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Triacylglycerol lipase (TGL) is an essential lipid metabolism enzyme that also plays a critical role in energy metabolism; however, how it regulates other life processes is unknown. To investigate the functional role of TGL in moth reproduction, males Sitotroga cerealella were used as a model. The TGL gene was cloned and analysed. The results showed that the open reading frame of TGL was 1968 bp long and contained three conserved regions. TGL gene expression was higher in the larval and early adult stages than in the pupal stage, with the highest levels observed in the fat body, testis and accessory glands during the early adult stage. Moreover, after TGL in male adults was silenced through RNAi, the protein content in male accessory glands remained unchanged, and the spermatophore transferred into females mated with TGL -silenced males became small and empty; meanwhile, the number of apyrene sperm in the spermatophore was significantly reduced due to the reduction of apyrene sperm in males, which eventually led to the significant reduction of egg-laying amount. All of the findings suggest that TGL regulates the amount of sperm in male moths as well as the morphology and quality of spermatophores transferred to females after mating with treated males, implying that TGL is critical for Sitotroga cerealella’s reproductive process.

Male ejaculates are often structurally complex, and this complexity is likely to influence key reproductive interactions between males and females. However, despite its potential evolutionary significance, the molecular underpinnings of ejaculate structural complexity have received little empirical attention. To address this knowledge gap, we sought to understand the biochemical and functional properties of the structurally complex ejaculates of Pieris rapae butterflies. Males in this species produce large ejaculates called spermatophores composed of an outer envelope, an inner matrix, and a bolus of sperm. Females are thought to benefit from the nutrition contained in the soluble inner matrix through increases in longevity and fecundity. However, the indigestible outer envelope of the spermatophore delays female remating, allowing males to monopolize paternity for longer. Here, we show that these two nonsperm-containing spermatophore regions, the inner matrix and the outer envelope, differ in their protein composition and functional properties. We also reveal how these divergent protein mixtures are separately stored in the male reproductive tract and sequentially transferred to the female reproductive tract during spermatophore assembly. Intriguingly, we discovered large quantities of female-derived proteases in both spermatophore regions shortly after mating, which may contribute to spermatophore digestion and hence, female control over remating rate. Finally, we report evidence of past selection on these spermatophore proteins and female proteases, indicating a complex evolutionary history. Our findings illustrate how structural complexity of ejaculates may allow functionally and/or spatially associated suites of proteins to respond rapidly to divergent selective pressures, such as sexual conflict or reproductive cooperation.

Bobtail squids (Family Sepiolidae) have a variety of sperm storage patterns, but their reproductive biology has not been studied in many species, especially those in Japanese waters. Two species, Austrorossia bipapillata (Subfamily Rossiinae) and Sepiolina nipponensis (Subfamily Heteroteuthinae) inhabit Suruga Bay in Japan. These were sampled approximately bimonthly by trawling at around 500 m between the end of September, 2020 and May, 2022. They were measured for body size (dorsal mantle length) and weighed for gonadosomatic index (GSI) calculation, and their sperm storage mechanism was investigated. The reproductive season occurs from May to October in A. bipapillata and from December to February in S. nipponensis. In both species, spermatangia were deposited inside the mantle cavity as implanted spermatangia. In A. bipapillata, 5.7 ± 6.5 spermatangia were deeply implanted in the opening of the oviduct, and in S. nipponensis 62.7 ± 61.5 spermatangia were attached to the left gill and surface of the connective-tissue capsule enclosing the digestive gland. The GSI was lower and there were fewer spermatophores stored in the spermatophoric sac of A. bipapillata males compared to S. nipponensis, leading us to suggest that it is exposed to weaker sperm competition than S. nipponensis, irrespective of similar sperm storage mechanism in the two species.

