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Functions of vitellogenins have been in the limelight of fish reproductive physiology research for decades. The Vtg system of acanthomorph teleosts consists of two complete forms of Vtgs (VtgAa and VtgAb) and an incomplete form, VtgC. Insufficient uptake and processing of Vtgs and their yolk proteins lead to inadequate oocyte hydration ensuing failure in acquisition of egg buoyancy and early developmental deficiencies. This review presents a summary of our studies on utilization of multiple Vtgs in species with different egg buoyancy characteristics, as examples. Studies of moronids revealed limited degradation of all three forms of lipovitellin heavy chain derived from their three respective forms of Vtg, by which they contribute to the free amino acid pool driving oocyte hydration during oocyte maturation. In later studies, CRISPR/Cas9 was employed to invalidate zebrafish type I, type II and type III Vtgs, which are orthologs of acanthamorph VtgAa, VtgAb and VtgC, respectively. Results revealed type I Vtg to have essential developmental and nutritional functions in both late embryos and larvae. Genomic disturbance of type II Vtg led to high mortalities during the first 24 h of embryonic development. Despite being a minor form of Vtg in zebrafish and most other species, type III Vtg was also found to contribute essentially to the developmental potential of zebrafish zygotes and early embryos. Apart from severe effects on progeny survival, these studies also disclosed previously unreported regulatory effects of Vtgs on fecundity and fertility, and on embryo hatching. We recently utilized parallel reactions monitoring based liquid chromatography tandem mass spectrometry to assess the processing and utilization of lipovitellins derived from different forms of Vtg in Atlantic halibut and European plaice. Results showed the Lv heavy chain of VtgAa (LvHAa) to be consumed during oocyte maturation and the Lv light chain of VtgAb (LvLAb) to be utilized specifically during late larval stages, while all remaining YPs (LvLAa, LvHAb, LvHC, and LvLC) were utilized during or after hatching up until first feeding in halibut. In plaice, all YPs except LvHAa, which similarly to halibut supports oocyte maturation, are utilized from late embryo to late larval development up until first feeding. The collective findings from these studies affirm substantial disparity in modes of utilization of different types of Vtgs among fish species with various egg buoyancy characteristics, and they reveal previously unknown regulatory functions of Vtgs in maintenance of reproductive assets such as maternal fecundity and fertility, and in embryonic hatching. Despite the progress that has been made over the past two decades by examining multiple Vtgs and their functions, a higher complexity of these systems with much greater diversity between species in modes of Vtg utilization is now evident. Further research is needed to reveal novel ways each species has evolved to utilize these complex multiple Vtg systems and to discover unifying principles for this evolution in fishes of diverse lineages, habitats and life history characteristics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The vitellogenin receptor (VgR) is essential for the uptake and transport of the yolk precursor, vitellogenin (Vg). Vg is synthesized in the fat body, released in the hemolymph, and absorbed in the ovaries, via receptor-mediated endocytosis. Besides its important role in the reproductive pathway, Vg occurs in nonreproductive worker honey bee, suggesting its participation in other pathways. The objective was to verify if the VgR occurs in the hypopharyngeal glands of Apis mellifera workers and how Vg is internalized by these cells. VgR occurrence in the hypopharyngeal glands was evaluated by qPCR analyses of VgR and immunohistochemistry in workers with different tasks. The VgR gene is expressed in the hypopharyngeal glands of workers with higher transcript levels in nurse honey bees. VgR is more expressed in 11-day-old workers from queenright colonies, compared to orphan ones. Nurse workers with developed hypopharyngeal glands present higher VgR transcripts than those with poorly developed glands. The immunohistochemistry results showed the co-localization of Vg, VgR and clathrin (protein that plays a major role in the formation of coated vesicles in endocytosis) in the hypopharyngeal glands, suggesting receptor-mediated endocytosis. The results demonstrate that VgR performs the transport of Vg to the hypopharyngeal glands, supporting the Ovary Ground Plan Hypothesis and contributing to the understanding of the role of this gland in the social context of honey bees., (© 2024 Wiley Periodicals LLC.)

The caligid ectoparasite, Caligus rogercresseyi, is one of the main concerns in the Chilean salmon industry. The molecular mechanisms displayed by the parasite during the reproductive process represent an opportunity for developing novel control strategies. Vitellogenin is a multifunctional protein recognized as a critical player in several crustaceans' biological processes, including reproduction, embryonic development, and immune response. This study aimed to characterize the C. rogercresseyi vitellogenins, including discovering novel transcripts and regulatory mechanisms associated with microRNAs. Herein, vitellogenin genes were identified by homology analysis using the reference sea louse genome, transcriptome database, and arthropods vitellogenin-protein database. The validation of expression transcripts was conducted by RNA nanopore sequencing technology. Moreover, fusion gene profiling, miRNA target analysis, and functional validation were performed using luciferase assay. Six putative vitellogenin genes were identified in the C. rogercresseyi genome with high homology with other copepods vitellogenins. Furthermore, miR-996 showed a putative role in regulating the Cr_Vitellogenin1 gene, which is highly expressed in females. Moreover, vitellogenin-fusion genes were identified in adult stages and highly regulated in males, demonstrating sex-related expression patterns. In females, the identified fusion genes merged with several non-vitellogenin genes involved in biological processes of ribosome assembly, BMP signaling pathway, and biosynthetic processes. This study reports the genome array of vitellogenins in C. rogercresseyi for the first time, revealing the putative role of fusion genes and miRNA regulation in sea lice biology., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Juvenile hormone (JH) acts in the regulation of caste differentiation between queens and workers (i.e., with or without reproductive capacity) during vitellin synthesis and oogenesis in social insects. However, the regulatory mechanisms have not yet been elucidated. Here, we identified a highly expressed microRNA (miRNA), miR-1175-3p, in the red imported fire ant, Solenopsis invicta. We found that miR-1175-3p is prominently present in the fat bodies and ovaries of workers. Furthermore, miR-1175-3p interacts with its target gene, broad-complex core (Br-C), in the fat bodies. By utilizing miR-1175-3p agomir, we successfully suppressed the expression of the Br-C protein in queens, resulting in reduced vitellogenin expression, fewer eggs, and poorly developed ovaries. Conversely, decreasing miR-1175-3p levels led to the increased expression of Br-C and vitellogenin in workers, triggering the "re-development" of the ovaries. Moreover, when queens were fed with JH, the expression of miR-1175-3p decreased, whereas the expression of vitellogenin-2 and vitellogenin-3 increased. Notably, the suppression of fertility in queens caused by treatment with agomir miR-1175-3p was completely rescued by the increased vitellogenin expression induced by being fed with JH. These results suggest the critical role of miR-1175-3p in JH-regulated reproduction, shedding light on the molecular mechanism underlying miRNA-mediated fecundity in social insects and providing a novel strategy for managing S. invicta., (© 2023 Institute of Zoology, Chinese Academy of Sciences.)

