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Delivering molecular tools into oocytes is essential for developmental and reproductive biology. Microinjection, the conventional method, is equipment intensive, often technically challenging and has a low yield, and is impractical in species with delicate oocytes or restricted spawning seasons. To overcome these limitations, we developed VitelloTag, a cost-effective, high-throughput system using vitellogeninderived fusion proteins to enable efficient cargo delivery via receptormediated endocytosis. We demonstrate its utility by delivering Cas9/sgRNA complexes in two distantly related species for gene knockout. [ABSTRACT FROM AUTHOR]

In addition to preventing precocious larval metamorphosis, juvenile hormone (JH), synthesized in corpora allata (CA), is known to stimulate female reproduction of insects. JH titer is extremely low or absent during metamorphosis, but thereafter rapidly increases in the previtellogenic stage and rises to a peak in the vitellogenic phase. However, the mechanisms underlying the biosynthesis of high levels of JH in adults remain unclear. We found in this study that 12 genes involved in JH synthesis pathway were highly expressed in the CA of adult locusts. By transcriptome analysis and quantitative real‐time – polymerase chain reaction validation, a total of 106 evolutionary conserved micro RNAs (miRNAs) and 163 species‐specific miRNAs were identified in locust CA. Dual‐luciferase assay revealed that 17 miRNAs bound to 10 JH synthesis genes (JHSGs) and downregulated their expression. These miRNAs were expressed in low levels during vitellogenic stage, which was oppositive from that of targeting JHSGs. Six miRNAs including miR‐971‐3p, miR‐31a, miR‐9‐5p, miR‐1‐3p, miR‐315, and miR‐282 were selected for function study. Co‐application of agomiRs resulted in significantly decreased levels of targeting JHSGs, accompanied by significantly reduced vitellogenin expression as well as arrested ovarian development. The data suggest that multiple miRNAs expressed synchronously at low levels in the vitellogenic phase, thereby ensuring the high levels of JHSG expression to facilitate JH biosynthesis required for JH‐dependent female reproduction. The findings provide important information for deciphering miRNA—messenger RNA modules for JH biosynthesis as well as JH regulation of insect metamorphosis and reproduction. [ABSTRACT FROM AUTHOR]

BACKGROUND: MicroRNA (miRNA) pathway genes have been widely reported to participate in several physiological events in insect lifecycles. The cigarette beetle Lasioderma serricorne is an economically important storage pest worldwide. However, the functions of miRNA pathway genes in L. serricorne remain to be clarified. Herein, we investigated the function of molting and reproduction of the miRNA pathway in L. serricorne. RESULTS: LsDicer‐1, LsArgonaute‐1, LsLoquacious and LsExportin‐5 were universally expressed in adults, whereas LsPasha and LsDrosha were mainly expressed in the pupae. The genes presented different patterns in various tissues. Silencing of LsDicer‐1, LsArgonaute‐1, LsDrosha and LsExportin‐5 resulted in a high proportion of wing deformities and molting defects. Silencing of LsDicer‐1, LsArgonaute‐1, LsPasha and LsLoquacious affected the development of the ovary and the maturation of oocytes, resulting in a significant decrease in fecundity. Further investigation revealed that the decreases in LsDicer‐1 and LsArgonaute‐1 expression destroyed follicular epithelia and delayed vitellogenesis and oocyte development. In addition, the expression levels of several miRNAs (let‐7, let‐7‐5p, miR‐8‐3p, miR‐8‐5p, miR‐9c‐5p, miR‐71, miR‐252‐5p, miR‐277‐3p, miR‐263b and Novel‐miR‐50) were decreased significantly after knockdown of these miRNA pathway core genes, indicating that they played important roles in regulating miRNA‐mediated gene expression. CONCLUSION: The results indicate that miRNA pathway genes play important roles in the molting, ovarian development and female fecundity of L. serricorne, and thus are potentially suitable target genes for developing an RNAi strategy against a major pest of stored products. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Mosquitoes such as Aedes aegypti must consume a blood meal for the nutrients necessary for egg production. Several transcriptome and proteome changes occur post‐blood meal that likely corresponds with codon usage alterations. Transfer RNA (tRNA) is the adapter molecule that reads messenger RNA codons to add the appropriate amino acid during protein synthesis. Chemical modifications to tRNA enhance codon decoding, improving the accuracy and efficiency of protein synthesis. Here, we examined tRNA modifications and transcripts associated with the blood meal and subsequent periods of vitellogenesis in A. aegypti. More specifically, we assessed tRNA transcript abundance and modification levels in the fat body at critical times post blood‐feeding. Based on a combination of alternative codon usage and identification of particular modifications, we discovered that increased transcription of tyrosine tRNAs is likely critical during the synthesis of egg yolk proteins in the fat body following a blood meal. Altogether, changes in both the abundance and modification of tRNA are essential factors in the process of vitellogenin production after blood‐feeding in mosquitoes. [ABSTRACT FROM AUTHOR]

While clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 technology has demonstrated remarkable promise as a gene-editing tool, its application in certain insects, such as the jewel wasp, Nasonia vitripennis , has been hindered by a lack of a tractable method for reagent delivery. Direct Parental (DIPA-) CRISPR recently emerged as a facile way to induce gene lesions because it involves adult injection with commercially available Cas9–sgRNA with no helper reagent. However, DIPA-CRISPR has so far been tested in only a few insects. Here, we have assessed the amenability of DIPA-CRISPR in N. vitripennis by targeting two eye pigmentation genes, cinnabar and vermilion , which function in the ommochrome pathway. Successful generation of lesions in both genes demonstrated the functionality of DIPA-CRISPR in N. vitripennis and its potential application to other genes, thereby expanding the range of insects suitable for this method. We varied two parameters, Cas9–sgRNA concentration and injection volume, to determine optimal injection conditions. We found that the larger injection volume coupled with either higher or lower reagent concentration was needed for consistent mutation production. However, DIPA-CRISPR yields an overall low mutation rate in N. vitripennis when compared to other tested insects, a characteristic that may be attributed to a proportionally low vitellogenic import efficiency in the jewel wasp. We discuss different factors that may be considered in determining when DIPA-CRISPR may be preferable over other reagent delivery methods. [ABSTRACT FROM AUTHOR]

Most cyprinid fish breed from spring to summer. On the other hand, the timing of initiation and duration of gametogenesis in preparation for reproduction vary widely depending on the species. It is unclear what factors determine the types of timing and duration of gametogenesis. In order to infer the factors, it is necessary to clarify the gametogenesis process of as many species as possible, categorize them, and explore commonalities and differences between species belonging to different types of gametogenesis. In the present study, we determined the maturity of Hemigrammocypris neglecta, a temporary water-spawning fish of the subfamily Oxygastrinae, based on histological observations of the gonads. Female vitellogenesis began in May and continued during the breeding season from May to August. This species was a multiple-spawner that repeatedly ovulates and spawns multiple times during the breeding season. Male spermatogenesis (meiosis) began in April, and a large number of spermatozoa were observed in the testes and sperm duct from May to August, indicating that reproduction is possible during this period. The timing of the initiation of gametogenesis in spring/summer-spawning cyprinid fishes has been roughly divided into three types: autumn type, winter/spring type, and breeding season type. The female H. neglecta was the breeding season type. There are few species that belong to this type, suggesting that the type may have evolved independently with reflect the reproductive ecology of each species. The male is the winter/spring type, a characteristic common to the subfamily Oxygastrinae. [ABSTRACT FROM AUTHOR]

The molecular mechanisms that govern the metabolic commitment to reproduction, which often occurs at the expense of somatic reserves, remain poorly understood. We identified the Caenorhabditis elegans F-box protein FBXL-5 as a negative regulator of maternal provisioning of vitellogenin lipoproteins, which mediate the transfer of intestinal lipids to the germline. Mutations in fbxl-5 partially suppress the vitellogenesis defects observed in the heterochronic mutants lin-4 and lin-29, both of which ectopically express fbxl-5 at the adult developmental stage. FBXL-5 functions in the intestine to negatively regulate expression of the vitellogenin genes; and consistently, intestine-specific over-expression of FBXL-5 is sufficient to inhibit vitellogenesis, restrict lipid accumulation, and shorten lifespan. Our epistasis analyses suggest that fbxl-5 functions in concert with cul-6, a cullin gene, and the Skp1-related gene skr-3 to regulate vitellogenesis. Additionally, fbxl-5 acts genetically upstream of rict-1, which encodes the core mTORC2 protein Rictor, to govern vitellogenesis. Together, our results reveal an unexpected role for a SCF ubiquitin-ligase complex in controlling intestinal lipid homeostasis by engaging mTORC2 signaling. [ABSTRACT FROM AUTHOR]

