Pupal Cues Increase Sperm Production but Not Testis Size in an Insect

Theoretic and empirical studies show that social surroundings experienced by male insects during their larval or adult stage can influence their testicular investment in diverse ways. Although insect pupae do not feed and crawl, they can communicate using sex-specific and/or non-sex specific cues. Yet, it is unknown, in any insect, whether and how male pupae can fine-tune their resource allocation to sperm production and testis size in response to socio-sexual environments. We investigated this question using a moth, Ephestia kuehniella, which produces fertile eupyrene sperm and unfertile apyrene sperm. We held male pupae individually or in groups with different sex ratios, and dissected adults upon eclosion, measured their testis size, and counted both types of sperm. We demonstrated that after exposure to conspecific pupal cues regardless of sex, male pupae increased production of eupyrenes and apyrenes at the same rate but kept testis size unchanged. We suggest that testis size is fixed after pupation because most morphological traits are formed during the larval stage, allowing little room for pupae to adjust testis size. Like adults, male pupae with fully grown testes have sufficient resources to produce more sperm of both types according to the perceived increase in sperm competition risk.

Pupal Cues Increase Sperm Production but Not Testis Size in an Insect

Theoretic and empirical studies show that social surroundings experienced by male insects during their larval or adult stage can influence their testicular investment in diverse ways. Although insect pupae do not feed and crawl, they can communicate using sex-specific and/or non-sex specific cues. Yet, it is unknown, in any insect, whether and how male pupae can fine-tune their resource allocation to sperm production and testis size in response to socio-sexual environment. We investigated this question using a moth, Ephestia kuehniella, which produces fertile eupyrene sperm and unfertile apyrene sperm. We held male pupae individually or in groups with different sex ratios, and dissected adults upon eclosion, measured their testis size, and counted both types of sperm. We demonstrated that after exposure to conspecific pupal cues regardless of sex, male pupae increased production of eupyrenes and apyrenes at the same rate but kept testis size unchanged. We suggest that testis size is fixed after pupation because most morphological traits are formed during the larval stage, allowing little room for pupae to adjust testis size. Like adults, male pupae with fully grown testes have sufficient resources to produce more sperm of both types according to the perceived increase of sperm competition risk.

Destination of apyrene sperm following migration from the bursa copulatrix in the monandrous swallowtail butterfly Byasa alcinous

Most male lepidopterans produce fertile eupyrene sperm and non-fertile apyrene sperm, both of which are transferred to the female in a spermatophore during mating. Apyrene sperm outnumbers eupyrene sperm and both sperm types migrate from the bursa copulatrix to the spermatheca after mating. While eupyrene sperm are maintained in the spermatheca until oviposition, the number of apyrene sperm decreases with time. It is unclear whether apyrene sperm disappear from all sperm storage organs in females because both sperm types are often observed in the spermathecal gland. To investigate this, the numbers of both sperm types were estimated in the spermatheca and spermathecal gland of female Byasa alcinous (a monandrous butterfly) 6, 12, 48, 96, and 192 h after mating terminated. Apyrene sperm arrived in the spermatheca earlier than eupyrene sperm; however, some eupyrene and apyrene sperm migrated to the spermathecal gland from the spermatheca at almost the same time. The number of apyrene sperm reached a peak 12 h after the termination of mating and then decreased with time in both the spermatheca and spermathecal gland. Our results suggest that the role of apyrene sperm might be completed early after arriving in the spermatheca of B. alcinous.

Dysfunction of dimorphic sperm impairs male fertility in the silkworm

Sperm, which have a vital role in sexual reproduction in the animal kingdom, can display heteromorphism in some species. The regulation of sperm dichotomy remains a longstanding puzzle even though the phenomenon has been widely documented for over a century. Here we use Bombyx mori as a model to study a form of sperm dimorphism (eupyrene and apyrene sperm), which is nearly universal among Lepidoptera. We demonstrate that B. mori Sex-lethal (BmSxl) is crucial for apyrene sperm development, and that B. mori poly(A)-specific ribonuclease-like domain-containing 1 (BmPnldc1) is required for eupyrene sperm development. BmSXL is distributed in the nuclei and cytoplasm of somatic cyst cells in a mesh-like pattern and in the cytoplasm of germ cells enclosed in spermatocysts and sperm bundles. Cytological analyses of dimorphic sperm in BmSxl mutants (∆BmSxl) showed deficient apyrene sperm with abnormal nuclei, as well as loss of motility associated with malformed mitochondrial derivatives. We define the crucial function of apyrene sperm in the process of fertilization as assisting the migration of eupyrene spermatozoa from bursa copulatrix to spermatheca. By contrast, BmPnldc1 deficiency (∆BmPnldc1) caused eupyrene sperm abnormalities and impaired the release of eupyrene sperm bundles during spermiation. Although apyrene or eupyrene sperm defects impaired fertility of the mutated males, double copulation of a wild-type female with ∆BmSxl and ∆BmPnldc1 males could rescue the sterility phenotypes induced by single copulation with either gene-deficient male. Our findings demonstrate the crucial functions of BmSxl and BmPnldc1 in the development of sperm dimorphism and the indispensable roles of nonfertile apyrene sperm in fertilization.