Chlorothalonil is a broad-spectrum organochlorine fungicide widely employed in agriculture to control fungal foliar diseases. This fungicide enters aquatic environments through the leaching process, leading to toxicity in non-target organisms. Organic contaminants can impact organism reproduction as they have the potential to interact with the neuroendocrine system. Although there are reports of toxic effects of chlorothalonil, information regarding its impact on reproduction is limited. The aim of the present study was to evaluate the influence of chlorothalonil on male reproductive physiology using the zebrafish (Danio rerio) as ecotoxicological model. Zebrafish were exposed for 7 days to two concentrations of chlorothalonil (0.1 and 10 μg/L) along with a control group (with DMSO - 0.001%). Gene expression of hypothalamus-pituitary-gonad axis components (gnrh2, gnrh3, lhr, fshr, star, hsd17b1, hsd17b3, and cyp19a1), as well as hepatic vitellogenin concentration were assessed. In sperm cells, reactive oxygen species (ROS) content, lipid peroxidation (LPO), mitochondrial functionality, and membrane integrity and fluidity were evaluated. Results indicate that exposure to the higher concentration of chlorothalonil led to a reduction in brain gnr2 expression. In gonads, mRNA levels of lhr, star, and hsd17b1 were decreased at both chlorothalonil concentrations tested. Similarly, hepatic vitellogenin concentration was reduced. Regarding sperm cells, a decreased ROS level was observed, without significant difference in LPO level. Additionally, a higher mitochondrial potential and lower membrane fluidity were observed in zebrafish exposed to chlorothalonil. These findings demonstrate that chlorothalonil acts as an endocrine disruptor, influencing reproductive control mechanisms, as evidenced by changes in expression of genes HPG axis, as well as hepatic vitellogenin concentration. Furthermore, our findings reveal that exposure to this contaminant may compromise the reproductive success of the species, as it affected sperm quality parameters., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Alkylphenols, such as nonylphenol and 4-tert-octylphenol (OP), are byproducts of the biodegradation of alkylphenol ethoxylates and present substantial ecological and health risks in aquatic environments and higher life forms. In this context, our study aimed to explore the effect of OP on reproductive endocrine function in both female and male zebrafish. Over a period of 21 days, the zebrafish were subjected to varying concentrations of OP (0, 0.02, 0.1, and 0.5 μg/L), based on the lowest effective concentration (EC10 = 0.48 μg/L) identified for zebrafish embryos. OP exposure led to a pronounced increase in hepatic vitellogenin (vtg) mRNA expression and 17β-estradiol biosynthesis in both sexes. Conversely, OP exhibits anti-androgenic properties, significantly diminishes gonadal androgen receptor (ar) mRNA expression, and reduces endogenous androgen (testosterone and 11-ketotestosterone) levels in male zebrafish. Notably, cortisol and thyroid hormone (TH) levels demonstrated concentration-dependent elevations in zebrafish, influencing the regulation of gonadal steroid hormones (GSHs). These findings suggest that prolonged OP exposure may result in sustained reproductive dysfunction in adult zebrafish, which is largely attributable to the intricate reciprocal relationship between hormone levels and the associated gene expression. Our comprehensive biological response analysis of adult zebrafish offers vital insights into the reproductive toxicological effects of OP, thereby enriching future ecological studies on aquatic systems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Vitellogenin (Vg) is a female-specific egg-yolk precursor protein, synthesized in the liver of fish in response to estrogens. In the present study, complete gene of phosvitinless vitellogenin (vgc) was sequenced, its 3D structure was predicted and validated by web-based softwares. The complete nucleotide sequence of vgc was 4126 bp which encodes for 1272 amino acids and showed the presence of three conserved domains viz. LPD_N, DUF1943 and DUF1944. The retrieved amino acid sequence of VgC protein was subjected to in silico analysis for understanding the structural and functional properties of protein. mRNA levels of multiple vg genes have also been quantified during annual reproductive cycle employing qPCR. A correlation has been observed between seasonal changes in gonadosomatic index with estradiol levels and hepatic expression of three types of vg genes (vga, vgb, vgc) during ovarian cycle of murrel. During preparatory phase, when photoperiod and temperature are low; low titre of E 2 in blood induces expression of vgc gene. A rapid increase in the levels of E 2 favours induction of vgb and vga genes in liver of murrel during early pre-spawning phase when photoperiod is long and temperature is high in nature. These results suggest that among three vitellogenin proteins, VgC is synthesized earlier than VgA and VgB during oogenesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In insects, vitellogenin (Vg) is generally viewed as a female-specific protein. Its primary function is to supply nutrition to developing embryos. Here, we reported Vg from the male adults of a natural predator, Chrysopa pallens. The male Vg was depleted by RNAi. Mating with Vg-deficient male downregulated female Vg expression, suppressed ovarian development and decreased reproductive output. Whole-organism transcriptome analysis after male Vg knockdown showed no differential expression of the known spermatogenesis-related regulators and seminal fluid protein genes, but a sharp downregulation of an unknown gene, which encodes a testis-enriched big protein (Vcsoo). Separate knockdown of male Vg and Vcsoo disturbed the assembly of spermatid cytoplasmic organelles in males and suppressed the expansion of ovary germarium in mated females. These results demonstrated that C. pallens male Vg signals through the downstream Vcsoo and regulates male and female reproduction., (© 2023 Royal Entomological Society.)

Females from many mosquito species feed on blood to acquire nutrients for egg development. The oogenetic cycle has been characterized in the arboviral vector Aedes aegypti, where after a bloodmeal, the lipid transporter lipophorin (Lp) shuttles lipids from the midgut and fat body to the ovaries, and a yolk precursor protein, vitellogenin (Vg), is deposited into the oocyte by receptor-mediated endocytosis. Our understanding of how the roles of these two nutrient transporters are mutually coordinated is however limited in this and other mosquito species. Here, we demonstrate that in the malaria mosquito Anopheles gambiae, Lp and Vg are reciprocally regulated in a timely manner to optimize egg development and ensure fertility. Defective lipid transport via Lp knockdown triggers abortive ovarian follicle development, leading to misregulation of Vg and aberrant yolk granules. Conversely, depletion of Vg causes an upregulation of Lp in the fat body in a manner that appears to be at least partially dependent on target of rapamycin (TOR) signaling, resulting in excess lipid accumulation in the developing follicles. Embryos deposited by Vg-depleted mothers are completely inviable, and are arrested early during development, likely due to severely reduced amino acid levels and protein synthesis. Our findings demonstrate that the mutual regulation of these two nutrient transporters is essential to safeguard fertility by ensuring correct nutrient balance in the developing oocyte, and validate Vg and Lp as two potential candidates for mosquito control., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Stryapunina et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Vitellogenesis is the most important process in animal reproduction, in which yolk proteins play a vital role. Among multiple yolk protein precursors, vitellogenin (Vtg) is a well-known major yolk protein (MYP) in most oviparous animals. However, the nature of MYP in the freshwater gastropod snail Biomphalaria glabrata remains elusive. In the current study, we applied bioinformatics, tissue-specific transcriptomics, ovotestis-targeted proteomics, and phylogenetics to investigate the large lipid transfer protein (LLTP) superfamily and ferritin-like family in B. glabrata. Four members of LLTP superfamily (BgVtg1, BgVtg2, BgApo1, and BgApo2), one yolk ferritin (Bg yolk ferritin), and four soma ferritins (Bg ferritin 1, 2, 3, and 4) were identified in B. glabrata genome. The proteomic analysis demonstrated that, among the putative yolk proteins, BgVtg1 was the yolk protein appearing in the highest amount in the ovotestis, followed by Bg yolk ferritin. RNAseq profile showed that the leading synthesis sites of BgVtg1 and Bg yolk ferritin are in the ovotestis (presumably follicle cells) and digestive gland, respectively. Phylogenetic analysis indicated that BgVtg1 is well clustered with Vtgs of other vertebrates and invertebrates. We conclude that, vitellogenin (BgVtg1), not yolk ferritin (Bg yolk ferritin), is the major yolk protein precursor in the schistosomiasis vector snail B. glabrata., (© 2024. The Author(s).)

In this study, a vitellogenin like1 gene (SfVg-like1) in Sogatella furcifera was identified. The open reading frame (ORF) encoded 1,321 amino acid sequence. Structure analysis reveals that the amino acid sequence of SfVg-like1 has 3 conserved LPD_N, DUF1943 and VWFD domains. Phylogenetic analyses showed that SfVg-like1 was clustered in the same branch with the Vg-like1 of Nilaparvata lugens (100% bootstrap value) compared with other Hemiptera insects Vgs associated with vitellogenesis. Temporo-spatial expression analyses showed that SfVg-like1 expressed during all stages, and in both genders. The relative expression levels of SfVg-like1 mRNA were higher in adults than in nymph developmental stages. The knockdown of SfVg-like1 gene resulted in the inhibition of the ovarian development in female adults, whereas the morphology of the testis in male adults was not been affected. The silence of SfVg-like1 could decrease the relative expression levels of target of rapamycin (SfTOR, GenBank MW193765) and vitellogenin (SfVg, GenBank MH271114) genes significantly in female adults. However, the knockdown of SfTOR or SfVg genes in female adults did not affect the transcript level of SfVg-like1. Therefore, it demonstrated that SfVg-like1 might locate on the upstream signaling pathways of SfTOR and SfVg. These results demonstrate that SfVg-like1 is essential for S. furcifera reproduction, and it could be the potential target for the control of this pest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America.)