Hilsa (Tenualosa ilisha), a highly prized edible fish, is consumed by over 250 million peopleworldwide. Estrogens are essential hormones required during reproduction and bind with estrogen receptors in target organs for biological activity. The two unique subtypes of the estrogen receptor found in fish are alpha (a) and beta (b) and exhibit distinctive roles in reproduction. The present study aimed to understand the breeding physiology of hilsa during the seasonal gonadal cycles through GSI seasonal variation, histological study, and molecular identification, characterization, and transcriptional modification of estrogen receptors in hilsa. Monthly GSI analysis from females showed that during September, the GSI value was maximum (22.01 ± 0.68), followed by the May GSI value (18.78 ± 0.97). Histological observation showed the formation of asynchronous gametes during the development of ovaries. The histological analysis revealed the formation of developing oocytes, nucleus, presence of primary oogonia, secondary oogonia, chromatin nucleolar oocytes, and early perinucleolar oocytes in the ovary (January-early July). Progressively, formations of the yolk vesicles, yolk granule stage, premature stage, and mature stage with post-ovulatory follicles were also identified. Linear observation was recorded during the monsoon season from July to November. Furthermore, partial length cDNAs for estrogen receptors were characterized, and their mRNA expression patterns demonstrate that ER-a expression significantly increased in September, followed by April and August in the ovary and liver tissue. Both the liver and ovary showed the highest mRNA expression of ER-b in September. The study revealed that ER-a expression was higher in the ovary as compared with liver tissue. Furthermore, we introduce threedimensional (3D) models depicting the hilsa estrogen receptors in complex with estradiol (E2), constructed through homology modeling. This investigation contributes valuable insights into the molecular characteristics of estrogen receptors in this teleost fish. Our present work provided preliminary evidence of estrogen receptors during reproduction in hilsa. [ABSTRACT FROM AUTHOR]

Interspecific hybridisation is common in aquaculture. The maturation of hybrids is an important issue for successive breeding and reducing the risk of genetic pollution if they escape from sea cages. This study examined the maturation of Kue-Tama, the hybrid of the longtooth grouper Epinephelus bruneus × giant grouper E. lanceolatus (LGGG) produced on 16 April 2014. We used 60 LGGG, five of which were treated with methyltestosterone (MT) and the others were not. We assessed the spermiation response (SpR) of all LGGG from March 2018 to May 2020 and sperm motility from September 2019 to May 2020. We also analysed seasonal fluctuations in plasma sex steroids (estradiol 17β [E2] and 11-ketotestosterone [11-KT]) and gonad histology from April 2018 to February 2019. We confirmed SpR in all MT-treated LGGG and in five out of 55 untreated LGGG. Some LGGG from both groups exhibited SpRs throughout the year. The spermatozoa from both groups showed vigorous motility. SpR in MT-treated LGGG occurred at low 11-KT levels (< 0.1 ng/ml), whereas that in untreated LGGG occurred at high levels (> 273.4 ng/ml). The E2 levels in a few LGGG without SpR increased significantly from September 2018 to February 2019. The April gonads were identified as ovaries without histological confirmation, whereas the July and September gonads were verified histologically. One of the three gonads collected in July 2019 contained vitellogenic oocytes. Our study demonstrated that male LGGG hybrids mature throughout the year and that females can undergo vitellogenesis in the subtropical waters and photoperiod conditions of Amami Islands. [ABSTRACT FROM AUTHOR]

The mosquito fat body is the principal source of yolk protein precursors (YPP) during mosquito egg development in female Aedes aegypti. To better understand the metabolic and signaling pathways involved in mosquito reproduction, we investigated changes in the mosquito fat body phosphoproteome at multiple time points after a blood meal. Using LC/MS, we identified 3570 phosphorylated proteins containing 14,551 individual phosphorylation sites. We observed protein phosphorylation changes in cellular pathways required for vitellogenesis, as well as proteins involved in primary cellular functions. Specifically, after a blood meal, proteins involved in ribosome synthesis, transcription, translation, and autophagy showed dynamic changes in their phosphorylation patterns. Our results provide new insight into blood meal-induced fat body dynamics and reveal potential proteins that can be targeted for interference with mosquito reproduction. Considering the devastating impact of mosquitoes on human health, worldwide, new approaches to control mosquitoes are urgently needed.

The expansion of agriculture and livestock in Argentina has significantly increased the use of pesticides, adversely impacting animal and human health. These chemical compounds, acting as xenoestrogens, can disrupt the reproductive physiology of wildlife. In this context, vitellogenin, a liver protein typically elevated in oviparous females during reproduction, can also be induced in males and immature females by exposure to xenoestrogens. The aim of this work was to study the production of vitellogenin in response to exogenous estrogens in male Salvator merianae lizards, a species widely distributed in Argentina. Eighteen adult tegus from the Universidad Nacional de Tucumán were used, distributed into three groups: males treated with 17 ?-estradiol, females in the vitellogenesis phase (positive control), and untreated males (negative control). Treated males received doses of 17 ?-estradiol every three days for two weeks, and blood samples were taken before and during treatment for analysis by SDS-PAGE, spectrophotometry, and immunohistochemistry. Results revealed a significant increase in triglyceride levels and the synthesis of a high molecular weight lipoprotein in treated males, a pattern similar to that observed in females during vitellogenesis and absent in negative controls. This pioneering study demonstrates the induction of hepatic vitellogenin in Salvator merianae males treated with estradiol, highlighting the potential utility of this species as a biomarker of environmental contamination by xenoestrogens.

Gonad maturity in fish is essential in fish farming because it is related to seed production. Sesame oil is one of the ingredients as a feed supplement to stimulate fish gonad maturation. The research aimed to obtain the best dose of sesame seed extract (Sesamum indicum L.) in feed to increase the gonad maturity of comet fish (Carassius auratus). This research had four treatment levels and three repetitions. The treatment levels in this study were: P0 Without giving sesame seed extract (Control), P1 Giving sesame seed extract 5 mL/kg feed, P2 giving sesame seed extract 10 mL/kg feed, P3 Giving sesame seed extract 15 mL/kg. The results of the six-week study showed that at the beginning of maintenance, they were in TKG I, in the 3rd week, they entered TKG III, and in the 6th week, P1, P2, and P3 entered TKG IV while P0 were still in TKG III. The highest gonad maturity index at each observation time occurred in treatment P2 with feed containing 10 mL/kg of sesame seed extract. In the 1st week, the highest gonad maturity index occurred at P2 at 7.80. In the 3rd week, the highest gonad maturity index occurred in P2 at 9.26%. In the 6th week, the highest gonad maturity index occurred in P2 at 11.17%. Fecundity at week 6 showed the highest result of P2 4,290. The level of gonad maturity with treatment given sesame extract was faster, with a maintenance time of 6 weeks. The results of this study illustrate that sesame extract can be used as a supplement to increase comet fish gonad maturity. [ABSTRACT FROM AUTHOR]

Spotted wolffish (Anarhichas minor) is a poorly understood species which is often captured as part of mixed demersal fisheries across its range. Abundance has declined in many regions and there is a need for greater knowledge on its biology. To improve our understanding of reproduction of A. minor, we investigated inter- and intra- annual differences in fecundity, the influence of condition on fecundity and time scale of ovary development. From 2006 to 2021, 150 females A. minor were sampled in Icelandic waters. Of these females, 73 were also used to estimate spawning time together with an additional 334 females from commercial catch and surveys from 2006 to 2023. Backwards extrapolation of oocyte size indicates that vitellogenesis begins in December and is likely completed after 8–10 months. There was no evidence of either intra- or inter-annual differences in fecundity, indicating that downregulation is minimal and that fecundity of A. minor is stable between years. A positive relationship between oocytes size and fish length was detected, while body condition and hepatosomatic index had only a small influence on fecundity in comparison with weight. There was a negative relationship between length and relative fecundity and the exponent of the fecundity–length relationship was lower than exponent of the weight–length relationship. Therefore, total egg production is likely not proportional to the spawning stock biomass of A. minor, and unusually, total egg production would decrease with increasing proportion of larger fish in the population. The spawning season was estimated to be from middle of August to middle of October with peak of spawning in September. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the effects of arginine (Arg) on the vitellogenesis and ovarian maturation of Chinese mitten crab. Six isonitrogenous and isolipidic diets (1.83, 2.52, 3.26, 3.96, 4.81 and 5.52 % Arg) were fed to adult female Chinese mitten crab (60.05 ± 4.33 g). The feeding period lasted for eleven weeks. The results showed that gonadosomatic index of crabs increased with increasing dietary Arg concentrations from 1.83 % to 2.52 % and significantly decreased from 2.52 % to 5.52 %. The quadratic regression analysis based on gonadosomatic index estimated that the optimal Arg requirement for adult female crabs was 2.60 %. Dietary supplementation with 2.52 % Arg significantly increased the crude protein concentration in the ovary. A dose of 2.52 % Arg significantly decreased the vitellogenesis-inhibiting hormone (VIH) content in the hemolymph. Arg (2.52 %) significantly upregulated the Vg and protein kinase A (PKA) gene expressions and increased the cyclic adenosine monophosphate (cAMP) concentration in the hepatopancreas. Moreover, dietary supplementation with 2.52 % Arg significantly decreased the cyclic guanosine monophosphate (cGMP) concentration and downregulated protein kinase G (PKG) and p38 mitogen-activated protein kinase (p38 MAPK) gene expression. Arg at 2.52 % significantly increased hemolymph Vg content and the gene expression of the vitellogenin receptor (VgR). In conclusion, Arg at 2.52 % improved vitellogenesis and ovarian maturation by reducing the VIH content and regulating Vg expression via the cGMP/cAMP pathway.

