Application of artificial insemination technique to eupyrene and/or apyrene sperm in Bombyx mori

The silkworm, Bombyx mori, has a dimorphic sperm system. The eupyrene sperm is the sperm to fertilize eggs and the apyrene sperm plays a crucial role for assisting fertilization. Heat-treated (33 degrees C for 96h) Daizo (DH) males, one of the strains in the silkworm, produce only eupyrene sperm, while in triploid males only apyrene sperm are functional. Though both types of males are found to be sterile, double copulation of the two males with a single female greatly increases fertility. Here we examined the fertilizing ability of eupyrene and apyrene sperm by means of an artificial insemination technique previously established in B. mori. Neither the eupyrene sperm collected from DH males, nor the apyrene sperm from triploid males have the ability to fertilize eggs. Artificial insemination with the mixture of eupyrene and apyrene sperm leveled up the frequency of fertilized eggs to more than 80%. When cryopreserved DH sperm (eupyrene sperm) were subjected to the same experiment, more than 95% fertilized eggs were obtained. These results confirmed that apyrene sperm play an important and indispensable role in fertilization in B. mori. Separate collection of functional eupyrene sperm and functional apyrene sperm and success of fertilization by means of the artificial insemination technique are applicable for further studies to elucidate the function of apyrene sperm.     

Significance of peristaltic squeezing of sperm bundles in the silkworm, Bombyx mori: elimination of irregular eupyrene sperm nuclei of the triploid

Silkworm (Lepidoptera) males produce dimorphic sperm: nucleate eupyrene sperm and anucleate apyrene sperm. The eupyrene sperm are ordinary sperm to fertilise the eggs, while the function of apyrene sperm remains uncertain. After meiosis, 256 sperm cells are enclosed by a layer of cyst cells, forming a sperm bundle. We have previously documented that the nucleus of eupyrene sperm anchors to the head cyst cell, which locates at the anterior apex of the bundle, by an acrosome tubule-basal body assembly. Neither the basal body attachment to the nucleus nor the acrosome is seen in apyrene sperm, and the nuclei remain in the middle region of the bundle. Peristaltic squeezing starts from the anterior of the bundles in both types of sperm, and cytoplasmic debris of the eupyrene sperm, and both the nuclei and debris of apyrene sperm, are eliminated at the final stage of spermatogenesis. Since the irregularity of meiotic division in apyrene sperm is known, we used triploid silkworm males that show irregular meiotic division even in eupyrene spermatocytes and are highly sterile. The irregular nuclei of the triploid are discarded by the peristaltic squeezing just as those of the apyrene sperm. Transmission electron microscopic observations disclose the abnormality in the acrosome tubule and in the connection to the basal body. The peristaltic squeezing of sperm bundles in the silkworm appears to be the final control mechanism to eliminate irregular nuclei before they enter female reproductive organs.     

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.     

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.     

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.     

Effects of a fertility-reducing baculovirus on sperm numbers and sizes in the Indian Meal Moth, Plodia interpunctella

1. A dose-dependent decrease in male fertility occurs in the Indian Meal Moth, Plodia interpunctella , when sub-lethally infected with granulovirus during the larval stage.
2. Here, the causes for this decline are investigated by examining eupyrene and apyrene sperm numbers and sizes produced by males across four levels of viral challenge.
3. The results could not explain how reduced male fertility is caused in this host–pathogen interaction. While a reduction in both eupyrene and apyrene sperm numbers from all virus-treated males was found, this was not significant and neither was there a difference in sperm lengths across the four treatments. There were also no differences in the variances of sperm numbers or lengths between the doses, and no associations between sperm numbers or lengths and body size were found.
4. A significant correlation between eupyrene and apyrene numbers was found, but this was independent of dose. Significant between-male variance in apyrene sperm lengths was found, indicating that individual males differ in the range of apyrene sperm sizes they produce.
5. It is suggested that further intracellular and behavioural study is needed to identify the causes of the granulovirus-induced reduction in fertility of P. interpunctella .     