Perchlorate, a widespread environmental contaminant originating from various industrial applications, agricultural practices, and natural sources, poses potential risks to ecosystems and human health. While previous studies have highlighted its influence on the thyroid endocrine system and its impact on gonadal maturation, reproduction, and sex hormone synthesis, the specific interplay between thyroid and steroid hormones, in this context, remains largely unexplored. Therefore, this study was undertaken to investigate the adverse effects and underlying mechanisms triggered by exposure to sodium perchlorate (SP) on reproductive endocrine activity in zebrafish. For 21 d, the fish were exposed to test SP concentrations (0, 3, 30, 300 mg/L), which were determined based on the exposure concentrations that induced various toxic effects in the fish, considering naturally occurring concentrations. Exposure to SP, except at 3 mg/L in males, significantly decreased the production of thyroid hormone (TH) in both female and male zebrafish. Moreover, gonadal steroid levels were markedly reduced in both sexes. The expression of hepatic vitellogenin (VTG) mRNA in female zebrafish was significantly decreased, whereas aromatase activity in male zebrafish was significantly elevated in the SP exposure groups. The reduced levels of THs and gonadal steroid hormones were strongly correlated. Abnormal responses to SP exposure led to reduced reproductive success in the 300 mg/L SP exposure group. These findings indicate that prolonged and continuous exposure to a specific concentration of SP may lead to long-term reproductive problems in zebrafish, primarily through hormonal imbalances and suppression of hepatic VTG mRNA expression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The present study evaluates the endocrine effect in flatfish through vitellogenin (vtg) gene expression and its association with pollutants data obtained from fish muscle and sediment from two regions in the Gulf of Mexico (GoM): Perdido Fold Belt (northwestern) and the Yucatan Peninsula (southeast). The results revealed induction of vtg in male flatfish in both geographical regions with different levels and patterns of distribution per oceanographic campaign (OC). In the Perdido Fold Belt, vtg was observed in male fish during four OC (carried out in 2016 and 2017), positively associated with Pb, V, Cd and bile metabolites (hydroxynaphthalene and hydroxyphenanthrene). In the Yucatan Peninsula, the induction of vtg in males was also detected in three OC (carried out in 2016 and 2018) mainly associated with Ni, Pb, Al, Cd, V and polycyclic aromatic hydrocarbons. Ultimately, estrogenic alterations could affect reproductive capacity of male flatfish in the GoM., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Microsporidia are a group of obligate intracellular parasites that infect almost all animals, causing serious human diseases and major economic losses to the farming industry. Nosema bombycis is a typical microsporidium that infects multiple lepidopteran insects via fecal-oral and transovarial transmission (TOT); however, the underlying TOT processes and mechanisms remain unknown. Here, we characterized the TOT process and identified key factors enabling N. bombycis to invade the ovariole and oocyte of silkworm Bombyx mori. We found that the parasites commenced with TOT at the early pupal stage when ovarioles penetrated the ovary wall and were exposed to the hemolymph. Subsequently, the parasites in hemolymph and hemolymph cells firstly infiltrated the ovariole sheath, from where they invaded the oocyte via two routes: (I) infecting follicular cells, thereby penetrating oocytes after proliferation, and (II) infecting nurse cells, thus entering oocytes following replication. In follicle and nurse cells, the parasites restructured and built large vacuoles to deliver themselves into the oocyte. In the whole process, the parasites were coated with B. mori vitellogenin (BmVg) on their surfaces. To investigate the BmVg effects on TOT, we suppressed its expression and found a dramatic decrease of pathogen load in both ovarioles and eggs, suggesting that BmVg plays a crucial role in the TOT. Thereby, we identified the BmVg domains and parasite spore wall proteins (SWPs) mediating the interaction, and demonstrated that the von Willebrand domain (VWD) interacted with SWP12, SWP26 and SWP30, and the unknown function domain (DUF1943) bound with the SWP30. When disrupting these interactions, we found significant reductions of the pathogen load in both ovarioles and eggs, suggesting that the interplays between BmVg and SWPs were vital for the TOT. In conclusion, our study has elucidated key aspects about the microsporidian TOT and revealed the key factors for understanding the molecular mechanisms underlying this transmission., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The red imported fire ant ( Solenopsis invicta Buren) is a social pest species with a robust reproductive ability that causes extensive damage. Identification of the genes involved in queen fertility is critical in order to better understand the reproductive biology and screening for the potential molecular targets in S. invicta . Here, we used the mRNA deep sequencing (RNA-seq) approach to identify differentially expressed genes (DEGs) in the transcriptomes of three reproductive caste types of S. invicta , including queen (QA) and winged female (FA) and male (MA) ants. The genes that were specific to and highly expressed in the queens were then screened, and the Vg2 and Vg3 genes were chosen as targets to explore their functions in oogenesis and fertility. A minimum of 6.08 giga bases (Gb) of clean reads was obtained from all samples, with a mapping rate > 89.78%. There were 7524, 7133, and 977 DEGs identified in the MA vs. QA, MA vs. FA, and FA vs. QA comparisons, respectively. qRT-PCR was used to validate 10 randomly selected DEGs, including vitellogenin 2 ( Vg2 ) and 3 ( Vg3 ), and their expression patterns were mostly consistent with the RNA-seq data. The S. invicta Vgs included conserved domains and motifs that are commonly found in most insect Vgs. SiVg2 and SiVg3 were highly expressed in queens and winged females and were most highly expressed in the thorax, followed by the fat body, head, and epidermis. Evaluation based on a loss-of-function-based knockdown analysis showed that the downregulation of either or both of these genes resulted in smaller ovaries, less oogenesis, and less egg production. The results of transcriptional sequencing provide a foundation for clarifying the regulators of queen fertility in S. invicta . The functions of SiVg2 and SiVg3 as regulators of oogenesis highlight their importance in queen fecundity and their potential as targets of reproductive disruption in S. invicta control.

Inadequate gonadal maturation and poor spawning performance increasingly threaten the sustainability of shrimp aquaculture. Unraveling the mechanisms regulating ovarian development and maturation hence is critical to address industry challenges. Vitellogenin (Vtg), a precursor of yolk protein found in the hepatopancreas and ovary of shrimp, plays a key role in facilitating shrimp's oocyte maturation and embryonic development after oviposition. This study found that FpVtg was specifically expressed in F. penicillatus hepatopancreas and ovary. FpVtg was localized predominantly in the oocyte cytoplasm and distributed uniformly in the hepatopancreas tissue. Silencing FpVtg led to apoptosis in both hepatopancreas and ovary tissues. Furthermore, FpVtg depletion upregulated the expression of ovarian peritrophin 1, ovarian peritrophin 2, serine proteinase inhibitor 6, and juvenile hormone esterase-like carboxylesterase 1, while downregulated that of vitellogenin, delta-9 desaturase, and insulin-like receptor. KEGG pathway analysis implicated such as PI3K-AKT signaling, RNA transport, ECM-receptor interaction, hippo signaling, oocyte meiosis, and apoptosis were enriched and involved in ovarian development. These findings have provided insights into the FpVtg's reproductive role and the associated regulatory genes and pathways in F. penicillatus. This knowledge can contribute to establishing strategies to improve the breeding and aquaculture production of F. penicillatus by elucidating its vitellogenesis regulation in redtail prawn and other penaeid species. Further characterization of the implicated pathways and genes will clarify the intricacies underlying ovarian maturation., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Vitellogenin (VTG) is a biomarker for possible endocrine activity of chemicals acting via the estrogen, androgen, or steroidogenesis pathways. VTG is assessed in standardised fish guideline studies conducted for regulatory safety assessment of chemicals. VTG data can be highly variable leading to concerns for potential equivocal, false positive and/or negative outcomes. Consequently, additional fish testing may be required to address uncertainties in the VTG response, and possibly erroneous/missed identification of endocrine activity. To better understand the technical challenges of VTG assessment and reporting for regulatory purposes, a survey was sent to 27 testing laboratories performing these analyses. The survey results from 16 respondents (6 from the UK, 3 from the USA, and 7 from the EU) were analysed and discussed in a follow-up webinar. High variability in background VTG concentrations was widely acknowledged and thought to be associated with fish batch, husbandry, laboratory practices, and several methodological aspects. These include sample collection and storage, VTG quantification, data handling, and the benchmarks used for data acceptability. Information gathered in the survey provides a basis for improving and harmonizing the measurement of VTG in fish, and an opportunity to reassess the suitability of current acceptability criteria in test guidelines., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rebeccca J Brown, Grace Panter reports financial support was provided by Bayer AG. E.R. Salinas, L. Weltje, J.R. Wheeler, Y. Wolf, B. Holmes, and L. Lagadic are affiliated to companies that develop and manufacture agrochemicals. Authors’ affiliations do not imply competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The US Environmental Protection Agency (USEPA) is faced with long lists of chemicals that require hazard assessment. The present study is part of a larger effort to develop in vitro assays and quantitative structure-activity relationships applicable to untested chemicals on USEPA inventories through study of estrogen receptor (ER) binding and estrogen-mediated gene expression in fish. The present effort investigates metabolic activation of chemicals resulting in increased estrogenicity. Phenolphthalin (PLIN) was shown not to bind rainbow trout (Oncorhynchus mykiss) ER (rtER) in a competitive binding assay, but vitellogenin (Vtg) expression was induced in trout liver slices exposed to 10 -4 and 10 -3.7 M PLIN. Phenolphthalein (PLEIN), a metabolite of PLIN, was subsequently determined to be formed when slices were exposed to PLIN. It binds rtER with a relative binding affinity to 17β-estradiol of 0.020%. Slices exposed to PLEIN expressed Vtg messenger RNA (mRNA) at 10 -4.3 , 10 -4 , and 10 -3.7 M, with no detectable PLIN present. Thus, Vtg expression noted in PLIN slice exposures was explained by metabolism to PLEIN in trout liver slices. A second model chemical, 4,4'-methylenedianiline (MDA), was not shown to bind rtER but did induce Vtg mRNA production in tissue slices at 10 -4.3 , 10 -4 , and 10 -3.7 M in amounts nearly equal to reference estradiol induction, thus indicating metabolic activation of MDA. A series of experiments were performed to identify a potential metabolite responsible for the observed increase in activity. Potential metabolites hydroxylamine-MDA, nitroso-MDA, azo-MDA, and azoxy-MDA were not observed. However, acetylated MDA was observed and tested in both ER-binding and tissue slice Vtg induction assays. Environ Toxicol Chem 2023;42:2747-2757. © 2023 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA., (© 2023 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Effects of dietary protein quality on insect development (not just growth) are unclear. Dietary amino acid blends matching yolk proteins support reproduction and juvenile development in Drosophila melanogaster. We matched amino acids to vitellogenin and tested development of juvenile male lubber grasshoppers, which do not produce vitellogenin. Last instars were fed classic dry diets with amino acids substituted for proteins. Matching amino acids to vitellogenin allowed molting to adulthood, while an unmatched isonitrogenous diet did not. Health on dry diets was poor, so we developed wet diets with agar, horse feed, and amino acids. Juveniles fed these diets matched to vitellogenin developed comparably to juveniles fed lettuce. However, wet diets with amino acids dissimilar to vitellogenin (low-quality) slowed development but maintained size at adulthood. We observed no compensatory feeding on low-quality diets. Theory suggests accumulation of proteins permits development. To detect a threshold, we started last juvenile instars on high-quality diets, then abruptly switched them to low-qualities diets. When switched to the poor-quality diet at 6d, grasshoppers molted at a similar age (∼17d) to grasshoppers continuously on the high-quality diet. Total hemolymph proteins levels were unaffected by the timing of diet switches. Last, methionine is essential but can be noxious at high levels. Diets with low-quality protein except for methionine slowed growth early but did not alter the time or size at molt. Overall, the feeding threshold is solely due to essential amino acids, and low-quality protein diets slowed development but did not affect adult size., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