The molecular mechanisms that govern the metabolic commitment to reproduction, which often occurs at the expense of somatic reserves, remain poorly understood. We identified the Caenorhabditis elegans F-box protein FBXL-5 as a negative regulator of maternal provisioning of vitellogenin lipoproteins, which mediate the transfer of intestinal lipids to the germline. Mutations in fbxl-5 partially suppress the vitellogenesis defects observed in the heterochronic mutants lin-4 and lin-29, both of which ectopically express fbxl-5 at the adult developmental stage. FBXL-5 functions in the intestine to negatively regulate expression of the vitellogenin genes; and consistently, intestine-specific over-expression of FBXL-5 is sufficient to inhibit vitellogenesis, restrict lipid accumulation, and shorten lifespan. Our epistasis analyses suggest that fbxl-5 functions in concert with cul-6, a cullin gene, and the Skp1-related gene skr-3 to regulate vitellogenesis. Additionally, fbxl-5 acts genetically upstream of rict-1, which encodes the core mTORC2 protein Rictor, to govern vitellogenesis. Together, our results reveal an unexpected role for a SCF ubiquitin-ligase complex in controlling intestinal lipid homeostasis by engaging mTORC2 signaling.

Hilsa (Tenualosa ilisha), a highly prized edible fish, is consumed by over 250 million people worldwide. Estrogens are essential hormones required during reproduction and bind with estrogen receptors in target organs for biological activity. The two unique subtypes of the estrogen receptor found in fish are alpha (α) and beta (β) and exhibit distinctive roles in reproduction. The present study aimed to understand the breeding physiology of hilsa during the seasonal gonadal cycles through GSI seasonal variation, histological study, and molecular identification, characterization, and transcriptional modification of estrogen receptors in hilsa. Monthly GSI analysis from females showed that during September, the GSI value was maximum (22.01 ± 0.68), followed by the May GSI value (18.78 ± 0.97). Histological observation showed the formation of asynchronous gametes during the development of ovaries. The histological analysis revealed the formation of developing oocytes, nucleus, presence of primary oogonia, secondary oogonia, chromatin nucleolar oocytes, and early perinucleolar oocytes in the ovary (January–early July). Progressively, formations of the yolk vesicles, yolk granule stage, premature stage, and mature stage with post-ovulatory follicles were also identified. Linear observation was recorded during the monsoon season from July to November. Furthermore, partial length cDNAs for estrogen receptors were characterized, and their mRNA expression patterns demonstrate that ER-α expression significantly increased in September, followed by April and August in the ovary and liver tissue. Both the liver and ovary showed the highest mRNA expression of ER-β in September. The study revealed that ER-α expression was higher in the ovary as compared with liver tissue. Furthermore, we introduce three-dimensional (3D) models depicting the hilsa estrogen receptors in complex with estradiol (E2), constructed through homology modeling. This investigation contributes valuable insights into the molecular characteristics of estrogen receptors in this teleost fish. Our present work provided preliminary evidence of estrogen receptors during reproduction in hilsa.

Antarctic krill oil has been proven to be able to promote the ovarian development of crustaceans, but its optimal application dose and potential regulatory mechanism in Macrobrachium rosenbergii are still unclear. In this study, five isonitrogenous and isolipidic diets with gradient additions of Antarctic krill oil (0%, 1.5%, 3%, 4.5%, and 6%) were served exposed to 8 weeks of feeding. The results show that 3–4.5% Antarctic krill oil supplementation significantly increases the weight gain rate and specific growth rate of M. rosenbergii (p < 0.05). In addition, 3–4.5% Antarctic krill oil supplementation significantly increased the content of hemolymph vitellogenin (VTG) and the levels of reproductive hormones, including methyl farnesoate (MF), estradiol (E2), and progesterone (P4) (p < 0.05). The differences in ovarian index, oocyte volume, yolk granule deposition in oocytes, and the transcription levels of VTG genes in hepatopancreas and ovarian tissues demonstrated that the addition of Antarctic krill oil significantly promoted ovarian development and vitellogenesis, especially at the 4.5% addition level. In terms of molecular signaling, this study confirms that the retinol metabolic signaling pathway, MF signaling pathway, steroid hormone signaling pathway, and ecdysone signaling pathway, along with their specific molecules, such as Farnesoic acid-O-methyltransferase (FAMeT), retinoid x receptor (RXR), ecdysone receptor (EcR), and estrogen-related receptor (ERR), are involved in the regulation of the ovarian development of M. rosenbergii by adding Antarctic krill oil at appropriate doses. The findings indicate that the supplementation of 4.5% Antarctic krill oil in the diet is optimal for stimulating the secretion of reproductive hormones in female M. rosenbergii, thereby promoting vitellogenesis and ovarian development.

The primary insect steroid hormone ecdysone requires a membrane transporter to enter its target cells. Although an organic anion-transporting polypeptide (OATP) named Ecdysone Importer (EcI) serves this role in the fruit fly Drosophila melanogaster and most likely in other arthropod species, this highly conserved transporter is apparently missing in mosquitoes. Here we report three additional OATPs that facilitate cellular incorporation of ecdysone in Drosophila and the yellow fever mosquito Aedes aegypti. These additional ecdysone importers (EcI-2, -3, and -4) are dispensable for development and reproduction in Drosophila, consistent with the predominant role of EcI. In contrast, in Aedes, EcI-2 is indispensable for ecdysone-mediated development, whereas EcI-4 is critical for vitellogenesis induced by ecdysone in adult females. Altogether, our results indicate unique and essential functions of these additional ecdysone importers in mosquito development and reproduction, making them attractive molecular targets for species- and stage-specific control of ecdysone signaling in mosquitoes.

Reproduction is of great importance for the continuation of the species. In insects, the fat body is the major tissue for nutrient storage and involved in vitellogenesis, which is essential for female reproduction. Here, 2 proteins, hexamerin and allergen, were separated from the fat bodies of adult female American cockroaches (Periplaneta americana) and identified as storage proteins, encoding for 733 amino acids with molecular weight of 87.88 kDa and 686 amino acids with molecular weight of 82.18 kDa, respectively. The encoding genes of these 2 storage proteins are mainly expressed in the fat body. RNA interference‐mediated knockdown of Hexamerin and Allergen in the early stage of the first reproductive cycle in females suppressed vitellogenesis and ovarian maturation, indicating that these storage proteins are involved in controlling reproduction. Importantly, the expression of Hexamerin and Allergen was repressed by knockdown of the juvenile hormone (JH) receptor gene Met and the primary response gene Kr‐h1, and was induced by methoprene, a JH analog, in both in vivo and in vitro experiments. Altogether, we have determined that hexamerin and allergen are identified as storage proteins and play an important role in promoting female reproduction in the American cockroach. The expression of their encoding genes is induced by JH signaling. Our data reveal a novel mechanism by which hexamerin and allergen are necessary for JH‐stimulated female reproduction. [ABSTRACT FROM AUTHOR]

Vitellogenin (Vtg) is a precursor of yolk proteins in egg-laying vertebrates and invertebrates and plays an important role in vitellogenesis and embryonic development. However, the Vtg family remains poorly characterized in Exopalaemon carinicauda, a major commercial mariculture species found along the coasts of the Yellow and Bohai Seas. In this study, 10 Vtg genes from the genomes of E. carinicauda were identified and characterized. Phylogenetic analyses showed that the Vtg genes in crustaceans could be classified into four groups: Astacidea, Brachyra, Penaeidae, and Palaemonidae. EcVtg genes were unevenly distributed on the chromosomes of E. carinicauda, and a molecular evolutionary analysis showed that the EcVtg genes were primarily constrained by purifying selection during evolution. All putative EcVtg proteins were characterized by the presence of three conserved functional domains: a lipoprotein N-terminal domain (LPD_N), a domain of unknown function (DUF1943), and a von Willebrand factor type D domain (vWD). All EcVtg genes exhibited higher expression in the female hepatopancreas than in other tissues, and EcVtg gene expression during ovarian development suggested that the hepatopancreas is the main synthesis site in E. carinicauda. EcVtg1a, EcVtg2, and EcVtg3 play major roles in exogenous vitellogenesis, and EcVtg3 also plays a major role in endogenous vitellogenesis. Bilateral ablation of the eyestalk significantly upregulates EcVtg mRNA expression in the female hepatopancreas, indicating that the X-organ/sinus gland complex plays an important role in ovarian development, mostly by inducing Vtg synthesis. These results could improve our understanding of the function of multiple Vtg genes in crustaceans and aid future studies on the function of EcVtg genes during ovarian development in E. carinicauda. [ABSTRACT FROM AUTHOR]