Variations in sperm number in relation to larval crowding and spermatophore size in the armyworm, Pseudaletia separata

1. Number of sperm and its relationship with larval rearing density were investigated in the armyworm Pseudaletia separata . Males that emerged from crowded larvae produced significantly more apyrene sperm than those from solitary larvae (375 700 ± 116 600 and 290 300 ± 99 600 at a mating with a 3-day old virgin, respectively), with no significant difference in number of eupyrene sperm between the two types being observed.
2. For both solitary- and crowded-type, the amount of fertile sperm the males produced at a mating exceeded the number needed to fertilize all of a female's eggs, suggesting that sperm competition may be a major selective force for keeping sperm numerous. The production of more apyrene sperm by crowded-type males may be an adaptation to cope with the increased sperm competition from rival males at high density.
3. The relationship between number of sperm and spermatophore size was also studied using solitary-type moths. Large spermatophores were found to have more eupyrene and apyrene sperm than small ones.     

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.     

Sperm morphology and arrangement along the male reproductive tract of the butterfly Euptoieta hegesia (Insecta: Lepidoptera)

Summary

The present study was undertaken to describe the morphological and organizational modifications that occur in apyrene and eupyrene spermatozoa along the male adult reproductive tract of the butterfly, Euptoieta hegesia. Testis, vas deferens, vesicula seminalis and ductus ejaculatorius were studied by transmission electron microscopy. In the testis, both sperm types are organized into cysts; apyrene sperm are devoid of extracellular structures while eupyrene ones have lacinate and reticular appendages. In the testis basal region, both sperm pass through an epithelial barrier and lose their cystic envelope. The eupyrene morphological and organizational modifications are more drastic than the apyrene ones. From the vas deferens to the ductus ejaculatorius, apyrene sperm are dispersed in the lumen and acquire several concentric layers that are formed by the folding of their abundant cell membrane. The apyrene distribution observed here suggests that their functions include eupyrene transportation. Eupyrene sperm, however, remain aggregated along the tract. In the vas deferens, they are covered by a filamentous material that develops into a homogeneous matrix surrounding the spermatozoa coat in the vesicula seminalis and the ductus ejaculatorius. Eupyrene sperm undergo complex morphological changes that include the loss of lacinate appendages and the formation of a dense and heterogeneous extracellular coat. The formation of the matrix and the coat in eupyrene extratesticular sperm is related to the loss of lacinate appendages. These changes are in general extracellular and are probably important for sperm maturation.     

Effect of fetal bovine serum on the enhancement of in-vitro cultivation of spermatocysts of the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae)

Like other Lepidoptera, the silkworm (Bombyx mori) has both nucleated eupyrene and anucleated apyrene sperm that are derived from the same spermatocysts. The former type is responsible for egg fertilization, while the function of the latter is still uncertain. Many hypotheses have been presented concerning the role of the apyrene sperm in mating and fertilization, but none is supported by a convincing experimental approach. The aim of the present study was to enhance the production of apyrene sperm in vitro by using different concentrations of fetal bovine serum (FBS), namely 20%, 30% and 40%, in the culture medium used for cultivating the naked spermatocysts isolated from the silkworm testes at 0 hr, 120 hr, and 192 to approximately 360 hr after the fourth molt. Cultivation of 0-hr spermatocysts was not successful. The development of spermatocysts into eupyrene and apyrene sperm bundles was slightly slower in vitro than in vivo. The overall growth percentage of both eupyrene and apyrene bundles was satisfactory when the spermatocysts were cultivated in TC-100 culture medium containing 30% FBS.     