This study was done to investigate the effects of thymol, fumagillin, oxalic acid (Api-Bioxal) and hops extract (Nose-Go) on Nosema sp. spore load, the expression of vitellogenin ( vg ) and superoxide-dismutase-1 ( sod-1 ) genes and mortality of bees infected with N. ceranae . Five healthy colonies were assigned as the negative control, and 25 Nosema sp. infected colonies were assigned to five treatment groups including: the positive control: no additive to sirup; fumagillin 26.4 mg/L, thymol 0.1 g/L, Api-Bioxal 0.64 g/L and Nose-Go 5.0 g/L sirup. The reduction in the number of Nosema sp. spores in fumagillin, thymol, Api-Bioxal and Nose-Go compared to the positive control was 54, 25, 30 and 58%, respectively. Nosema sp. infection in all infected groups increased ( p  < .05) Escherichia coli population compared to the negative control. Nose-Go had a negative effect on lactobacillus population compared to other substances. Nosema sp. infection decreased vg and sod-1 genes expression in all infected groups compared to the negative control. Fumagillin and Nose-Go increased the expression of vg gene, and Nose-Go and thymol increased the expression of sod-1 gene than the positive control. Nose-Go has the potential to treat nosemosis if the necessary lactobacillus population is provided in the gut.

Vitellogenin (VTG), a biomarker for endocrine activity, is a mechanistic component of the regulatory assessment of potential endocrine-disrupting properties of chemicals. This review of VTG data is based on changes reported for 106 substances in standard fish species. High intra-study and inter-laboratory variability in VTG concentrations was confirmed, as well as discrepancies in interpretation of results based on large differences between fish in the dilution water versus solvent control, or due to the presence of outlier measurements. VTG responses in fish were ranked against predictions for estrogen receptor agonist activity and aromatase inhibition from bioactivity model output and ToxCast in vitro assay results, respectively. These endocrine mechanisms explained most of the VTG responses in the absence of systemic toxicity, the magnitude of the VTG response being proportional to the in vitro potency. Interpretation of the VTG data was sometimes confounded by an alternative endocrine mechanism of action. There was evidence for both false positive and negative responses for VTG synthesis, but overall, it was rare for substances without endocrine activity in vitro to cause a concentration-dependent VTG response in fish in the absence of systemic toxicity. To increase confidence in the VTG results, we recommend improvements in the VTG measurement methodologies and greater transparency in reporting of VTG data (including quality control criteria for assay performance). This review supports the application of New Approach Methodologies (NAMs) by demonstrating that endocrine activity in vitro from mammalian cell lines is predictive for in vivo VTG response in fish, suggesting that in vitro mechanistic data could be used more broadly in decision-making to help reduce animal testing., Competing Interests: Declaration of competing interest The employment affiliation of the authors is listed on the cover page. Rebecca J. Brown and Grace H. Panter are affiliated to wca environment Ltd., an independent consultancy providing objective advice on the risk assessment of chemicals in the environment and the workplace. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rebecca J Brown and Grace H. Panter both report financial support was provided by Bayer AG. Natalie Burden is affiliated to NC3Rs, an independent scientific organisation, tasked by Government with supporting the UK science base through the application of the 3Rs. Edward R. Salinas, Lennart Weltje, James R. Wheeler, Yvonne Wolf, and Laurent Lagadic are affiliated to companies that develop and manufacture agrochemicals, some of which having been included in the present review., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The specific functions and essentiality of type II vitellogenin (Vtg2) in early zebrafish development were investigated in this study. A vtg2-mutant zebrafish line was produced and effects of genomic disturbance were observed in F2 females and F3 offspring. No change in vtg2 transcript has been detected, however, Vtg2 abundance in F2 female liver was 5×, and in 1 hpf F3 vtg2-mutant embryos was 3.8× less than Wt (p < 0.05). Fecundity was unaffected while fertilization rate was more than halved in F2 vtg2-mutant females (p < 0.05). Hatching rate was significantly higher in F3 vtg2-mutant embryos in comparison to Wt embryos. Survival rate declined drastically to 29% and 18% at 24 hpf and 20 dpf, respectively, in F3 vtg2-mutant embryos. The introduced mutation caused vitelline membrane deficiencies, significant mortalities at early embryonic stages, and morphological abnormalities in the surviving F3 vtg2-mutant larvae. Overrepresentation of histones, zona pellucida proteins, lectins, and protein degradation related proteins in F3 vtg2-mutant embryos provide evidence to impaired mechanisms involved in vitellin membrane formation. Overall findings imply a potential function of Vtg2 in acquisition of vitellin membrane integrity, among other reproductive functions, and therefore, its essentiality in early zebrafish embryo development., (© 2023. The Author(s).)

Vitellogenin (Vg) is the precursor of vitellin, which is an important female-specific protein stored in oocytes as the major nutrient and energy sources for embryogenesis in oviparous animals. In this study, we performed comprehensive genome-wide analysis of Vg gene family in the prawn Macrobrachium rosenbergii, and eight Vg genes designated as MrVg1a, MrVg1b and MrVg2-7 were identified. MrVg1a clusters with the previously described MrVg1b near the end of chromosome 46 and MrVg2 is on the chromosome 42 while MrVg3-7 cluster on the chromosome 23. All the putative MrVg proteins are characterized by the presence of three conserved functional domains: LPD-N, DUF1943 and vWD. Phylogenetic analysis revealed that MrVg1a shares 93% identity with MrVg1b and groups together into a branch while MrVg2-7 group into another branch, suggesting that MrVg1a, 1b and MrVg2-7 might diversify from a common ancestral gene. All the corresponding MrVg transcripts especially for MrVg1 exhibit high expression in the female hepatopancreas at late vitellogensis stage but extremely low in the ovaries except MrVg5, indicating that hepatopancreas is the major site of MrVgs synthesis. In the male, interestingly, MrVg5 and MrVg6 are also highly expressed in the testis, suggesting their potential involvement in testicular development. Bilateral ablation of eyestalk significantly upregulate all the MrVgs mRNA in the female hepatopancreas and the MrVg1 in ovary, but have no effect on the expression of MrVg2-7 in the ovary, demonstrating that eyestalk hormones could promote the ovarian development mostly by inducing the synthesis of MrVgs in the hepatopancreas but rarely in the ovary. Our results provide new insights into the prawn MrVgs family and improve our understanding of the potential role for each member of the family in the gonadal development of M. rosenbergii., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

This study investigated the effects of different levels of hypoxia on the reproductive system of yellow catfish. Yellow catfish (Pelteobagrus fulvidraco) were exposed to three dissolved oxygen concentration levels: normoxia (6.5 ± 0.2 mg/L), moderate hypoxia (MH, 3.8 ± 0.3 mg/L) and severe hypoxia (SH, 1.9 ± 0.2 mg/L) for 30 days. The gonadosomatic index of males, not females, significantly decreased in the SH group. In the SH group, for the females, the ratio of vitellogenic follicles significantly decreased, whereas the number of atretic follicles significantly increased. In male fish, a significantly reduced number of spermatozoa was observed in both the MH and SH groups. Elevated apoptosis levels in the testes and ovaries were observed only in the SH group. Serum 17β-estradiol and vitellogenin levels in females and testosterone levels in males significantly decreased in the SH group. The concentration of 11-ketotestosterone in males significantly decreased in both the MH and SH groups. In female fish, dysregulated expression of the hypothalamic-pituitary-gonadal (HPG) axis, steroidogenesis genes, and hepatic genes related to vitellogenesis were observed only in the SH group. However, in male fish, moderate hypoxia altered the expression of HPG genes, including gnrh1, lhcgr, and amh. Moreover, the MH group significantly altered the expression of steroidogenesis genes like star, 17β-hsd, and cyp17a1. The results of this study suggest that severe hypoxia can cause reproductive defects in female and male yellow catfish. Moreover, the reproductive system of male yellow catfish is more sensitive to moderate hypoxia than that of female catfish. Our findings contribute to our understanding of the response of the teleost reproductive system to long-term hypoxia., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2023. Published by Elsevier Inc.)

