

Peristaltic squeezing of sperm bundles at the late stage of spermatogenesis in the silkworm, Bombyx mori

Silkworm (Lepidoptera) males produce dimorphic sperm, nucleate eupyrene sperm, and anucleate apyrene sperm. The eupyrene sperm is the ordinary sperm fertilizing eggs, while the function of the apyrene sperm, which are about four times as numerous as the eupyrene sperm, is still uncertain. We found the peristaltic phenomenon at the very late stage of spermatogenesis. Peristalsis occurs in both eupyrene and apyrene sperm bundles. Through peristaltic action, cytoplasm of the eupyrene sperm and both cytoplasm and nuclei of the apyrene sperm are discarded from the posterior end of the sperm bundles. Peristaltic squeezing seems to be a process to eliminate the irregular nuclei of apyrene sperm while preserving the nuclei of eupyrene sperm.     

Immunocytochemical localization of tubulins in spermatids and spermatozoa of Euptoieta hegesia (Lepidoptera: Nymphalidae)

A comparative analysis of the distribution of tubulin types in apyrene and eupyrene sperm of Euptoieta hegesia butterflies was carried out, also verifying the presence of tubulin in lacinate appendages of the eupyrene sperm. Ultrathin sections of LR White embedded spermatids and spermatozoa were labeled for alpha, beta, gamma, alpha-acetylated and alpha-tyrosinated tubulins. Apyrene and eupyrene spermatids show the same antibody recognition pattern for tubulins. All tubulin types were detected in axonemal microtubules. Alpha and gamma tubulins were also detected on the cytoplasmic microtubules. However, for beta and tyrosinated tubulins only scattered labeling was detected on cytoplasmic microtubules and acetylated tubulin was not detected. In apyrene and eupyrene spermatozoa only the axoneme labeling was analyzed since cytoplasmic microtubules no longer exist in these cells. Alpha, beta and tyrosinated tubulins were easily detected on the apyrene and eupyrene axoneme; gamma tubulin was strongly marked on eupyrene axonemes but was scattered on the apyrene ones. Acetylated tubulin appeared with scattered labeling on the axoneme of both sperm types. Our results demonstrate significant differences in tubulin distribution in apyrene and eupyrene axonemal and cytoplasmic microtubules. Extracellular structures, especially the lacinate appendages, were not labeled by antibodies for any tubulin.     

Precocious reprogramming of eupyrene-apyrene spermatogenesis commitment induced by allatectomy of the penultimate larval instar of the moth Actias selene

A possible causal relationship between the switch-over from eupyrene to apyrene spermatogenesis and pupation in Lepidoptera was examined in Actias selene. Precocious pupation, 11 days earlier than in the controls, was induced by allatectomy on the first day of the penultimate larval instar. The course of the eupyrene spermatogenesis, until nuclear elongation in the spermatid, is not related to pupation. In both allatectomized and control individuals, eupyrene metaphases appear 8 days after ecdysis of the fourth larval instar. Nuclear elongation, however, is triggered in the allatectomized individuals earlier than in the controls, probably by the premature decline of the juvenile hormone titre following allatectomy. Apyrene commitment, on the other hand, is directly related to pupation, as apyrene spermatogenesis begins 2 days after pupation in both control and allatectomized individuals.     

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.     

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.     

Double copulation of a female with sterile diploid and polyploid males recovers fertility in Bombyx mori

Silkworm males produce dimorphic sperm, nucleate eupyrene sperm and anucleate apyrene sperm. Apyrene sperm have been speculated to have an assisting role in fertilisation. However, the coexistence of eupyrene and apyrene sperm in the testis and female reproductive organs has made it difficult to define the role of apyrene sperm. Polyploid males are highly sterile. Microscopic observation revealed that the elimination of eupyrene nuclei by peristaltic squeezing caused the sterility of polyploids. Heat-shock applied to pupae of Daizo males (DH) also induced high sterility due to the lack of normal apyrene sperm. When eupyrene sperm of sterile DH males and apyrene sperm of sterile polyploid males were mixed by double copulation, a remarkable increase in fertility of the double-mated females was observed. This finding strongly suggests that the apyrene sperm are indispensable in fertilisation of the silkworm and that polyploid apyrene sperm function as a substitute for diploid sperm. We established an experimental system in which we can separate the two types of sperm for further studies on their functions without chemical and/or mechanical treatments.     