Interspecific and intraspecific comparisons of ejaculates in the cotton bollworm <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> and the tobacco budworm <Emphasis Type="Italic">H. assulta</Emphasis>

The evolution of reproductive isolation is a prerequisite in the formation of new species. Although there are numerous studies on ejaculates in lepidopteran insects, ejaculate comparisons among sibling species have not been adequately addressed to understand possible reproductive barriers to hybridization. Here, we examined the interspecific and intraspecific variations of ejaculates in the sibling noctuid moths Helicoverpa armigera and Helicoverpa assulta. We found that there were considerable variations in the number of apyrene and eupyrene sperm and the length of eupyrene sperm. Male pupal mass explained not only a significant proportion of the variation in apyrene sperm number in both H. armigera and H. assulta, but also a significant proportion of the variation in eupyrene sperm number in H. assulta. There was a significant positive relationship between the number of eupyrene sperm and the number of apyrene sperm in both species. No difference in the length of eupyrene sperm was found between them; however, ejaculates of H. armigera had many more eupyrene sperm than H. assulta had. In H. armigera, large males generally mated with large females. The evolutionary consequences of these differences are discussed in this paper.     

Glucose and ecdysteroid increase apyrene sperm production in in vitro cultivation of spermatocysts of Bombyx mori

Two types of sperm, nucleate eupyrene and anucleate apyrene, occur in the silkworm as in other lepidopteran species. Hormones and other substances have been assumed to play important roles in sperm dimorphism. We established an in vitro cultivation system for silkworm spermatocytes, and found that apyrene sperm are not produced when spermatocytes from larval testes are cultivated, though eupyrene spermatocytes develop normally into mature sperm. Based on the fact that ecdysteroid titers increase rapidly and peak 1 day after spinning, and that the amount of glycogen reaches its peak 1 day before the spinning stage, we studied the effects of adding glucose and/or 20-hydroxyecdysone to the culture medium. The experiments disclosed a significant additive effect of both substances on apyrene sperm production.     

Adult Responses to Larval Population Size in the Almond Moth, Cadra cautella

Juvenile population size may affect the potential for future mating opportunities and therefore potentially sperm competition; this may favour ontogenetic adjustments in sperm production. Theory predicts that males should optimize their ejaculatory investment in accordance with the risk of sperm competition. Evidence for these theories is typically revealed in males of highly polyandrous species. Whether such responses to environmental cues exist for females, or are maintained in mildly polyandrous species in which most females do not re-mate, is unknown. Male lepidopterans produce normal, fertilizing sperm (eupyrene) and non-fertilizing (apyrene) sperm. Apyrene sperm are associated with reduced female receptivity, suggesting a role in sperm competition. We tested the effect of juvenile population size on life-history parameters and reproductive investment in the mildly polyandrous almond moth, Cadra cautella , a species in which current male ejaculate traits suggest previous selection for paternity protection consistent with a sperm-competitive environment. Larvae were reared at high (H) or low population sizes (L). We recorded larval development time, adult longevity and male gametic investment. Our results show a response by adults to signals in the juvenile environment. H males transferred more apyrene, but not eupyrene sperm. We also examined potential trade-offs between somatic characters and reproductive behaviours. Larval duration was longer for H individuals, females and heavier individuals. Further, H females and L males lived longer than L females. Our data are consistent with the theory that males should adjust their reproductive investment in accordance with sperm competition risk.     

The structural mechanism of trypsin-induced intrinsic motility in Manduca sexta spermatozoa in vitro

Lepidopteran males produce eupyrene (nucleate) and apyrene (anucleate) spermatozoa, but in the female only eupyrene spermatozoa leave the spermatheca and fertilize the eggs. Both kinds of spermatozoa lack intrinsic motility in the male genital duct. They become motile in the spermatophore, in a process involving proteases from the male duct. In vitro, trypsin induces immotile spermatozoa to become motile. We studied the changes spermatozoa of Manduca sexta undergo during trypsin-induced motility and found that (a) they mimick rather closely those occurring in vivo during normal sperm maturation in genital ducts and (b) they are time- and dose-dependent. As in vivo, they comprise, successively, (a) disappearance of an extracellular matrix that maintains the integrity of eupyrene bundles in the seminal vesicle, (b) dispersion of the eupyrene bundles and intermingling of eupyrene and apyrene spermatozoa and (c) "hatching" of eupyrene spermatozoa from individual enclosing envelopes that are formed in the seminal vesicle. "Hatching" may not directly be related to motility since eupyrene spermatozoa become motile before "hatching" and motile apyrene spermatozoa never "hatch". Rather "hatching" may be related to the capacitation of eupyrene spermatozoa to either leave the spermatheca or fertilize the eggs, or both, as neither apyrene spermatozoa, nor those eupyrene spermatozoa that fail to "hatch", leave the spermatheca.     