Several adverse outcome pathways (AOPs) have linked molecular initiating events like aromatase inhibition, androgen receptor (AR) agonism, and estrogen receptor (ER) antagonism to reproductive impairment in adult fish. Estrogen receptor agonists can also cause adverse reproductive effects, however, the early key events (KEs) in an AOP leading to this are mostly unknown. The primary aim of this study was to develop hypotheses regarding the potential mechanisms through which exposure to ER agonists might lead to reproductive impairment in female fish. Mature fathead minnows were exposed to 1 or 10 ng 17α-ethynylestradiol (EE2)/L or 10 or 100 µg bisphenol A (BPA)/L for 14 d. The response to EE2 and BPA was contrasted with the effects of 500 ng/L of 17β-trenbolone (TRB), an AR agonist, as well as TRB combined with the low and high concentrations of EE2 or BPA tested individually. Exposure to 10 ng EE2/L, 100 µg BPA/L, TRB, or the various mixtures with TRB caused significant decreases in plasma concentrations of 17β-estradiol. Exposure to TRB alone caused a significant reduction in plasma vitellogenin (VTG), but VTG was unaffected or even increased in females exposed to EE2 or BPA alone or, in most cases, in mixtures with TRB. Over the course of the 14-d exposure, the only treatments that clearly did not affect egg production were 1 ng EE2/L and 10 µg BPA/L. Based on these results and knowledge of hypothalamic-pituitary-gonadal axis function, we hypothesize an AOP whereby decreased production of maturation-inducing steroid leading to impaired oocyte maturation and ovulation, possibly due to negative feedback or direct inhibitory effects of membrane ER activation, could be responsible for causing adverse reproductive impacts in female fish exposed to ER agonists., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

Background: Culex quinquefasciatus, a cosmopolitan, domestic, and highly anthropophilic mosquito, is a vector of pathogenic arboviruses such as West Nile virus and Rift Valley virus, as well as lymphatic filariasis. The current knowledge on its reproductive physiology regarding vitellogenin expression in different tissues is still limited., Objectives: In this study, we analysed the transcriptional profiles of vitellogenin genes in the fat body and ovaries of C. quinquefasciatus females during the first gonotrophic cycle., Methods: C. quinquefasciatus ovaries and/or fat bodies were dissected in different times during the first gonotrophic cycle and total RNA was extracted and used for reverse transcription polymerase chain reaction, quantitative real time-PCR, and in situ hybridisation., Findings: We confirmed the classical descriptions of the vitellogenic process in mosquitoes by verifying that vitellogenin genes are transcribed in the fat bodies of C. quinquefasciatus females. Using RNA in situ hybridisation approach, we showed that vitellogenin genes are also transcribed in developing ovaries, specifically by the follicle cells., Main Conclusions: This is the first time that vitellogenin transcripts are observed in mosquito ovaries. Studies to determine if Vg transcripts are translated into proteins and their contribution to the reproductive success of the mosquito need to be further investigated.

Plasticizers are broadly classified as phthalate or nonphthalate. Recently, acetyl tributyl citrate (ATBC), an environmentally friendly nonphthalate plasticizer, was revealed to have the ability to disrupt thyroid hormone activity in fish species. Therefore, we aimed to assess whether ATBC exhibits any sex hormone (i.e., androgenic or estrogenic) activities. First, we examined the effects of ATBC on gonadal sex differentiation. Subsequently, we analyzed the different expression of biomarker genes that respond to endocrine-disrupting chemicals (EDCs) with sexual hormone activity in the liver. We observed normal testes and ovaries after both XX and XY medakas were exposed to ATBC, indicating that ATBC is not an EDCs with strong sex hormone activity and that it does not induce intersex (testis-to-ova or ovo-to-testis) or sex changes in Japanese medaka. The vitellogenin 1 (vtg1) and vitellogenin 2 (vtg2) mRNA expression levels in the liver of XX medakas were significantly reduced compared with those in the control group, whereas the expression levels of these genes in the liver of XY medakas remained unchanged. Finally, we examined the changes in the expression of biomarker genes that respond to EDCs with sex hormone activity in the gonads. The expression levels of biomarker genes did not differ significantly from that of the control group, although the expression levels of gsdf mRNA tended to increase while that of aromatase mRNA tended to decrease in the ovary of XX medakas following ATBC exposure. Conversely, the expression levels of gsdf and aromatase mRNAs in the testis of XY medakas remained unchanged. These results suggest that ATBC does not exhibit estrogenic activity, although it may have weak androgenic activity or no sexual hormone activity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Honeybee diseases are a serious threat to beekeeping and pollination. Transgenerational immune priming (TGIP) has been attracting increasing attention as a promising strategy to protect honeybee colonies from infections. This study investigated whether feeding honeybees (Apis mellifera) with a heat-killed pathogen cocktail can provide them with transgenerational immunity to these pathogens. We found that vitellogenin (Vg) and defensin-1 were highly upregulated in nurse bees upon feeding them with a cocktail of heat-killed Ascosphaera apis and Paenibacillus larvae (A + P cocktail). Pathogen-pattern-recognition receptor genes in the Toll signaling pathway were upregulated in nurse bees upon ingestion of the A + P cocktail. In the nurse bees of the hives supplied with the A + P cocktail, Vg was upregulated in the fat body, and the defensin-1 expression and Vg uptake in the hypopharyngeal glands were induced. Consequently, the major proteins in royal jelly were upregulated. In addition, defensin-1 was upregulated in the queen larvae and young worker larvae in these hives. In correlation, the young worker larvae showed high pathogen resistance to P. larvae infection. Thus, our findings imply that introduction of a heat-killed pathogen cocktail into hives is an efficient strategy for conferring honeybees with social immunity through TGIP., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Chicken egg yolk granules (EYG) were the precipitated component of egg yolk after water dilution and centrifugation. Compared with egg yolk, EYG are rich in proteins, phospholipids, and minerals. In this study, an integrated proteomic analysis was carried out to in-depth mapping of the proteome, phosphoproteome, and N-glycoproteome of EYGs. After hydrolysis of the EYG total protein, the hydrolyzed peptides or the enriched phosphopeptides/glycopeptides were identified by liquid chromatography-tandem mass spectrometry. A total of 125 phosphorylation sites from 36 phosphoproteins and 244 N-glycosylation sites from 100 N-glycoproteins were identified in EYG. All 3 vitellogenins (precursors of egg yolk high-density lipoprotein) were heavily phosphorylated and N-glycosylated, of which 37 phosphorylation sites and 32 N-glycosylation sites were identified on vitellogenins-2. A Total of 30 N-glycosylation sites were identified on apolipoprotein-B (precursor of egg yolk low-density lipoprotein), but no phosphorylation site was identified. These phosphorylation and N-glycosylation of EYG proteins provide new insights for understanding the assembly structure and functional characteristics of EYG, thus contributing to its development and utilization., Competing Interests: DISCLOSURES The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in the present study., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Vitellogenin (Vg) is the major precursor of the egg-yolk proteins, which mainly acts as an energy reserve molecule for providing nutrients during embryonic development. Vg also plays an immune function in vertebrates such as fish, but there are few studies on the immune function of Vg in invertebrates. In the present study, a Vg homologue (CgVg) was identified and characterized in oyster Crassostrea gigas. There are three domains in the CgVg protein, including a Vitellogenin&#95;N domain, a domain of unknown function 1943 (DUF1943) and a von Willebrand factor type D domain (VWD). The mRNA transcripts of CgVg were detected in all tested tissues with high expression in the gonad, hepatopancreas and haemocytes, which was 466.29-, 117.15- and 57.49-fold (p < 0.01) of that in adductor muscle, respectively. After Vibrio splendidus stimulation, the mRNA expression level of CgVg in haemocytes increased significantly at 6, 12 and 24 h, which was 1.97-, 3.58- and 1.3-fold (p < 0.01) of that in the seawater group, respectively. The immunofluorescence assay showed that positive signals of CgVg protein were mainly located at the cytoplasm of haemocytes. The recombinant protein of DUF1943 domain (rDUF1943) and VWD domain (rVWD) was able to bind lipopolysaccharide (LPS), mannose (MAN), peptidoglycan (PGN) and poly (I:C), as well as Gram-positive bacteria (Staphylococcus aureus and Micrococcus luteus), Gram-negative bacteria (Escherichia coli and V. splendidus) and fungi (Pichia pastoris). rDUF1943 exhibited stronger agglutination activity towards S. aureus, M. luteus, E. coli, V. splendidus and P. pastoris, while agglutination was only observed in the rVWD group towards P. pastoris. The rVWD inhibited the growth of E. coli, S. aureus and V. splendidus, while no antibacterial activity was detected in rDUF1943 group. Collectively, CgVg not only functioned as a pattern recognition receptor (PRR) to bind various microorganisms and PAMPs, but also as an immune effector participating in the clearance of invaders, in which DUF1943 and VWD domain were mainly responsible for agglutinating and inhibiting microorganism respectively., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Vitellogenin (VTG), an egg yolk precursor, is abnormally produced by male and juvenile oviparous species after exposure to estrogens. Plasma VTG in loggerhead sea turtles (Caretta caretta) helped us understand their reproductive maturation and investigate it as a biomarker of contaminant exposure. The presence of VTG was screened in plasma from 404 loggerheads from the northwestern Atlantic Ocean using a freshwater turtle antibody in western blots. The concentrations of VTG were semiquantified using band intensities calibrated to results from a loggerhead antibody enzyme-linked immunoassay. The detection and concentrations of VTG were in (from highest to lowest): nesting females, in-water adult females, subadult females, smaller females, unknown sex, and males. Loggerheads from this region begin vitellogenesis at ≅77 cm straight carapace length. We classified VTG expression as abnormal in nine male or juvenile turtles. Organochlorine contaminant (OC) concentrations were measured in blood and/or fat biopsies of some turtles. One abnormal VTG female had the second highest fat polychlorinated biphenyl (PCB) and 4,4'-dichlorodiphenyldichloroethylene concentrations compared among 43 VTG-negative juveniles. The nine VTG-abnormal turtles had average blood PCB concentrations 8.5% higher, but not significantly different, than 46 VTG-negative juveniles (p = 0.453). In turtles less than 77 cm, blood PCB concentrations were significantly, but weakly, correlated with semiquantified VTG concentrations (tau = 0.1, p = 0.004). Greater blood OC concentrations were found in adult females than in males, which motivated the creation of a conceptual model of OC, VTG, and hormone concentrations across a reproductive cycle. A decision tree is also provided incorporating VTG as a sexing tool. Abnormal VTG expression cannot conclusively be linked to endocrine disruption caused by these OC concentrations. Studies should further investigate causes of abnormal VTG expression in wild sea turtles. Environ Toxicol Chem 2023;42:1309-1325. © 2023 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA., (© 2023 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