Dimorphic sperm formation by Sex-lethal

Sex is determined by diverse mechanisms and master sex-determination genes are highly divergent, even among closely related species. Therefore, it is possible that homologs of master sex-determination genes might have alternative functions in different species. Herein, we focused on Sex-lethal (Sxl), which is the master sex-determination gene in Drosophila melanogaster and is necessary for female germline development. It has been widely shown that the sex-determination function of Sxl in Drosophilidae species is not conserved in other insects of different orders. We investigated the function of Sxl in the lepidopteran insect Bombyx mori. In lepidopteran insects (moths and butterflies), spermatogenesis results in two different types of sperm: nucleated fertile eupyrene sperm and anucleate nonfertile parasperm, also known as apyrene sperm. Genetic analyses using Sxl mutants revealed that the gene is indispensable for proper morphogenesis of apyrene sperm. Similarly, our analyses using Sxl mutants clearly demonstrate that apyrene sperm are necessary for eupyrene sperm migration from the bursa copulatrix to the spermatheca. Therefore, apyrene sperm is necessary for successful fertilization of eupyrene sperm in B. mori. Although Sxl is essential for oogenesis in D. melanogaster, it also plays important roles in spermatogenesis in B. mori. Therefore, the ancestral function of Sxl might be related to germline development.

Evolutionary Proteomics Reveals Distinct Patterns of Complexity and Divergence between Lepidopteran Sperm Morphs

Spermatozoa are one of the most strikingly diverse animal cell types. One poorly understood example of this diversity is sperm heteromorphism, where males produce multiple distinct morphs of sperm in a single ejaculate. Typically, only one morph is capable of fertilization and the function of the nonfertilizing morph, called parasperm, remains to be elucidated. Sperm heteromorphism has multiple independent origins, including Lepidoptera (moths and butterflies), where males produce a fertilizing eupyrene sperm and an apyrene parasperm, which lacks a nucleus and nuclear DNA. Here we report a comparative proteomic analysis of eupyrene and apyrene sperm between two distantly related lepidopteran species, the monarch butterfly (Danaus plexippus) and Carolina sphinx moth (Manduca sexta). In both species, we identified ∼700 sperm proteins, with half present in both morphs and the majority of the remainder observed only in eupyrene sperm. Apyrene sperm thus have a distinctly less complex proteome. Gene ontology (GO) analysis revealed proteins shared between morphs tend to be associated with canonical sperm cell structures (e.g., flagellum) and metabolism (e.g., ATP production). GO terms for morph-specific proteins broadly reflect known structural differences, but also suggest a role for apyrene sperm in modulating female neurobiology. Comparative analysis indicates that proteins shared between morphs are most conserved between species as components of sperm, whereas morph-specific proteins turn over more quickly, especially in apyrene sperm. The rapid divergence of apyrene sperm content is consistent with a relaxation of selective constraints associated with fertilization and karyogamy. On the other hand, parasperm generally exhibit greater evolutionary lability, and our observations may therefore reflect adaptive responses to shifting regimes of sexual selection.

Field and Laboratory Studies on the Ecology, Reproduction, and Adult Diapause of the Asian Comma Butterfly, Polygonia c-aureum L. (Lepidoptera: Nymphalidae)

Adult diapause and reproduction of a nymphalid butterfly, Polygonia c-aureum L., were investigated in field and laboratory examinations. Laboratory studies showed that old virgin male butterflies of non-diapausing generations had heavy accessory glands and simplex, which were suppressed in diapausing generations. The number of eupyrene sperm bundles in the duplex increased with adult age, whereas testis size decreased with age. Field examinations indicated that reproductive development of both sexes of diapausing generations in autumn was suppressed, and developed in spring. We attempted to estimate the physiological age of wild-caught males, as adult male age can be estimated from the testis size. We also attempted to determine whether or not wild male butterflies had mated from the development of the accessory glands and simplex, as well as the number of eupyrene sperm bundles in the duplex, by comparing unmated males with mated males. Field examinations suggest that almost all females in a population of non-diapausing generations mated and showed a tendency toward polyandry, while in the diapausing generation, in spring, monoandry rather than polyandry predominated. This suggests a different mating strategy between non-diapausing and diapausing generations.

Sperm morph and remating frequency in the Indian meal moth, Plodia interpunctella

All Lepidoptera produce two sperm types: normal, nucleated ‘eupyrene’ sperm and anucleate ‘apyrene’ sperm. One hypothesis for the evolution of apyrene sperm suggests that they act to reduce female remating rate. Apyrene sperm require less resources to produce than do eupyrene sperm, and could delay remating by females by acting as a ‘cheap filler’, packing the spermatheca and thereby reducing receptivity. This would reduce the risk of sperm competition, giving a potential adaptive advantage to the male producing these sperm. This leads to the prediction that the probability of a female remating should correlate with the number of stored apyrene sperm, which has previously been supported by experiments using the green-veined white butterfly, Pieris napi . We repeated this experiment using the Indian meal moth, Plodia interpunctella . We find that in this species, eupyrene, not apyrene sperm number is the best predictor of female remating probability, indicating that the ‘cheap filler’ hypothesis for the function of apyrene sperm is not well supported in Pl. interpunctella .

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.