The ultrastructure of oogenic cells and mature oocytes has been described in a deep-sea clam, Calyptogena pacifica, for the first time. Specimens were collected in June 2018 from the Piip volcano in the Bering Sea (55.3821° N, 167.2613° E). Female germ cells developed in the branched gonadal tubules. The pattern of oogenic development is similar to that in littoral species. Although somatic accessory cells do not form any follicles around developing oocytes, these cells have a well-developed endoplasmic reticulum, which could be a source of yolk precursors and be involved in vitellogenesis. Lipid droplets appear in early oocytes, but they disappear later, suggesting that developing oocytes use these lipid droplets. At the onset of vitellogenesis, developing oocytes accumulate lipids in the ooplasm facing the gonadal tubule wall. Yolk granules, in contrast, appear at later stages of oogenesis. Mature oocytes, which are 200 µm in diameter, are filled with a significant volume of lipid droplets (occupying 24% of ooplasm) and a smaller volume of yolk granules (~ 8%). The mature oocyte's features, i.e., the large cell size (~ 200 µm in diameter) and the high abundance of ooplasmic inclusions, are typical of species with lecithotrophic development. The large volume of lipid droplets would enhance buoyancy of the oocytes and provide embryos and larvae with nutrients during dispersal. Symbiotic bacteria were found in accessory cells, in vitellogenic oocytes, and within remnants of degenerating oocytes. [ABSTRACT FROM AUTHOR]

Methyl farnesoate (MF), a crucial sesquiterpenoid hormone, plays a pivotal role in the reproduction of female crustaceans, particularly in the vitellogenesis process. Despite extensive research on its functions, the molecular mechanisms that regulate MF levels during the vitellogenic phase remain largely elusive. This study investigates the roles of microRNAs (miRNAs), significant post-transcriptional regulators of gene expression, in controlling MF levels in the swimming crab Portunus trituberculatus. Through bioinformatic analysis, four miRNAs were identified as potential regulators targeting two genes encoding Carboxylesterases (CXEs), which are key enzymes in MF degradation. Dual luciferase reporter assays revealed that let-7b and miR-141 suppress CXE1 and CXE2 expression by directly binding to their 3′ UTRs. In vivo overexpression of let-7b and miR-141 significantly diminished CXE1 and CXE2 levels, consequently elevating hemolymph MF and enhancing vitellogenin expression. Spatiotemporal expression profile analysis showed that these two miRNAs and their targets exhibited generally opposite patterns during ovarian development. These findings demonstrate that let-7b and miR-141 collaboratively modulate MF levels by targeting CXEs, thus influencing vitellogenesis in P. trituberculatus. Additionally, we found that the expression of let-7b and miR-141 were suppressed by MF, constituting a regulatory loop for the regulation of MF levels. The findings contribute novel insights into miRNA-mediated ovarian development regulation in crustaceans and offer valuable information for developing innovative reproduction manipulation techniques for P. trituberculatus. [ABSTRACT FROM AUTHOR]

The present study aimed to assess the effects of thermal manipulation on the vitellogenesis and final oocyte maturation (FOM) in pikeperch. Two groups of fish were stocked in two separate tanks of the climate chamber. One group was stocked at 10-month age (THERMAL), while the other was continually kept under stable photothermal conditions until 19-month-age (CONTROL) and then transferred to a climate chamber. The progress of vitellogenesis was assessed via evaluation of the oocyte diameter at the mid- and late-autumn phases, and the gonadosomatic index (GSI) at the late-autumn phase. Finally, females from the CONTROL group were hormonally stimulated before (WARMING) and after (STABLE) increase of the temperature from wintering to spawning, and the FOM progress, ovulation and plasma levels of 17 α, 20β dihydroxy progesterone (DHP) were assessed. Significantly larger oocytes at the mid-autumn phase (878.8 ± 40.1 µm vs 836.5 ± 46.5 µm) as well as the GSI at the end of the autumn phase (10.5 ± 1.7% vs 7.6 ± 1.1%) were noticed in THERMAL fish. Significantly faster FOM was seen in fish under the WARMING post-stimulation regime, and these fish had higher DHP levels at the moment of hormonal stimulation (5.4 ± 1.4 ng/ml vs 3.8 ± 1.2 ng/ml). According to the obtained results, it appears that photothermal induction of fish at a younger stage might have a positive impact on the first spawning, while the WARMING thermal regime seems to be more efficient in stimulating the FOM in fish upon first wintering. [ABSTRACT FROM AUTHOR]

The reproductive cycle of Crotalus durissus is markedly seasonal and synchronous between individuals. The start of vitellogenesis occurs at the end of the summer and coincides with copulation. However, given that the copulation is dissociated from ovulation, sperm storage is obligatory in females. In viperids, sperm storage in the female reproductive tract is reported to occur in two regions: (1) the posterior infundibulum, which presents sperm storage glands; and (2) the nonglandular uterus where sperm is stored in crypts by means of the uterine muscular twisting (UMT). The mechanisms that allow the survival of sperm in the female reproductive tract of snakes are still unknown. In this study, we investigated five regions of the reproductive tract of C. durissus, searching for the presence of spermatozoa and sperm storage structures in different oviductal portions. Additionally, we used histological techniques to verify the occurrence of hypertrophy of the infundibular and uterine glands during the processes of vitellogenesis, as well as histochemical techniques to investigate the nature of the secretion produced in the nonglandular uterus and posterior infundibulum. Storage sperm were observed in the nonglandular uterus and although the posterior infundibulum had storage receptacles, sperm were not observed in that region. Both sperm storage regions presented granules testing positive for acidic and neutral polysaccharides, in vitellogenic and previtellogenic females. This presence of guaranteeing conditions for sperm storage. Histochemical analysis revealed the possible storage capacity of sperm in the nonglandular uterus. In addition, the UMT was observed in all the females with storage sperm, which assures the maintenance of sperm in the nonglandular uterus until ovulation. [ABSTRACT FROM AUTHOR]

The insulin superfamily comprises a group of peptides with diverse physiological functions and is conserved across the animal kingdom. Insulin‐like peptides (ILPs) of crustaceans are classified into four major types: insulin, relaxin, gonadulin, and androgenic gland hormone (AGH)/insulin‐like androgenic gland factor (IAG). Of these, the physiological functions of AGH/IAG have been clarified to be the regulation of male sex differentiation, but those of the other types have not been uncovered. In this study, we chemically synthesized Maj‐ILP1, an ILP identified in the ovary of the kuruma prawn Marsupenaeus japonicus, using a combination of solid‐phase peptide synthesis and regioselective disulfide bond formation reactions. As the circular dichroism spectral pattern of synthetic Maj‐ILP1 is typical of other ILPs reported thus far, the synthetic peptide likely possessed the proper conformation. Functional analysis using ex vivo tissue incubation revealed that Maj‐ILP1 significantly increased the expression of the yolk protein genes Maj‐Vg1 and Maj‐Vg2 in the hepatopancreas and Maj‐Vg1 in the ovary of adolescent prawns. This is the first report on the synthesis of a crustacean ILP other than IAGs and also shows the positive relationship between the reproductive process and female‐dominant ILP. [ABSTRACT FROM AUTHOR]

Lipid accumulation in ovaries is essential for reproductive success in decapods. Understanding of the mechanism of ovarian lipid accumulation is still in its infancy. In this study, we investigated lipid compositions of ovaries and hepatopancreas throughout vitellogenesis and hemolymph in the late-vitellogenic stage in the mud crab Scylla paramamosain. Total lipid content in ovaries consecutively increased from the early to late stage, and the main accumulated lipids were phosphatidylcholine (PC), alkenylglycerophosphoethanolamine (alkenyl-PE), triacylglycerol (TG), cholesteryl ester, and cholesterol. The lipid composition of hemolymph indicated that PC, alkenyl-PE, diacylglycerol (DG), ether DG, monoacylglycerol, and cholesterol were the main lipids transferred to ovaries from hepatopancreas. In hepatopancreas, the total lipid content appeared to increase during vitellogenesis and the late stage was significantly different from early and mid-stages. TG was the predominant lipid class that slightly decreased from the early to mid-stage, but dramatically increased in the late stage. In contrast, PC and alkenyl-PE significantly increased from the early to mid-stage and remained constantly in relatively low proportion until the late stage. These findings suggested PC and alkenyl-PE in the hepatopancreas likely play critical roles in regulating lipid transfer. Esterified fatty acids in ovaries and hepatopancreas were mainly C16:0, C18:0, 16:1, C18:1, C18:2, C20:4, C20:5, and C22:6, of which highly unsaturated fatty acids (HUFA) primarily occurred in alkenyl-PE and phosphatidylserine (PS) but not PC. In hemolymph, PC was abundant in HUFA besides of saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA), while PS was predominantly composed of SFA and MUFA, implying a transformation of HUFA between phospholipids prior to transfer. These findings highlight the importance of phospholipid metabolism in the hepatopancreas to ovarian lipid accumulation. Our study provides novel insights into the mechanism of ovarian lipid accumulation in decapods.