In insects, the biogenic amine tyramine (TA) has been shown to control several physiological processes. Recently, the involvement of the type 1 tyramine receptor (TAR1) in reproductive processes has been demonstrated in different insects. Here, we investigate the putative role of Rhodnius prolixus TAR1 (RpTAR1) in reproduction in female R. prolixus. RpTAR1 transcript was highly expressed in tissues associated with egg development. Moreover, after a blood meal, which is the stimulus for full egg development, RpTAR1 transcript was upregulated in the ovaries and in the fat body. After RNAi-mediated RpTAR1 knockdown, an ovarian phenotype characterized by the absence or reduction of egg production was observed. Furthermore, protein and Vg accumulation in the fat body was observed, suggesting an impairment in protein release from the fat body into the hemolymph. However, even though fewer eggs were produced and laid, there was no difference in hatching ratio of those laid, in comparison to the controls, indicating that the overall low protein uptake by the ovaries did not influence the viability of individual eggs produced. Interestingly, the eggs from dsTAR1-treated insects appeared more red, indicating a higher content of RHBP compared to the control. A higher colocalization between Vg and Rab11, a marker for the recycling endosome pathway, was observed after dsTAR1 injection, suggesting that a more active lysosome degradation pathway in response to the Vg accumulation may occur. In addition to the Vg accumulation in the fat body, dsTAR1 treatment altered JH pathway. However, it remains to be elucidated whether this event is either directly related to the RpTAR1 downregulation or for a consequence to the Vg accumulation. Lastly, the RpTAR1 action on Vg synthesis and release in the fat body was monitored in the presence or absence of yohimbine, the antagonist of TAR1, in an ex-vivo experiment. Yohimbine antagonises the TAR1 stimulated release of Vg. These results provide critical information concerning the role of TAR1 in Vg synthesis and release in R. prolixus. Furthermore, this work opens the way for further investigation into innovative methods for controlling R. prolixus., Competing Interests: Declaration of competing interest The authors declare no potential conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Vitellogenins (Vgs) are critical for the development and fecundity of insects. As such, these essential proteins can be used by plants to reliably sense the presence of insects. We addressed this with a combination of molecular and chemical analyses, genetic transformation, bioactivity tests, and insect performance assays. The small N-terminal subunit of Vgs of the planthopper Nilaparvata lugens (NlVgN) was found to trigger strong defense responses in rice when it enters the plants during feeding or oviposition by the insect. The defenses induced by NlVgN not only decreased the hatching rate of N. lugens eggs, but also induced volatile emissions in plants, which rendered them attractive to a common egg parasitoid. VgN of other planthoppers triggered the same defenses in rice. We further show that VgN deposited during planthopper feeding compared with during oviposition induces a somewhat different response, probably to target the appropriate developmental stage of the insect. We also confirm that NlVgN is essential for planthopper growth, development, and fecundity. This study demonstrates that VgN in planthopper eggs and saliva acts as a reliable and unavoidable elicitor of plant defenses. Its importance for insect performance precludes evolutionary adaptions to prevent detection by rice plants., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

This study was conducted to investigate a comprehensive effect of 17α-ethinylestradiol (EE2) in zebrafish (Danio rerio) with the emphasis on endocrine disruption, oxidative stress and detoxification processes at different levels. Adult male triploid zebrafish were exposed to EE2 administered in feed at two concentrations - 10 and 1000 μg/kg for six weeks. The estrogenic potential of EE2 was evaluated using an analysis of vitellogenin, gene expression focused on reproductive disorders and gonad histological examination. The alterations in antioxidant and detoxification status were assessed using analyses of enzyme activities and changes in transcriptional levels of selected genes. The most significant changes were observed especially in fish exposed to a high concentration of EE2 (i.e., 1000 μg/kg). Such high concentration caused extensive mortality (25 %) mainly in the second half of the experiment followed by a highly significant decrease in the length and body weight. Similarly, highly significant induction of vitellogenin level and vtg1 mRNA expression (about 43,000-fold compared to the control) as well as a significant downregulation of gonad aromatase expression (cyp19a1a) and histological changes in testicular tissue were confirmed in this group. In the group exposed to environmentally relevant concentration of EE2 (i.e., 10 μg/kg), no significant differences in vitellogenin were observed, although all fish were positive in the detection of vitellogenin compared to control, where only 40 % of individuals were positive. In addition, the high concentration of EE2 resulted in significant alterations in most monitored antioxidant and detoxifying enzymes with the exception of catalase, followed by strongly significant upregulation in mRNA expression of gsr, gpx1a, cat and cyp1a genes. Furthermore, a significant decrease in the glutathione reductase activity was recorded in fish exposed to 10 μg EE2/kg. To our knowledge, this is the first study which reports the effects of subchronic per oral exposure to EE2 in adult triploid zebrafish., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Binding immunoglobulin protein (BiP, also known as GRP78), a chaperone and master regulator of the unfolded protein response (UPR) pathway, plays an essential role in several flavivirus infections, but its functional role in regulating dengue virus replication in the mosquito remains largely unknown. We here demonstrated the interaction between a dengue virus serotype 2 (DENV2) and BiP in Aedes aegypti and report the discovery of a novel functional role of BiP in mosquito vitellogenesis. Silencing Ae. aegypti BiP (AaBiP) expression resulted in the significant inhibition of DENV2 viral genome replication, viral protein production, and infectious viral particle biogenesis. Co-immunoprecipitation assays showed that the DENV2 non-structural protein 1 (NS1) interacts with the AaBiP protein, and silencing AaBiP expression led to enhanced DENV2 NS1 aggregation, indicating that AaBiP plays a role in viral protein stability. A kinetic study focusing on pulse treatment of MG132, a proteasome inhibitor, in AaBiP-silenced mosquitoes showed that DENV2 NS1 was drastically elevated, which further suggests that AaBiP-mediated viral protein degradation is mediated by proteasomal machinery. Silencing of AaBiP also resulted in a reduction in mosquito fertility and fecundity. Depletion of AaBiP inhibited mosquito vitellogenesis due to the reduction of vitellogenin mRNA and elevated aggregation of vitellogenin protein post blood meal, further suppressing ovary development and fecundity. Overall, our results suggest that AaBiP is a dual-function protein with roles in both the regulation of dengue virus replication and mosquito reproduction. Our findings will be useful in the establishment of more efficient strategies for vector-borne disease control., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Most arthropods are famous for their large reproductive capacity, with the ovary playing a vital role in the process. The study of the regulatory mechanisms of ovarian development may have the potential for a reproduction-based pest management strategy. GATA-binding transcription factors (GATAs) as important regulatory factors mediate many physiological processes, including development, immunity, insecticide resistance and reproduction. The Pannier (pnr), a member of GATA family, was confirmed to be involved in ovarian development of Bactrocera dorsalis in our previous study. However, the direct evidence of pnr regulating the fly ovarian development is still lacking., Results: We used CRISPR/Cas9 to create Bdpnr loss-of-function mutations. Homozygous Bdpnr -/- mutants were nonviable, with most individuals dying during embryogenesis, some surviving to the larval stages, and the remaining few dying during pupation. In contrast, heterozygous individuals reached the adult stage, but ovarian development was disrupted, with concomitant decreases in egg laying and hatching rates. We also found that two genes encoding vitellogenin proteins (BdVg1 and BdVg2) and the vitellogenin receptor (BdVgR) were significantly down-regulated in heterozygous mutants compared to wild-type controls., Conclusion: These results indicate that Bdpnr is required for embryonic and post-embryonic development, including the formation of ovaries. Bdpnr could therefore be considered as a molecular target for tephritid fly pest control. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Microplastics not only accumulate in the bodies of fishes and cause damage to the organs, but also cause many other problems, such as reduced reproductive capacity, by acting directly or indirectly on the hypothalamus-pituitary-gonad axis (HPG axis). In this study, we investigated the changes in HPG axis-related genes in male medaka (Oryzias latipes) exposed to fiber-type microplastics. We confirmed the progression of vitellogenesis, a sign of endocrine disruption, in male fish. In the microfiber-exposed group, microfiber accumulation was confirmed in the gills and intestines. One week after exposure to two different concentrations of microfibers (500 and 1,000 fibers/L), the fish showed increased expression of gonadotropin-releasing hormone (GnRH) and luteinizing hormone receptor (LH-R) mRNA. From day 10 of exposure to the microfibers, there was an increase in the expression of the gonadotropin-inhibitory hormone (GnIH) mRNA and a decrease in the expression of GnRH and LH-R mRNA. There was an increase in the cytochrome P450 aromatase (CYP19a) mRNA expression and plasma estradiol (E 2 ) concentration in the 1,000 fibers/L exposure group. High vitellogenin (VTG) mRNA expression was confirmed seven days after exposure in the 1,000 fibers/L group, which was consistent with the VTG mRNA expression signals detected in the liver using in situ hybridization. These results suggest that microfiber ingestion may cause short-term endocrinal disruption of the HPG axis in male medaka, which in turn may interfere with their normal maturation process., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Environmental estrogens have generated great concern because of their potential threat to aquatic organisms; however, the commonly used vitellogenin (Vtg) biomarker detection methods are not capable of detecting estrogenic activity below 10 ng/L 17β-estradiol. In this study, we developed multiple immunoassays based on Japanese flounder (Paralichthys olivaceus) choriogenin (Chg), a highly sensitive biomarker of environmental estrogens. Chg genes (ChgL and ChgH) of Japanese flounder were cloned for the first time, and a recombinant ChgL protein with a molecular weight of approximately 52 kDa was prepared using a prokaryotic expression system and purified using Ni-affinity column chromatography. Subsequently, specific monoclonal antibodies against ChgL were prepared and used to develop sandwich enzyme-linked immunosorbent assays (ELISAs), which had a detection range of 3.9-250 ng/mL and detection limit of 1.9 ng/mL. An immunofluorescence method was also established and used to visually detect ChgL induction in the tissues. In addition, a lateral flow immunoassay for ChgL that could detect estrogen activity within 10 min was developed. Finally, the reliability of the immunoassays was examined by measuring ChgL induction in the plasma and tissues of Japanese flounder exposed to 0, 2, 10, and 50 ng/L 17α-ethinylestradiol (EE 2 ). The results showed that 2 ng/L EE 2 notably increased ChgL levels in the plasma, demonstrating that ChgL is more sensitive than Vtg to environmental estrogens; 50 ng/L EE 2 induced obvious Chg induction in the sinusoidal vessels of the liver. Conclusions taken together, this study provides reliable methods for sensitive and rapid detection of estrogenic activity in aquatic environments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The maintenance of proteostasis is essential for cellular and organism healthspan. How proteostasis collapse influences reproductive span remains largely unclear. In Caenorhabditis elegans, excess accumulation of vitellogenins, the major components in yolk proteins, is crucial for the development of the embryo and occurs throughout the whole body during the aging process. Here, we show that vitellogenin accumulation leads to reproduction cessation. Excess vitellogenin is accumulated in the intestine and transported into the germline, impairing lysosomal activity in these tissues. The lysosomal function in the germline is required for reproductive span by maintaining oocyte quality. In contrast, autophagy and sperm depletion are not involved in vitellogenin accumulation-induced reproductive aging. Our findings provide insights into how proteome imbalance has an impact on reproductive aging and imply that improvement of lysosomal function is an effective approach for mid-life intervention for maintaining reproductive health in mammals., (© 2023. Science China Press.)