Impact of male mating history on the temporal sperm dynamics of Choristoneura rosaceana and C. fumiferana females

In the oblique-banded leafroller, Choristoneura rosaceana, and the spruce budworm, C. fumiferana, male reproductive performance decreases with consecutive matings. While the onset time of mating did not vary, the time spent mating was longer in mated than in virgin males. Furthermore, a decline observed in the spermatophore mass with successive matings was associated with a concomitant decline in its apyrene and eupyrene spermatozoa content. In the hours following mating, spermatozoa migrate from the spermatophore, located in the bursa copulatrix, to the spermatheca. Regardless of the male's previous mating history, the number of apyrene sperm dropped rapidly in the days following mating whereas the number of eupyrene spermatozoa declined gradually. As the temporal pattern of sperm movement was similar in all treatments, females mated with previously-mated males would suffer from sperm shortage sooner than those mated with virgins. Large C. rosaceana females stored more apyrene spermatozoa in their spermatheca than small ones, irrespective of the time after mating or male mating history, while only large females mated with once-mated males received more apyrene sperm and accessory gland secretions than small ones mated with virgin or twice-mated males. The results obtained in this study are discussed in relation with their potential impact on the reproductive success of both sexes.     

Juvenile socio-sexual experience determines lifetime sperm expenditure and adult survival in a polygamous moth,Ephestia kuehniella

Male animals often adjust their sperm investment in response to sperm competition environment. To date, only a few studies have investigated how juvenile sociosexual settings affect sperm production before adulthood and sperm allocation during the first mating. Yet, it is unclear whether juvenile sociosexual experience (1) determines lifetime sperm production and allocation in any animal species; (2) alters the eupyrene : apyrene sperm ratio in lifetime ejaculates of any lepidopteran insects, and (3) influences lifetime ejaculation patterns, number of matings and adult longevity. Here we used a polygamous moth, Ephestia kuehniella, to address these questions. Upon male adult emergence from juveniles reared at different density and sex ratio, we paired each male with a virgin female daily until his death. We dissected each mated female to count the sperm transferred and recorded male longevity and lifetime number of matings. We demonstrate for the first time that males ejaculated significantly more eupyrenes and apyrenes in their lifetime after their young were exposed to juvenile rivals. Adult moths continued to produce eupyrene sperm, contradicting the previous predictions for lepidopterans. The eupyrene : apyrene ratio in the lifetime ejaculates remained unchanged in all treatments, suggesting that the sperm ratio is critical for reproductive success. Male juvenile exposure to other juveniles regardless of sex ratio caused significantly shorter adult longevity and faster decline in sperm ejaculation over successive matings. However, males from all treatments achieved similar number of matings in their lifetime. This study provides insight into adaptive resource allocation by males in response to juvenile sociosexual environment.     

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.     

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.     

Panning for sperm gold: Isolation and purification of apyrene and eupyrene sperm from lepidopterans

We describe a simple and straightforward procedure for the purification and separation of apyrene and eupyrene forms of lepidopteran sperm. The procedure is generally applicable to both butterfly and moth species with results varying according to the relative amounts of sperm produced and size of sperm storage organs. The technique relies upon inherent differences between eupyene sperm bundles and free apyrene sperm morphology. These differences allow for separation of the sperm morphs by repeated “panning” of sperm bundles into the center of a plastic dish. The purified eupyrene sperm bundles can then be removed and apyrene sperm collected from the supernatant by centrifugation. Efficacy of the purification process was confirmed by light microscopy and gel electrophoresis of the resulting fractions. Both one- and two-dimensional gel electrophoresis identified significant protein differences between the fractions further suggesting that the panning procedure effectively separated eurpyrene from apyrene sperm. The panning procedure should provide a convenient and accessible technique for further studies of sperm biology in lepidopterans.