Gustavus, a positive regulator in arthropod reproduction, features a conserved SPRY and a C-terminal SOCS box domain and belongs to the SPSB protein family. The SPSB family, encompassing SPSB1 to SPSB4, plays pivotal roles in higher animals, including immune response, apoptosis, growth, and stress responses. In Neocaridina denticulata sinensis, alternative splicing yielded two NdGustavus isoforms, NdGusX1 and NdGusX2, with distinct expression patterns-high in ovaries and muscles, respectively, and across all ovarian germ cells. These isoforms showed similar expression dynamics during embryogenesis and significant upregulation post-copper ion exposure (P < 0.05). The in situ hybridization result elucidated that NdGusX1 and NdGusX2 were expressed across the germ cell spectrum in the ovary, with NdGusX1 showing enhanced expression in oogonia and primary oocytes. In addition, RNA interference revealed functional complementation in ovaries and potential functional differentiation in muscles. Knockdown of NdGusX1 and NdGusX2 potentially disrupted endogenous vitellogenin synthesis, regulating vitellogenesis and reducing mature oocyte volume, affecting follicular cavity occupation. This study provides a theoretical framework for understanding the biological functions of the SPSB family in crustacean ovarian maturation.

Delivering molecular tools into oocytes is essential for developmental and reproductive biology. Microinjection, the conventional method, is equipment-intensive, often technically challenging, and 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 vitellogenin-derived fusion proteins to enable efficient cargo delivery via receptor-mediated endocytosis. We demonstrate its utility by delivering Cas9/sgRNA complexes in two distantly related species for gene knockout.

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.

Female mosquitoes produce eggs in gonadotrophic cycles that are divided between a previtellogenic and vitellogenic phase. Previtellogenic females consume water and sugar sources like nectar while also being attracted to hosts for blood feeding. Consumption of a blood meal activates the vitellogenic phase, which produces mature eggs and suppresses host attraction. In this study, we tested the hypothesis that neuropeptide Y-like hormones differentially modulate host attraction behavior in the mosquito Aedes aegypti. A series of experiments collectively indicated that enteroendocrine cells (EECs) in the posterior midgut produce and release neuropeptide F (NPF) into the hemolymph during the previtellogenic phase which stimulates attraction to humans and biting behavior. Consumption of a blood meal, which primarily consists of protein by dry weight, down-regulated NPF in EECs until mature eggs developed, which was associated with a decline in hemolymph titer. NPF depletion depended on protein digestion but was not associated with EEC loss. Other experiments showed that neurons in the terminal ganglion extend axons to the posterior midgut and produce RYamide, which showed evidence of increased secretion into circulation after a blood meal. Injection of RYamide-1 and -2 into previtellogenic females suppressed host attraction, while coinjection of RYamides with or without short NPF-2 also inhibited the host attraction activity of NPF. Overall, our results identify NPF and RYamide as gut-associated hormones in A. aegypti that link host attraction behavior to shifts in diet during sequential gonadotrophic cycles.

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.

To enhance our understanding of teleost reproductive physiology, we identified six Sichuan bream (Sinibrama taeniatus) vitellogenin genes (vtg1-6) and characterized their sequence structures. We categorized them into type Ⅰ (vtg1,4,5 and 6), type Ⅱ (vtg2) and type Ⅲ (vtg3) based on differences in their subdomain structure. The promoter sequence of vtgs has multiple estrogen response elements, and their abundance appears to correlate with the responsiveness of vtg gene expression to estrogen. Gene expression analyses revealed that the vitellogenesis of Sichuan bream involves both heterosynthesis and autosynthesis pathways, with the dominant pathway originating from the liver. The drug treatment experiments revealed that 17β-estradiol (E2) tightly regulated the level of vtg mRNA in the liver. Feeding fish with a diet containing 100 μg/g E2 for three weeks significantly induced vtg gene expression and ovarian development, leading to an earlier onset of vitellogenesis. Additionally, it was observed that the initiation of vtg transcription required E2 binding to its receptor, a process primarily mediated by estrogen receptor alpha in Sichuan bream. The findings of this study provide novel insights into the molecular information of the vitellogenin gene family in teleosts, thereby contributing to the regulation of gonadal development in farmed fish.