Integration between animal reproduction and symbiont inheritance is fundamental in symbiosis biology, but the underlying molecular mechanisms are largely unknown. Vitellogenin (Vg) is critical for oogenesis, and it is also a pathogen pattern recognition molecule in some animals. Previous studies have shown that Vg is involved in the regulation of symbiont abundance and transmission. However, the mechanisms by which an insect and its symbiont contribute to the function of Vg and how Vg impacts the persistence of insect-microbe symbiosis remain largely unclear. Symbionts are transovarially transmitted via maternal inheritance of the bacteriocytes in the whitefly Bemisia tabaci. Surprisingly, Vg is localized in bacteriocytes of whiteflies. Vg could be synthesized in whitefly bacteriocytes by the gene Vg expressed in these cells or exported into bacteriocytes from hemolymph via the Vg receptor. We further found that the juvenile hormone and " Candidatus Portiera aleyrodidarum" (here termed Portiera ) control the level and localization of Vg in whiteflies. Immunocapture PCR revealed interactions between Vg and Portiera . Suppressing Vg expression reduced Portiera abundance as well as whitefly oogenesis and fecundity. Thus, we reveal that Vg facilitated the persistence of whitefly-bacteriocyte symbiont associations. This study will provide insight into the key role of Vg in the coevolution of insect reproduction and symbiont inheritance. IMPORTANCE Intracellular heritable symbionts have been incorporated into insect reproductive and developmental biology by various mechanisms. All Bemisia tabaci species harbor the obligate symbiont Portiera in specialized insect cells called bacteriocytes. We report that the whitefly juvenile hormone and Portiera determined vitellogenin (Vg) localization in bacteriocytes of whiteflies. In turn, Vg affected whitefly fecundity as well as fitness and transmission of the symbiont. Our findings show that Vg, a multifunctional protein, is indispensable for symbiont integration into the reproduction and development of insects. This reflects the outcome of long-term coevolution of the insect-microbe symbiosis.

In the viviparous teleost species belonging to the family Goodeidae, intraovarian embryos absorb maternal supplements while they grow during the gestation period. They take up the components via trophotaeniae, a hindgut-derived placental structure. Our previous study using a goodeid species Xenotoca eiseni revealed that intraovarian embryos absorb the yolk protein vitellogenin (Vtg) via the trophotaenia. However, another group indicated yolk components accumulate in the intestinal lumen of X. eiseni embryos. Here, we investigated whether the intestinal duct is capable of protein uptake, as is the trophotaenia. Immunohistochemical studies indicated that endogenous vitellogenin is detected in the intestinal epithelial cells of the intraovarian embryo. Tracer analysis using FITC-Vtg also indicated that intestinal tissues can take up protein. The endocytosis-related genes expressed in trophotaenia were also detected in the intestinal tissues of the embryo. Lipid transporter genes which are not expressed in the trophotaenia were detected in the embryonic intestine. This evidence suggests that the intraovarian embryo of X. eiseni possesses two distinct sites for uptake of the maternal proteins. However, the presumed functions of the embryonic intestine and trophotaenia might be not identical. The study provides a new perspective on how mother-to-embryo matrotrophic interactions have changed in the evolution of viviparous teleosts., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Bisphenol A (BPA) is an endocrine disruptor that is present in freshwater and marine environments. However, conclusive evidence for the toxicity of chronic BPA exposure to marine fishes remains lacking. Therefore, we investigated the influence of BPA on male marine medaka (Oryzias melastigma). BPA exposure induced formation of testis-ova at 2610 µg/L, and male-type anal fins became more female type in a concentration-dependent manner. Some males with female-type anal fins had normal testes, indicating that anal fin shape is more sensitive to BPA. Gonadal soma-derived factor (gsdf) expression decreased after BPA exposure in the 746 and 2610 µg/L exposure groups, although the changes were not statistically significant. Additionally, liver vitellogenin (vtg) expression increased in a dose-dependent manner and was significantly higher in all exposure groups. vtg and gsdf are likely to be useful biomarkers for the impact of estrogenic endocrine disrupters in O. melastigma., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Oocyte maturation is critical for insect reproduction. Vitellogenesis, the timely production and uptake of vitellogenin (Vg), is crucial for female fecundity. Vg is synthesized in fat body and absorbed by the oocytes through endocytosis during insect oogenesis. In the silkworm, Bombyx mori, we discovered that a nucleus-enriched long-noncoding RNA (lncRNA) lncR26319 regulates Endophilin A (EndoA) - a member of the endophilin family of endocytic proteins - through competitive binding to miR-2834. The lncR26319-miR-2834-EndoA axis was required for Vg endocytosis in the silkworm; loss of EndoA or overexpression of miR-2834 significantly reduced egg numbers in virgin moths. In addition, accumulation of miR-2834 resulted in pupal and adult deformation and reduced fecundity in females. The expression of Vg, 30-kDa (30K) protein, and egg-specific protein (Esp) decreased after knockdown of EndoA or overexpression of miR-2834, while knockdown of miR-2834 had an opposite effect on the expression of Vg, 30K protein gene, and Esp. These results suggest that the lncR26319-miR-2834-EndoA axis contributes to the endocytic activity in the Vg uptake and leads to the normal progression of oogenesis in the silkworm. Thus, miR-2834 and EndoA are crucial for female reproduction and could be potential targets for new pest management strategies in lepidopterans., (© 2022 The Authors. Insect Science published by John Wiley & Sons Australia, Ltd on behalf of Institute of Zoology, Chinese Academy of Sciences.)