Harpiosquilla raphidea, the largest of the mantis shrimps, is a commercially important crustacean species widely distributed in many countries of the Pacific Ocean. However, no data are currently available regarding the molecular mechanisms that regulate reproduction in this species. To address this knowledge gap, we performed transcriptome sequencing (RNA-Seq) of previtellogenic and vitellogenic ovaries of female H. raphidea, and compared the expression patterns of transcripts from the two resulting libraries to identify genes involved in ovarian development. A total of 418,635,748 clean reads were retrieved after removing adapter sequences and filtering out low-quality data. The reads were assembled into 242,861 unigenes with an average length of 585.27 base pairs (bp) and an N50 of 829 bp. A search of all unigenes against the NR (non-redundant protein), SwissProt, KEGG, GO, and COG databases yielded 53,111; 25,460; 27,255; 26,793; and 3838 unigene matches, respectively). Of the 53,111 unigenes identified in the NR database, 30,441 could be functionally annotated, 59.49% of which were significantly matched with the transcripts of L. vannamei. A total of 13,867 full-length transcripts and 11,441 partial transcripts were retrieved. In addition, using all-unigenes as a reference, a total of 12,765 simple sequence repeats (SSRs) were identified. DESeq2 analysis identified a total of 962 differentially expressed genes (DEGs) between previtellogenic and vitellogenic ovaries, 456 of which were upregulated and 506 downregulated. Five unigenes were validated by quantitative real-time PCR (qPCR). The results of both the RNA-Seq and qPCR analysis revealed that the H. raphidea vitellogenin gene (HrVtg) was upregulated in vitellogenic ovaries. The open reading frame of HrVtg was found to be 7455 bp long, corresponding to a polypeptide of 2485 amino acids. The important functional genes and pathways identified here provide a valuable dataset for understanding the molecular mechanisms controlling ovarian development in H. raphidea.

This study evaluated growth, survival, and oogenesis before and after 17β-estradiol (E2) treatments. During the growth period, C. viridis reached a total length of 65 ± 4.2 cm and a weight of 2.5 ± 0.66 kg BW. To induce sex change in males, we implanted silastic tubing loaded with 0 (control), 0.5, or 1.5 mg of E2 kg−1, at days 0, 30, 60, and 90. Blood samples were collected before treatment (day 0) and on days 30, 60, 90, and 120. Initially, 23% of the selected males expressed milt. At 60 days post-treatment 13% in the control and 4.3% in both, 0.5 and 1.5 mg E2 kg−1 BW treatments. After 120 days we observed histological evidence of sex change in 50% of the histology-tagged fish in the 1.5 mg E2 kg−1 treatment. The gonadosomatic index and condition factor did not differ significantly (P > 0.05) between the control and the treatments. However, the mean individual fish weight differed significantly across all sampling times (P

The incidence of early onset puberty in female aquaculture fish species is less well known than in males and, it merits to be taken into account in order to properly address its outcomes. The goal of this study was to compare the growth performance and reproductive physiology between 2-year-old female sea bass with early or late onset puberty. Fish from seven fertilized egg cohorts of sea bass were considered. Animals were individually tagged and histologically identified when terminally sampled over the first and second year of life. Ovaries from 1-year-old females were in the chromatin nucleolus (CN) or perinucleolar stage (PN), whereas 2-year-old females showed oocytes in the PN stage and early vitellogenesis (EV) (GSI < 0.7%) or late vitellogenesis (LV) (GSI of 4.45 ± 0.55%). Accordingly, 2-year-old females with less advanced reproductive development were considered as fish with late onset puberty (PN, EV), in contrast to their counterparts that were considered to be fish with early onset puberty (LV). Non-spawning females with early onset puberty were observed. The early onset puberty occurred in 18.1 ± 6.4% of fish in the population, although it was variable among the cohorts. Body size of early pubertal females was usually larger than that of fish with late onset puberty (28.7% heavier in weight and 7.9% greater in fork length). Differences in circulating levels of the insulin-like growth factor-1 (Igf-1) and those of 17-beta estradiol (E2), follicle-stimulating hormone (Fsh) and vitellogenin (Vtg) one year before spawning, significantly contributed to explaining the total variance associated with the early onset puberty in this species. This study provides valuable information on the interplay that these factors might have at the onset of early puberty in fish and, in turn, its potential use as key indicators of this trait in the female sea bass.

Information regarding the endocrine regulation of oocyte maturation in cultured species is essential for the artificial seed production. To elucidate endocrine mechanisms that underlie oocyte maturation of the white-streaked grouper, Epinephelus ongus, a commercially important fish species, we collected and compared data linked to the ovaries of captive and wild white-streaked grouper specimens, and plasma steroids data were obtained using captive fish. This data allowed for a comparative analysis of the ovarian development of both groups; the comparison was necessary for the acquisition of physiological patterns universally representative of the species at hand. The dynamics of the gonadosomatic index (GSI) and oocyte development of both groups confirmed that vitellogenesis progressed toward the last quarter moon, final oocyte maturation began the day before the last quarter moon, and spawning occurred after the last quarter moon. These results indicate that the changes in ovarian development and GSI in wild and captive fish were synchronous, and that the endocrine changes observed in captive fish represent those that occur in wild fish. The changes in estradiol 17β (E2) levels in captive fish increased and decreased during the vitellogenic and final oocyte maturation phases, respectively. These dynamics were similar to those of GSI. We found that 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) was consistently detected at concentrations higher than those of 17α, 20β-21-trihydroxy-4-pregnen-3-one (20β-S); this trend was constant across all stages of ovarian development. In contrast, 20β-S was detected at lower concentrations than DHP in all captive individuals, but at higher levels after the onset of final oocyte maturation. These results suggest that 20β-S and/or DHP may be maturation-inducing hormones (MIH) in this species. Our results also highlight the importance of integrative analyses of wild and captive individuals to comprehensively elucidate the endocrine regulation of cultured species of interest to improve aquaculture management practices.

Maternal transfer of selenium (Se) to developing fish eggs during vitellogenesis can cause larval deformity and mortality. Previous studies have shown wide variation among fish species in both the magnitude of maternal transfer (exposure) and the egg Se concentration causing effects (sensitivity). We studied maternal transfer and effects of Se on early life stage development, survival, and growth of redside shiner (Richardsonius balteatus), a small‐bodied cyprinid that has been reported to have relatively high ovary:muscle Se concentration ratios. Gametes were collected from lentic areas in southeast British Columbia (Canada) with a range of dietary Se concentrations related to weathering of waste rock from coal mining. Eggs were fertilized and reared in the laboratory from hatch to the onset of exogenous feeding. Larvae were assessed for survival, length, weight, Se‐characteristic deformities, and edema. Eggs from a total of 56 females were collected, with egg Se concentrations from 0.7 to 28 mg/kg dry weight. Maternal transfer varied among sites, with egg:muscle Se concentration ratios ranging from <1 to >4. We also found that sampling residual ovaries can overestimate Se concentrations in ripe eggs by up to a factor of 5.7. A correlation between larval weight and egg Se concentration was identified, although the relationship was weak (r2 < 0.1) and appeared to be a site effect. No other relationships were observed between larval endpoints and egg Se concentrations up to the highest concentration tested, indicating that the effects threshold for this species may be >28 mg/kg dry weight in eggs. These data indicate that redside shiner is less sensitive to maternally transferred Se than most other tested fish species. Environ Toxicol Chem 2023;42:2350–2357. © 2023 SETAC [ABSTRACT FROM AUTHOR]