Diet significantly affects reproductive outcomes across species, yet the precise effects of macronutrient compositions beyond caloric intake on reproductive aging are understudied. Existing literature presents conflicting views on the fertility impacts of nutrient-rich versus nutrient-poor developmental diets, underscoring a notable research gap. This study addresses these gaps by examining effects of isocaloric diets with varied protein-to-carbohydrate ratios during both developmental and adult stages on reproductive aging of a large, outbred Drosophila melanogaster population (n = ∼2100). Our results clearly demonstrate an age-dependent dietary impact on reproductive output, initially dominated by the developmental diet, then by a combination of developmental and adult diets in early to mid-life, and ultimately by the adult diet in later life. Importantly, we found that the effects of developmental and adult diets on reproductive output are independent, with no significant interaction. Further investigations into the mechanisms revealed that the effect of developmental diet on fecundity is regulated via ovarioles formation and vitellogenesis; while, the effect of adult diet on fecundity is mostly regulated only via vitellogenesis. These insights resolve disputes in the literature about dietary impacts on fertility and offer valuable perspectives for optimizing fertility strategies in improving public health and conservation efforts in this changing world.

Modern insects have inhabited the earth for hundreds of millions of years, and part of their successful adaptation lies in their many reproductive strategies. Insect reproduction is linked to a high metabolic rate that provides viable eggs in a relatively short time. In this context, an accurate interplay between the endocrine system and the nutrients synthetized and metabolized is essential to produce healthy offspring. Lipids guarantee the metabolic energy needed for egg formation and represent the main energy source consumed during embryogenesis. Lipids availability is tightly regulated by a complex network of endocrine signals primarily controlled by the central nervous system (CNS) and associated endocrine glands, the corpora allata (CA) and corpora cardiaca (CC). This endocrine axis provides hormones and neuropeptides that significatively affect tissues closely involved in successful reproduction: the fat body, which is the metabolic center supplying the lipid resources and energy demanded in egg formation, and the ovaries, where the developing oocytes recruit lipids that will be used for optimal embryogenesis. The post-genomic era and the availability of modern experimental approaches have advanced our understanding of many processes involved in lipid homeostasis; therefore, it is crucial to integrate the findings of recent years into the knowledge already acquired in the last decades. The present chapter is devoted to reviewing major recent contributions made in elucidating the impact of the CNS/CA/CC-fat body-ovary axis on lipid metabolism in the context of insect reproduction, highlighting areas of fruitful research.

Sea spiders (Pycnogonida) are marine chelicerates. Current pycnogonid phylogeny based on molecular data remains uncertain and contradicts traditional morphological perspectives. To resolve this conflict, understanding their inner anatomy is crucial. The reproductive system of sea spiders shows promise as a source of phylogenetic signal, yet our knowledge in this area is limited. This study presents the first description of the whole female reproductive system of a sea spider at the ultrastructural level. We suggest a more detailed functional regionalization of the ovary based on the ovarian wall ultrastructure and distribution of oocyte developmental stages. Meiosis begins in the germarium, and oocytes progress to the vitellarium through a transportational zone. Vitellogenic oocytes extend through the vitellarium wall, connected with it by a stalk - specialized cells. Balbiani bodies are present in early vitellogenic oocytes but dissipate later. The formation of the vitelline envelope, yolk, and fertilization envelope involves functionally diverse RER vesicles. The study also identifies a reproductive sinus as a separate haemocoel compartment that may enhance nutrient concentration near vitellogenic oocytes. Additionally, oviduct and gonopore glands are described in the female of P. femoratum, although their specific functions and prevalence in other sea spider species remain unclear.

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.