Vitamin D receptors and vitamin D3-metabolizing enzymes have been found to be highly expressed in the ovaries and spermatophores of fish. However, the role of vitamin D3 on fish gonadal development has rarely been reported. In this study, 2-month-old female zebrafish were fed with different concentrations of vitamin D3 diets (0, 700, 1400, and 11 200 IU/kg) to investigate the effects of vitamin D3 on ovarian development. The diet with 0 IU/kg vitamin D3 resulted in elevated interstitial spaces, follicular atresia, and reproductive toxicity in zebrafish ovaries. Supplementation with 700 and 1400 IU/kg of vitamin D3 significantly increased the oocyte maturation rate; upregulated ovarian gonadal steroid hormone synthesis capacity; and elevated plasma estradiol, testosterone, and ovarian vitellogenin levels. Furthermore, the current study identified a vitamin D response element in the cyp19a1a promoter and demonstrated that 1.25(OH)2D3-vitamin D response directly activated cyp19a1a production through activating the vitamin D response element. In conclusion, this study shows that an appropriate concentration of vitamin D3 can promote zebrafish ovarian development and affect vitellogenin synthesis through the vdr/cyp19a1a/er/vtg gene axis. Summary Sentence Vitamin D3 can promote the maturation of fish oocytes and improve the quality of embryos. Graphical Abstract

Polyandry influences sexual behaviour and supports sexual selection in moths, incl. Noctuids. In polyandry, a female mates with multiple males within a few days. Copulation is a multi-phase process, as sperm are packed into spermatophores and transferred to the female genitalia. This process is costly, since spermatophore production and transfer is time-consuming. When this process is repeated, it results in sexual selection and enhances heterozygosity. Pest and migrating Noctuids are often polyandrous. Polyandry is common in the “pest clade” of Noctuidae (e.g., Euxoa, Dichagyris). We hypothesised that the asymmetric allocation of sensory and stimulating functions on the genitalia enhance sexual selection and promote speciation. In polyandrous species of the subtribe Poliina, we show that the evolution of the secondary asymmetry of male external genitalia is connected to an increase in species diversity. Some species inhabiting temperate forests were found to be moderately (Orthosia) or highly (Conistra) polyandrous, in connection with their different life cycles and despite similar reproductive timing. Based on fecundity data of some common species, we hypothesised that habitat generalists should be more polyandrous than habitat specialists. However, our data are insufficient to determine whether ecology and/or phylogeny influence the level of polyandry. Other factors, such as the connection between the sex ratio and the level of polyandry, should be surveyed in future studies, considering their relevance to plant protection.

In butterflies and moths, male‐killing endosymbionts are transmitted from infected females via their eggs, and the male progeny then perish. This means that successful transmission of the parasite relies on the successful mating of the host. Paradoxically, at the population level, parasite transmission also reduces the number of adult males present in the final population for infected females to mate with. Here we investigate if successful female mating when males are rare is indeed a likely rate‐limiting step in the transmission of male‐killing Spiroplasma in the African Monarch, Danaus chrysippus. In Lepidoptera, successful pairings are hallmarked by the transfer of a sperm‐containing spermatophore from the male to the female during copulation. Conveniently, this spermatophore remains detectable within the female upon dissection, and thus, spermatophore counts can be used to assess the frequency of successful mating in the field. We used such spermatophore counts to examine if altered sex ratios in the D. chrysippus do indeed affect female mating success. We examined two different field sites in East Africa where males were often rare. Surprisingly, mated females carried an average of 1.5 spermatophores each, regardless of male frequency, and importantly, only 10–20% remained unmated. This suggests that infected females will still be able to mate in the face of either Spiroplasma‐mediated male killing and/or fluctuations in adult sex ratio over the wet–dry season cycle. These observations may begin to explain how the male‐killing mollicute can still be successfully transmitted in a population where males are rare. African Queens or Monarchs can still find males even in the face of extensive male‐killing. This finding has unexpected implications for the fitness and maintenance of the male‐killing endosymbiont Spiroplasma.

The genus Hemicyclops Boeck, 1873 is known for its association with various marine invertebrates, including cnidarians, crustaceans, polychaetes, and sponges, with some species also occurring in planktonic communities. Here, we report the first association of Hemicyclops with the scleractinian coral Galaxea fascicularis (Linnaeus, 1767) (Scleractinia, Euphylliidae). Hemicyclops cyanus sp. nov. is described based on a female specimen collected from this coral host in the lagoon (depth 10 m) of Dongsha Atoll, Pratas Islands, South China Sea. The new species is readily distinguished from its congeners by its characteristic genital double-somite, which bears prominent anterolateral expansions, and by the flexed, elongated exopodal segment of leg 5, which is more than three times longer than wide. In H. cyanus sp. nov. , the paired spermatophores attached to the female are fused into a butterfly-shaped, highly modified complex with large lateral wings and a central tube into which the female urosome is inserted. To aid in species identification, we present the first comparative plate with schematic illustrations of the genital double-somites for the group of 25 species, including the type species.