Apyrene sperm from the triploid donors restore fecundity of cryopreserved semen in Bombyx mori

Female moths of Bombyx mori were artificially inseminated with cryopreserved semen. The fertility of inseminated females varied from 0% to 76.9% depending on the strain. Addition of fresh semen from triploid males, which are infertile but whose semen includes intact apyrene sperm, greatly improved fecundity of cryopreserved semen from normal males. Frozen apyrene sperm from the triploid donors also improved the fecundity of females, inseminated with cryopreserved normal semen, but less than fresh semen from triploid males. Fertilization success in B. mori requires the presence of both, intact eupyrene and apyrene sperm. Our results show that eupyrene sperm tolerate the cryopreservation process better than apyrene sperm. Hence, we recommend to add apyrene sperm from the triploid donors as helper sperm routinely to cryopreserved semen in artificial insemination. This may advance the application of cryopreservation as a routine technique to maintain silkworm resources. The technique may also be applicable to other moth and butterfly species which, like B. mori, possess eupyrene and apyrene sperm.     

Circadian rhythm of sperm release in the gypsy moth,Lymantria dispar: ultrastructural study of transepithelial penetration of sperm bundles

Release of mature bundles of spermatozoa from the testis into the vas deferens is a critical but poorly understood step in male insect reproduction. In moths, the release of sperm bundles is controlled by a circadian clock which imposes a temporal gate on the daily exit of bundles through the terminal epithelium-a layer of specialized epithelial cells separating testis follicles from the vas deferens. The sequence of cellular events associated with the daily cycle of sperm release was investigated by scanning and transmission electron microscopy. In the hours preceding sperm release, there is a solid barrier between the testis and the vas deferens formed by the interdigitation of cytoplasmic processes of adjacent terminal epithelial cells. At the beginning of the sperm release cycle, sperm bundles protrude through this barrier while the terminal epithelial cells change their shape and position relative to the bundles. Subsequently, the cyst cells enveloping the sperm bundles break down and spermatozoa move out of the testis through the exit channels formed between the epithelial cells. Afterwards, cyst cell remnants and other cellular debris are released into the vas deferens lumen, and the epithelial barrier is reconstructed due to phagocytic activity of its cells. These data provide a foundation on which to build an understanding of the cellular mechanisms of clock-controlled sperm release in insects.     

Eupyrene sperm migrates to spermatheca after apyrene sperm in the swallowtail butterfly, Papilio xuthus L. (Lepidoptera: Papilionidae)

When swallowtail butterflies, Papilio xuthus, are mated by the hand-pairing method, both types of sperm, eupyrene and apyrene sperm, are transferred from the male to the spermatheca via the spermatophore in the bursa copulatrix. This mechanism is demonstrated by two different kinds of experiments. The first set of experiments employed interrupted copulation, and the second set was examination of the sperm in the spermatophore and spermatheca after the termination of copulation. The sperm was transferred 30 min after the start of copulation. The eupyrene sperm was still in the bundle; the number of the bundles ranged from 9 to 108 (mean, 42.7; n = 27). The bundles were gradually released after the completion of copulation, and the free eupyrene spermatozoa then remained in the spermatophore at least 2 h before migrating to the spermatheca. On the other hand, about 160 000 apyrene spermatozoa were transferred to the spermatophore and remained there for more than 1 h. We observed 11 000 apyrene spermatozoa in the spermatheca 12 h after the completion of copulation, but most of this type of sperm disappeared shortly thereafter. In contrast, the eupyrene sperm arrived in the spermatheca more than 1 day after the completion of copulation and remained there at least 1 week. Therefore, these findings suggest that apyrene sperm migrate from the spermatophore to the spermatheca earlier than eupyrene sperm. Accordingly, if females mated multiply, the time difference might avoid the mixing of sperm. In addition, the predominance of sperm from the last mating session may occur not in the bursa copulatrix but in the spermatheca. Received: January 7, 2000 / Accepted: May 24, 2000