Bursicon, a neuropeptide hormone comprising two subunits-bursicon (burs) and partner of burs (pburs), belongs to the cystine-knot protein family. Bursicon heterodimers and homodimers bind to the lucine-rich G-protein coupled receptor (LGR) encoded by rickets to regulate multiple physiological processes in arthropods. Notably, these processes encompass the regulation of female reproduction, a recent revelation in Tribolium castaneum. In this study we investigated the role of burs/pburs/rickets in mediating female vitellogenesis and reproduction in a hemipteran insect, the whitefly, Bemisia tabaci. Our investigation unveiled a synchronized expression of burs, pburs and rickets, with their transcripts persisting detectable in the days following eclosion. RNAi-mediated knockdown of burs, pburs or rickets significantly suppressed the transcript levels of vitellogenin (Vg) and Vg receptor in the female whiteflies. These effects also impaired ovarian maturation and female fecundity, as evidenced by a reduction in the number of eggs laid per female, a decrease in egg size and a decline in egg hatching rate. Furthermore, knockdown of burs, pburs or rickets led to diminished juvenile hormone (JH) titers and reduced transcript level of Kruppel homolog-1. However, this impact did not extend to genes in the insulin pathway or target of rapamycin pathway, deviating from the results observed in T. castaneum. Taken together, we conclude that burs/pburs/rickets regulates the vitellogenesis and reproduction in the whiteflies by coordinating with the JH signaling pathway. [ABSTRACT FROM AUTHOR]

Simple Summary: In Coleoptera, successful female reproduction partially relies on effective vitellogenesis, characterized by vitellogenin (Vg) synthesis in the fat body, secretion into hemolymphs, transport through intercellular channels in follicular epithelium, endocytosis mediated by Vg receptor (VgR), and absorption and storage by mature oocytes. In two representative Coleoptera species, Leptinotarsa decemlineata (Chrysomelidae) and Henosepilachna vigintioctopunctata (Coccinellidae), we performed RNA interference targeting ecdysone receptor (EcR) or ultraspiracle (usp) genes. Depletion of the expression level of EcR or usp inhibited oocyte development and dramatically repressed the transcription of Vg in fat bodies. Our findings indicate that 20E signaling plays an indispensable role in the stimulation of Vg synthesis and uptake in the two beetle species. Coleoptera is the largest taxa of animals by far. The robust reproductive capacity is one of the main reasons for such domination. Successful female reproduction partially relies on effective vitellogenesis. However, the hormone regulation of vitellogenesis remains to be explored. In the present paper, in vitro culture of Leptinotarsa decemlineata 1-day-old adult fat bodies in the 20E-contained median did not activate juvenile hormone production and insulin-like peptide pathways, but significantly stimulated the expression of two LdVg genes, in a cycloheximide-dependent pattern. In vivo RNA interference (RNAi) of either ecdysone receptor (LdEcR) or ultraspiracle (Ldusp) by injection of corresponding dsRNA into 1-day-old female adults inhibited oocyte development, dramatically repressed the transcription of LdVg genes in fat bodies and of LdVgR in ovaries; application of JH into the LdEcR or Ldusp RNAi L. decemlineata females did not restore the oocyte development, partially rescued the decreased LdVg mRNA levels but over-compensated LdVgR expression levels. The same RNAi experiments were performed in another Coleoptera species, Henosepilachna vigintioctopunctata. Little yolk substances were seen in the misshapen oocytes in the HvEcR or Hvusp RNAi ovaries, in contrast to larger amounts of yolk granules in the normal oocytes. Correspondingly, the transcript levels of HvVg in the fat bodies and ovaries decreased significantly in the HvEcR and Hvusp RNAi samples. Our results here show that 20E signaling is indispensable in the activation of vitellogenesis in the developing oocytes of the two beetle species. [ABSTRACT FROM AUTHOR]

We observed the presence of follicular cells (FC) in the ovaries of Penaeus subtilis (n = 1198), which led us to classify the development of germ cells into six phases: oogonia, previtellogenic oocytes, primary and secondary vitellogenic oocytes, mature oocytes and atretic oocytes. The FC changes their shape according to the development of germ cells and showed a different distribution along the ovary, which allowed differentiating vitellogenic oocytes into primary and secondary. We also observed that the postovulatory follicles (POF) are composed of follicular cells. The presence of POF in penaeids ovaries is rarely reported, but allows the differentiation between spent and resting stages, commonly grouped in reproductive biology research. Furthermore, observation of ovarian lining was useful to differentiate immature females from females that had spawned at least once. Thus, ovarian development was classified into six stages: immature, early developing, advanced developing, ripe, spent and resting. The distribution and shape variations of FC, ovarian lining features and presence of POF were considered crucial for the classification of ovarian maturation stages. The methods developed here may improve estimates of their reproductive cycle, size at first maturity and spawning season, which are important variables in future studies of the reproductive dynamics. [ABSTRACT FROM AUTHOR]

The Mediterranean Sea is one of the busiest areas worldwide in terms of maritime activity and faces considerable anthropogenic disturbances, such as pollution by hydrocarbons and heavy metals. This study evaluated the environmental status of three sites on the Algerian coast using the annelid polychaete Perinereis cultrifera as a biomonitoring sentinel species. It included different reproductive parameters such as oocyte diameter, frequency distributions, sexual maturity index, biochemical quantitative parameters represented by the vitellogenins and vitellins, and a histological study of the coelome during the reproductive period. The results showed that the females collected from El-Kala (healthy site) have the highest diameter 304.1 ± 24.3 μm during April but the values were increased in Annaba 290.0 ± 21.0 μm and Skikda 245.3 ± 26.7 μm. Significant differences were observed between worms from the three study sites during the three months of the study. Moreover, the size-frequency indicated that a relatively high proportion of females containing mature oocytes was found in April in females collected at El-Kala 52.4%, 46.4% in females collected at Annaba and 36.7% in females collected at Skikda. In addition, regarding the sexual maturity index, the highest values were recorded in April (3.8, 2.9, and 1.9) at the El-Kala, Annaba, and Skikda study sites. However, the biochemical analysis demontrated that the highest mean concentration of vitellogenin was in females of El-Kala (5.3 ± 0.3 µg/mg of coelomic fluid) in April, and the lowest mean concentration was measured in females of Skikda (1.5 ± 0.2 µg/mg of coelomic fluid). Therefore, the highest mean concentration of vitellin was measured in females of El-Kala (4.4 ± 0.2 µg/mg of oocytes) in April, while the lowest mean concentration of vitellin was measured in females of Skikda (1.02 ± 0.2 µg/mg of oocytes) in May. Significant differences were observed between worms from the three sites during the three months of the study. Furthermore, histological observations revealed that oogenesis was asynchronous, with oocytes at different stages of vitellogenesis simultaneously present. The structure of the oocytes of females collected at El-Kala showed intense vitellogenic activity due to the presence of very dense yolk cells. In contrast, the oocytes of the females collected from the two polluted sites (Annaba and Skikda) were small, vitellogenesis presented low intensity, and yolk cells were less frequent at the periphery and less uniform at the cytoplasmic mass; the nucleus was smaller, indicating a slower vitellogenic activity. These observations confirm the previously obtained results.

Repeated blood feedings are required for adult female mosquitoes to maintain their gonadotrophic cycles, enabling them to be important pathogen carriers of human diseases. Elucidating the molecular mechanism underlying developmental switches between these mosquito gonadotrophic cycles will provide valuable insight into mosquito reproduction and could aid in the identification of targets to disrupt these cycles, thereby reducing disease transmission. We report here that the transcription factor ecdysone-induced protein 93 (E93), previously implicated in insect metamorphic transitions, plays a key role in determining the gonadotrophic cyclicity in adult females of the major arboviral vector Aedes aegypti Expression of the E93 gene in mosquitoes is down-regulated by juvenile hormone (JH) and up-regulated by 20-hydroxyecdysone (20E). We find that E93 controls Hormone Receptor 3 (HR3), the transcription factor linked to the termination of reproductive cycles. Moreover, knockdown of E93 expression via RNAi impaired fat body autophagy, suggesting that E93 governs autophagy-induced termination of vitellogenesis. E93 RNAi silencing prior to the first gonadotrophic cycle affected normal progression of the second cycle. Finally, transcriptomic analysis showed a considerable E93-dependent decline in the expression of genes involved in translation and metabolism at the end of a reproductive cycle. In conclusion, our data demonstrate that E93 acts as a crucial factor in regulating reproductive cycle switches in adult female mosquitoes.