The present study investigated the effects 17α-methyltestosterone on the reproductive performance and immune response of broodstock tiger shrimp Penaeus monodon transgenic F1. This study evaluated reproduction, mature spawners, spermatophore weight and density, and total haemocyte count (THC) immune response. Males aged 13 months were divided into four groups. Groups 1, 2 and 3 were injected 17α-methyltestosterone (17α-MT) at the dose of 100, 150, and 200 ng/100 g body weight three times with an interval of one week. Group 4 was eyestalk ablation. The specimen was kept in a concrete maturation tank for 10 tons. Approximately two weeks after the final injection (on the 35 th day), the male subjects were collected, their weights were recorded, and their spermatophores (sperm capsules) were removed. THC was observed before, then on the 7 th , 14 th , and 35 th days of the post-rearing period. 17α -MT induction using dose-dependent increases the percentage of the mature spawner, spermatophore weight, spermatozoa density, and higher total hemocyte count (p>0.05) than eyestalk ablation. The THC injected with 17α-MT at three doses was higher (p<0.05) than ablation on the 7 th and 14 th days. Unlike on the 35 th day, the highest THC was observed at a dose of 200 ng/100 g bw (p>0.05) among all treatments.

Grapholita molesta is a globally significant fruit pest. Females achieve maximal reproductive output through efficient sperm utilization following a single copulation. Post-mating maturation of eupyrene sperm is a critical step in reproductive success. Here, we report that a male accessory gland-derived serine protease (named GmAGSP1) is essential for this process. GmAGSP1 was only distantly related to other identified sperm-activating SPs, and its transcript was highly expressed in the AG at 48 h after emergence. RNAi-mediated knockdown of GmAGSP1 in males did not affect courtship rate, copulation duration, or mating frequency, whereas male fertility decreased significantly. Mating with GmAGSP1-knockdown males markedly impaired eupyrene sperm maturation in the spermatophores, with phenotypes including failure of eupyrene sperm bundles to dissociate normally and marked reduction in viability of the dissociated eupyrene sperm. Finally, untargeted metabolomic analysis preliminarily demonstrated marked alterations in multiple metabolic pathways within the spermatophore following mating with GmAGSP1-knockdown males. This study advances our understanding of the regulatory mechanism of “sperm activation in the spermatophore’s metabolic microenvironment mediated by male AG-derived SP” while providing critical insights for the development of novel genetic control strategies targeting G. molesta.

The Caribbean spiny lobster Panulirus argus, a long-lived decapod crustacean, is fished throughout its geographical range and generates close to 1 billion US dollars annually. Fisheries across this range are fully- or over-exploited, spurring management strategies such as closed seasons and minimum size limitations. The objective of this study was to determine the minimum legal size and appropriate closed season for the lobster population of Saint Kitts and Nevis by assessing and comparing the functional, physiological and morphological determinants of sexual maturity. Such a comparison has never been attempted for this species. Females did not reach functional maturity until 87.8 mm carapace length (CL) based on the presence of a spermatophore and eggs, but reached morphological maturity based on the presence of ovigerous setae at 72.5 mm CL. Physiological maturity was intermediate at 82.8 mm CL. Determinants of functional maturity overestimate the size at sexual maturity, while the presence of ovigerous setae underestimates it. The gonadosomatic index, a reliable method of assessing physiological maturity in female P. argus, can be used to monitor changes in reproductive activity in both males and females. Gross assessment of the ovary may be used to determine size at physiological maturity, and functional maturity can be used to calculate peak periods of reproduction. The peak physiological and functional maturity for P. argus occurs from March to May. As a result, we propose a closed season from March to June and suggest assessment of reproductive dynamics in order to determine minimum legal size for harvesting P. argus.