Full-length cDNAs of the Broad-Complex (BR-C) from Riptortus pedestris were cloned. Moreover, Kr-h1 and BR-C expression levels in apo-symbiotic and symbiotic host insects were compared to verify whether they are modulated by Burkholderia gut symbionts. Interestingly, Kr-h1 expression level was significantly increased in symbiotic females. To determine how Kr-h1 affects fecundity in insects, the biosynthesis of two reproduction-associated proteins, hexamerin-α and vitellogenin, was investigated in R. pedestris females. Hexamerin-α and vitellogenin expression at the transcriptional and translational levels decreased in Kr-h1-suppressed symbiotic females, subsequently reduced egg production. These results suggest that Burkholderia gut symbiont modulates Kr-h1 expression to enhance ovarian development and egg production of R. pedestris by increasing the biosynthesis of the two proteins., (© 2022 Wiley Periodicals LLC.)

Aquatic biota is increasingly being exposed to chemical pollutants due to human activities and the relationship between the level of environmental pollution and fish reproduction is a continuously ongoing issue. The vitellogenin (Vtg) protein synthesis can be induced in the liver of juvenile and male fish after stimulation of the estrogen receptor and therefore, Vtg has been used as a biomarker of xenoestrogen exposure in several fish species. The current study reported the first physicochemical characterization of Vtg from Oreochromis niloticus. Adult male fish were exposed to 17α-ethinylestradiol for Vtg induction. Purified vitellogenin from plasma showed low stability at 25 and 4 °C in saline conditions, and good stability in acidic (low pH) or in heated conditions. The 3D modeling provided useful information on the structure of O. niloticus Vtg showing conserved structural features. According to bioinformatics and experimental results, there are important structural differences between the two chemical forms of Vtg (VtgAb and VtgC) in a phylogenetic context. The present results add information about the development of ecotoxicological immunoassays to study the endocrine disruption in O. niloticus improving the Vtg performance as a biomarker of reproduction in fish., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Eusociality has been commonly observed in distinct animal lineages. The reproductive division of labor is a particular feature, achieved by the coordination between fertile and sterile castes within the same nest. The sociogenomic approach in social hymenopteran insects indicates that vitellogenin (Vg) has undergone neo-functionalization in sterile castes. Here, to know whether Vgs have distinct roles in nonreproductive castes in termites, we investigated the unique characteristics of Vgs in the rhinotermitid termite Reticulitermes speratus. The four Vgs were identified from R. speratus (RsVg1-4), and RsVg3 sequences were newly identified using the RACE method. Molecular phylogenetic analysis supported the monophyly of the four termite Vgs. Moreover, the termites Vg1-3 and Vg4 were positioned in two different clades. The  dN/dS ratios indicated that the branch leading to the common ancestor of termite Vg4 was under weak purifying selection. Expression analyses among castes (reproductives, workers, and soldiers) and females (nymphs, winged alates, and queens) showed that RsVg1-3 was highly expressed in fertile queens. In contrast, RsVg4 was highly expressed in workers and female nonreproductives (nymphs and winged adults). Localization of RsVg4 messenger RNA was confirmed in the fat body of worker heads and abdomens. These results suggest that Vg genes are functionalized after gene duplication during termite eusocial transition and that Vg4 is involved in nonreproductive roles in termites., (© 2022 Wiley Periodicals LLC.)

To reduce the use of intact animals for chemical safety testing, while ensuring protection of ecosystems and human health, there is a demand for new approach methodologies (NAMs) that provide relevant scientific information at a quality equivalent to or better than traditional approaches. The present case study examined whether bioactivity and associated potency measured in an in vitro screening assay for aromatase inhibition could be used together with an adverse outcome pathway (AOP) and mechanistically based computational models to predict previously uncharacterized in vivo effects. Model simulations were used to inform designs of 60-h and 10-21-day in vivo exposures of adult fathead minnows (Pimephales promelas) to three or four test concentrations of the in vitro aromatase inhibitor imazalil ranging from 0.12 to 260 µg/L water. Consistent with an AOP linking aromatase inhibition to reproductive impairment in fish, exposure to the fungicide resulted in significant reductions in ex vivo production of 17β-estradiol (E2) by ovary tissue (≥165 µg imazalil/L), plasma E2 concentrations (≥74 µg imazalil/L), vitellogenin (Vtg) messenger RNA expression (≥165 µg imazalil/L), Vtg plasma concentrations (≥74 µg imazalil/L), uptake of Vtg into oocytes (≥260 µg imazalil/L), and overall reproductive output in terms of cumulative fecundity, number of spawning events, and eggs per spawning event (≥24 µg imazalil/L). Despite many potential sources of uncertainty in potency and efficacy estimates based on model simulations, observed magnitudes of apical effects were quite consistent with model predictions, and in vivo potency was within an order of magnitude of that predicted based on in vitro relative potency. Overall, our study suggests that NAMs and AOP-based approaches can support meaningful reduction and refinement of animal testing. Environ Toxicol Chem 2023;42:100-116. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA., (© 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

The vitellogenin is composed by polypeptides that are precursors of egg yolk proteins that provides embryo and larvae nutrition. The mRNA encoding for vitellogenin Ab (Vtg-Ab; 4,536 bp long and 1,512 amino acids) were obtained by RNA-Seq library sequencing of pirarucu gonads. The Vtg-Ab sequences had high homology with Vtgs of other three teleosts species of the order Osteoglossiformes. The transcript of ovarian Vtg was identified based on structural criteria, and so we classify the Vtg of pirarucu as Vtg-Ab due to the truncated or shortened phosvitin (N-terminal end) and phosvitinless domain (C-terminal end). The Vtg-Ab of pirarucu present two major deletions with 133 amino acids in the Lipovitellin I domain and 89 amino acids in the truncated or shortened Phosvitin domain, both located in the N-terminal end region. The three-dimensional (3-D) structure Vtg-Ab protein shows the presence of a typical 4α-helices bundle protein that runs in anti-parallel. In general, the characterization of Vtg-Ab may be the useful elucidation of the hormonal regulation of vitellogenesis and improve the production of pirarucu for broodstock management in aquaculture and preparation of Vtg antibody production (species-specific) for sex identification., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Currently, endocrine disruptors (EDs) can be found in all the environmental compartments. To understand the effects of estrogenic EDs (EEDs), adults of Cyprinodon variegatus have been classically used as a marine model. However, it is during development that exposure to contaminants may generate permanent consequences. Thus, the aim of this study was to verify the effects produced by acute exposure to 17α-ethinylestradiol (EE2) in C. variegatus larvae. Quantitative PCR (qPCR) results revealed the induction of vtg and zp gene expression on exposure to 1000 ng/L EE2 and the induction of vtgc, zp2, zp3 and cyp19a2, and inhibition of vtgab, wap and cyp1a1 on exposure to 100 ng/L EE2. Lower concentrations inhibited the gene expression of vtgab and wap (50 ng/L), cyp1a1 (25 ng/L) and zp2 (12.5 ng/L). These alterations in gene expression allow us to affirm that larvae of C. variegatus are an efficient and sensitive model for biomonitoring EEDs., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: ECOTOX group reports financial support was provided by European Union through the European Regional Development Fund (ERDF), GLAUKOS Project. ECOTOX group reports article publishing charges was provided by CISUG University of Vigo. ECOTOX group reports equipment, drugs, or supplies was provided by Marine Research Centre (CIM) of the University of Vigo. ECOTOX group reports financial support was provided by European Union (Horizon 2020 research and innovation programme)., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Mosquito-borne diseases threaten human health, but mosquito control faces various challenges, such as resistance to chemical insecticides. Thus, there is an urgent need for more effective and environment-friendly control agents. Capsaicin can downregulate the mTOR signaling pathway of tumor cells. The TOR signaling pathway can mediate the expression of vitellogenin (Vg) to regulate the fecundity of insects. Whether capsaicin has the potential to inhibit fecundity of mosquitoes by regulating TOR pathway and Vg expression is currently unclear., Methods: Anopheles stephensi were fed with blood of mice administered capsaicin by gavage or sugar containing capsaicin followed by a blood feeding with normal mice. Then, the engorged female mosquitoes were tubed individually and underwent oviposition. The eggs and individuals in the subsequent development stages, including larvae, pupae, and emerging adults, were counted and compared between the capsaicin treatment and control groups. Additionally, total RNA and protein were extracted from the engorged mosquitoes at 24 h post blood feeding. Real-time PCR and western blot were performed to detect the transcriptional level and protein expression of the key fecundity-related molecules of mosquitoes. Finally, TOR signaling pathway was inhibited via rapamycin treatment, and changes in fecundity and the key molecule transcription and protein expression levels were examined to verify the role of TOR signaling pathway in the effect of capsaicin on mosquito fecundity., Results: The laid and total eggs (laid eggs plus retained eggs) of An. stephensi were significantly reduced by feeding on the blood of capsaicin-treated mice (P < 0.01) or capsaicin-containing sugar (P < 0.01) compared with those in the control group. Moreover, the transcription and protein expression or phosphorylation levels of fecundity-related molecules, such as Akt, TOR, S6K, and Vg, were significantly decreased by capsaicin treatment. However, the effects disappeared between control group and CAP group after the TOR signaling pathway was inhibited by rapamycin., Conclusions: Capsaicin can decrease the fecundity of An. stephensi by inhibiting the TOR signaling pathway. These data can help us to not only understand the effect of capsaicin on the reproductive ability of An. stephensi and its underlying mechanism, but also develop new efficient, safe, and pollution-free mosquito vector control agents., (© 2022. The Author(s).)