Abstract Specimens of Cnemidocarpa amphora were collected monthly from the Arabian Gulf from September 2017 to August 2018. Parts of their gonads were prepared for histological testing. The gonads’ diameters varied by month. Each gonad contained many ovarian follicles with different morphologies and was surrounded by several testicular follicles. The ovarian and testicular follicles were separate, although the latter were always present near the former. Repeated measures ANOVA tests were conducted to investigate monthly changes in oocyte stages. In squirts measuring 12–13 cm in length, the gonads measured 30–50 mm from July to August; 20–25 mm from September to October; 15–20 mm from November to February; and 25–40 mm from March to June. Oogonia budded from the germinal epithelium with diameters of 20–30 µm. Previtellogenic oocytes measuring 70–120 µm were characterized by the deposition of small granules of protein around the nucleus, a continuous layer of follicular cuboidal epithelium, and scattered vacuoles in the ooplasm. The measurement of gonads and oocyte diameters were performed by image analysis (Image scope 2.3, Image Line, Inc.) and stage micrometer. The vitellogenic oocytes measured 130–220 µm and the follicular epithelium consisted of flattened and cuboidal layers. Beneath the vitelline membrane, scattered test cells appeared in the ooplasm and different granules of protein and MPS were deposited in the ooplasm. In the later phase, lipid droplets began to appear in the ooplasm. Yolk bodies formed after the impregnation of various granules together and the oocyte was ready to be shed. Before spawning, a yolk membrane appeared above the ooplasm. Post-vitellogenic oocytes, in which the homogeneity of ooplasm was restored, underwent gradual lysis and entered the atretic phase. Different stages of sperm development were present year-round in different follicles of the same squirt; hence, the testes were always mature.

Vitellogenesis is the most important stage of ovarian maturation in fish, involving the synthesis and transport of essential yolk substances and their complex mechanisms and coordination process. The liver is the main tissue involved in the vitellogenesis of oviparous animals, but studies of vitellogenesis in fish rarely include the liver and ovary as a whole. The aim of this study was to explore the molecular mechanism and associated regulatory factors behind vitellogenesis in Sichuan bream (Sinibrama taeniatus). The different stages of oogenesis were first identified by successive histological observations. Then, ovary and liver tissues that developed to 115 days (stage II, previtellogenesis stage), 165 days (stage III, vitellogenesis stage) and 185 days (stage IV, late-vitellogenesis stage) were collected for transcriptomic and metabolomic analyses, and serum testosterone (T), 17β-estradiol (E2), vitellogenin (Vtg), triiodothyronine (T3), and thyroxine (T4) levels were measured at the corresponding stages. We found that energy redistribution during vitellogenesis is mainly regulated through glycolysis, fatty acid biosynthesis and the citrate cycle pathway. In the liver, energy metabolism was promoted by activating glucolipid metabolic pathways to provide sufficient ATP, but at the same time, the ovary tends to retain nutrients rather than decompose them to produce energy. In addition, we have identified several key factors involved in the metabolism of neutral lipids, polar lipids, amino acids and vitamins, which are involved in the assembly and transport of important yolk nutrients. The initiation of vitellogenesis was found to be associated with a surge in serum E2 levels, but the sustained increase in Vtg levels in the late stage may be due more to upregulation of the estrogen receptor. These results provide valuable information about the regulation of ovarian development in cultured fish.

Current methods for gene editing in insects rely on embryonic microinjection, which can be challenging for non-specialist laboratories. Recently, an alternative method known as "direct parental" CRISPR (DIPA-CRISPR) was developed. This method involves injecting commercial Cas9 protein and single-guide RNA into adult females, which can efficiently introduce mutations into developing oocytes. However, its versatility has not been fully explored, particularly in insects that have the most derived, polytrophic meroistic ovaries. In this study, we successfully applied DIPA-CRISPR to the yellow fever mosquito Aedes aegypti, which has polytrophic meroistic ovaries. Following adult injection of Cas9 ribonucleoproteins (Cas9 RNPs) targeting the kynurenine 3-monooxygenase gene, we recovered gene-edited G0 individuals. Injection at 24 h after blood-feeding resulted in the highest gene editing efficiency (3.5%), confirming that a key parameter of DIPA-CRISPR is the stage in which the adult females are injected. Together with our previous study, we demonstrated that DIPA-CRISPR is applicable to all three types of insect ovaries (i.e., panoistic, telotrophic, and polytrophic), which indicates that DIPA-CRISPR is a generalizable approach for insect gene editing. [ABSTRACT FROM AUTHOR]

Vitellogenesis is the main event of oocyte growth in oviparous animals, which is mainly manifested by the accumulation of vitellogenin (VTG). The accumulation of vitellogenin depends mainly on the absorption of exogenous vitellogenin, which enters oocyte through endocytosis mediated by its receptor (VGR). We investigated the expression and localization of VTG and VGR during gonad development of Pampus argenteus. The qPCR results show that vtgs were not expressed in male fish, but in the ovary and liver of female fish; the expression levels went up at first and then down. The expression levels of vgr in the testis were low and only 1%–3% of that in ovary. ELISA results show that during the ovarian development of P. argenteus, VTG in liver, serum, and ovary all showed a trend from increasing to decreasing. However, VTG in liver peaked in Stage IV, and in serum and ovary peaked in Stage V, reflecting changes in the characteristics of VTG in the liver (synthesis), blood (transport), and ovaries (accumulation). During gonad development, VGR in the ovaries first increased and then decreased, reaching a peak in Stage V, in contrast to vgr mRNA expression. The VGR content in the testis was extremely low and stable, consistent with vgr mRNA. Immunohistochemistry results show that the location and intensity of VTG and VGR positive signals were synchronized with the changes of their protein content, which revealed that VTG was mainly synthesized in the liver cytoplasm, secreted into the blood, and transported to ovary in Stage III. VGR is highly expressed in oocytes in Stage II. In Stage III, a large amount of VTG reaches the ovary, when VGR begins to translate and is subsequently transported to the plasma membrane of the oocyte. Therefore, the positive signal of VGR was stronger near the plasma membrane of oocytes in Stages I and II. By using qPCR, ELISA, and immunohistochemistry, the synthesis, transport, and accumulation of vitellogenin were elucidated and the mechanism of its endocytosis on egg membrane mediated by VTG during the development of P. argenteus was revealed preliminarily. [ABSTRACT FROM AUTHOR]

The pharaoh cuttlefish, Sepia pharaonis, has become a commercially cultured cephalopod in coastal southeast China. However, information on the reproductive histology of this species remains limited. To describe its reproductive development, this study investigated the ovarian structure and oogenesis of S. pharaonis from hatchlings to the postbreeding stage using anatomical methods and histological techniques. The results showed that oogenesis in the ovary was asynchronous and morphologically variable, with immature oocytes attached to the gonadal cord and encircling the ovarian stroma in sequence. After maturation, the egg was free in the ovarian cavity and was discharged through the fallopian tube to combine with the sperm. Furthermore, serial histological dissections of the ovaries demonstrated that oocyte development was asynchronous. Based on egg size and morphology, follicular cell morphology, and yolk formation, oogenesis in S. pharaonis was divided into five distinct stages: The oogonia stage, protoplasm growth stage, follicular penetration stage, vitellogenesis stage, and resorbing stage. Moreover, based on the appearance of follicular cells in the protoplasm growth stage and their disintegration and disappearance in the vitellogenesis stage, it can be inferred that follicular cells secrete yolk substances and participate in the formation of egg membranes. Through the dynamic observation and description of the ovary development and oogenesis, these results provide an important foundation for studies of the regulatory mechanisms of oogenesis in this species, enriching the theory of cephalopod reproductive biology and improving artificial reproduction technology. [ABSTRACT FROM AUTHOR]

Endoparasitoid wasps introduce venom into their host insects during the egg‐laying stage. Venom proteins play various roles in the host physiology, development, immunity, and behavior manipulation and regulation. In this study, we identified a venom protein, MmRho1, a small guanine nucleotide‐binding protein derived from ovary in the endoparasitoid wasp Microplitis mediator and found that knockdown of its expression by RNA interference caused down‐regulation of vitellogenin and juvenile hormone, egg production, and cocoons formation in the female wasps. We demonstrated that MmRho1 entered the cotton bollworm's (host) hemocytes and suppressed cellular immune responses after parasitism using immunofluorescence staining. Furthermore, wasp MmRho1 interacted with the cotton bollworm's actin cytoskeleton rearrangement regulator diaphanous by yeast 2‐hybrid and glutathione s‐transferase pull‐down. In conclusion, this study indicates that MmRho1 plays dual roles in wasp development and the suppression of the host insect cellular immune responses. [ABSTRACT FROM AUTHOR]

The eyestalk of crustaceans is a vital neuroendocrine organ complex that regulates various important physiological processes, especially molting and reproduction. The mandibular organ-inhibiting hormone (MOIH), an important member of the crustacean hyperglycemic hormone (CHH) neuropeptide family, mediates vitellogenesis and reproduction. In this study, EsMOIH was isolated from the eyestalk of Eriocheir sinensis. Sequence analysis suggested that EsMOIH belongs to CHH subfamily II and contains a conserved CHH/molt-/vitellogenesis-inhibiting hormone (MIH/VIH) domain and six conserved cysteines forming three disulfide bonds. The findings corroborated the results of phylogenetic analysis. Quantitative polymerase chain reaction (qPCR) analysis suggested that EsMOIH was ubiquitously expressed in all the examined tissues, but was highly specifically expressed in the hepatopancreas. The hepatopancreatic transcription of EsMOIH decreased significantly at stages III and IV, but increased thereafter and was highest at stage V. EsMOIH transcription was strongly negatively correlated with the hepatopancreatic expression of vitellogenin (EsVg), suggesting that EsMOIH could partake in inhibiting vitellogenesis in the hepatopancreas of E. sinensis. The RNA interference (RNAi)-mediated specific knockdown of EsMOIH significantly reduced EsMOIH expression in vitro and in vivo, but markedly increased EsVg expression in the ovary and hepatopancreas. Altogether, the results suggested that EsMOIH partakes in inhibiting vitellogenesis in E. sinensis. [ABSTRACT FROM AUTHOR]

To improve the quality of reproduction in Eurasian perch, Perca fluviatilis L., which is a promising candidate for Eurasian freshwater aquaculture that is currently cultivated in recirculating aquaculture systems (RAS), investigating the hormones that mediate and affect reproduction in this species is indispensable. The literature defines a group of four major corticosteroids (11-deoxycorticosterone, 11-deoxycortisol, corticosterone and cortisol) that might mediate critical stages of reproduction in female perch. Unfortunately, neither the basic roles nor the kinetics of these four corticosteroids throughout the reproductive cycle of female perch have been well defined to date. In this study, we therefore elucidated the plasma kinetics of these four corticosteroids during the reproductive cycle of domesticated female perch while monitoring the expression of the different receptors and enzymes that mediate their production and possible functions. Additionally, we performed an in vitro experiment during late vitellogenesis to investigate the possible direct roles of these steroids during that stage. Our results revealed that these four corticosteroids were detectable throughout the reproductive cycle, and the levels of most of them (11-deoxycorticosterone, 11-deoxycortisol, and cortisol) fluctuated significantly depending on the stage of reproduction. 11-Deoxycorticosterone and 11-deoxycortisol exhibited their highest levels, 1.8 ng/ml and 58 ng/ml, respectively, at the beginning of the reproductive cycle. By the end of the reproductive cycle, 11-deoxycortisol and cortisol plasma levels exhibited a surge, reaching 58 ng/ml and 150 ng/ml, respectively. During the perch reproductive cycle, the corticosteroid receptor complex is not regulated only at the hormone level, as the expression levels of all corticosteroid receptor genes showed a progressive and similar decline. I n vitro exposure of vitellogenic oocytes to some of these corticosteroids (11-deoxycorticosterone and 11-deoxycortisol) induced an increase in yolk globule diameter and a decrease in the density of yolk globules, which indicates the involvement of both of these hormones in yolk globule coalescence. Taken together, these results implicate corticosteroids in the reproductive cycle, although the related cellular mechanisms remain to be investigated. • Plasma cortisol, 11-deoxycorticosterone and 11-deoxycortisol fluctuate throughout the whole reproductive cycle of Eurasian perch. • Like cortisol, plasma 11-deoxycortisol is measured at very high levels in Eurasian perch and shows elevation at the end of the reproductive period. • The gene expression of the gonadal receptors of four main corticosteroids drops progressively throughout the reproductive cycle of Eurasian perch. • Some corticosteroids (11-deoxycorticosterone and 11-deoxycortisol) appear to be possible mediators of yolk globules coalescence during vitellogenesis. [ABSTRACT FROM AUTHOR]

Simple Summary: Exopalaemon carinicauda is a typical representative of crustaceans. However, the mechanism of ovary early development remains obscure. The purpose of this study is to identify and verify important genes related to ovary development by transcriptome sequencing and two-color fluorescent in situ hybridization, respectively. The result would provide clues for further study of ovary development in crustaceans. Eyestalk ablation is an effective method to promote ovarian development in crustaceans. Herein, we performed transcriptome sequencing of ovary and hepatopancreas tissues after eyestalk ablation in Exopalaemon carinicauda to identify genes related to ovarian development. Our analyses led to the identification of 97,383 unigenes and 190,757 transcripts, with an average N50 length of 1757 bp. In the ovary, four pathways related to oogenesis and three related to oocyte rapid growth were enriched. In the hepatopancreas, two vitellogenesis-associated transcripts were identified. Furthermore, short time-series expression miner (STEM) and gene ontology (GO) enrichment analyses revealed five terms related to gamete generation. In addition, two-color fluorescent in situ hybridization results suggested that dmrt1 might play a vital role in oogenesis during the early stage of ovarian development. Overall, our insights should support future studies focusing on investigating oogenesis and ovarian development in E. carinicauda. [ABSTRACT FROM AUTHOR]

The mud crab, Scylla paramamosain, has abundant nutrients in the ovary, where numerous lipids accumulate during ovarian maturation. However, the mechanism behind the accumulation of lipids in the ovary of mud crab during ovarian maturation is largely unknown. This study conducted a comparative transcriptome analysis of the ovaries of mud crabs at various stages of ovarian maturation. A total of 63.69 Gb of clean data was obtained, with a Q30 of 93.34%, and 81,893 unigenes were identified, including 10,996 differentially expressed genes (DEGs). After KEGG enrichment of these DEGs, MAPK signaling pathway was significantly enriched during vitellogenesis. Moreover, the expression levels of genes involved in carbohydrate, amino acid, and lipid metabolism were found to be higher during vitellogenesis. The two genes (Sp-Eip75B and Sp-Eip78C) that are homologous to the vertebrate gene PPARγ in the PPAR signaling pathway, were identified. Additionally, genes in MAPK signaling pathway might regulate lipid metabolism through PPAR signaling pathway based on Protein-Protein Interaction (PPI) network. These findings suggest that MAPK signaling pathway plays a critical role in lipid metabolism in the ovary during vitellogenesis, which provides new insights into the mechanism of lipid accumulation during ovarian maturation in mud crabs. [ABSTRACT FROM AUTHOR]

Commonly, female European eels are injected weekly with pituitary extract (PE) from carp (CPE) or salmon (SPE) to induce sexual maturation. However, a PE is a mixture of gonadotropins and other hormones that are not specific for eel and rapidly cleared from circulation. The aim of this study was therefore to test the effects of highly stable eel-specific recombinant gonadotropins (rGTHs) on oocyte development in vitro and in vivo in European eels. For the in vitro trial, the dose–effect responses of maturing eel oocytes on CPE and recombinant luteinizing hormone (rLH) were studied before and after 12 and 18 h of incubation. For the in vivo experiment, sexual maturation was stimulated by treatment with (i) CPE, (ii) recombinant follicle-stimulating hormone (rFSH) followed by CPE and (iii) rFSH followed by rLH. For the in vitro experiment, the expression of the nuclear progestin receptor 2 (pgr2) was induced by rLH, implying that rLH was preparing the oocyte for ovulation. For the in vivo experiment, the females treated with rGTHs had high gonadosomatic index (GSI) values (rFSH-CPE: 75, 77; rFSH-rLH: 80) in comparison with the females injected with CPE (50–60), suggesting that rFSH strongly induced vitellogenic growth. Larvae were produced for all treatment groups and for the first time by rGTH treatment alone but dose and timing still need optimization. [ABSTRACT FROM AUTHOR]

This study aims to determine the vitelogenesis activity by FSH hormone induction in the early stage of gonadal maturation. The treatament is a differences FSH dose with indicators of vitelogenesis activity were estradiol-17β levels in the blood, Hepatosomatic index (HSI) and Gonadosomatic Index (GSI). The study used a treatment A : Dose FSH at 1.0 ml/kg; B: Dose FSH at 1.25 ml/kg; C : Dose FSH at 1.5 ml/kg; control: nothing induction. The results showed that there was an effect of FSH dose on the estradiol 17β levels, HSI and GSI, there ongoing vitelogenesis activity. The trend of the GSI and HSI has a close correlation to levels of estradiol 17β, with the best dose of the hormone is 1.25 ml/kg resulting levels of esradiol 17β amounting to 63.35 pg / ml and the GSI and HSI are 3.45% and 1.48%. Furthermore, the best treatment successively resulted in increased levels of estradiol 17β, HSI and GSI in treatment C of 41.77 pg/ml, 1.48%, 3.24%; A : 28.01 pg / ml, 1.14 %, 3.42%;. FSH is suitable for reproductive domestication in Channa marulioides. [ABSTRACT FROM AUTHOR]