Insights from different reproductive gene knockdowns via RNA interference in the lady beetle Eriopis connexa: Establishing a new model for molecular studies on natural enemies.

Insect pest control can be achieved by the application of RNA interference (RNAi), a key molecular tool in functional genomics. Whereas most RNAi research has focused on insect pests, few studies have been performed on natural enemies. Validating the efficacy of RNAi in natural enemies is crucial for assessing its safety and enabling molecular research on these organisms. Here, we assessed the efficacy of RNAi in the ladybird beetle Eriopis connexa Germar (Coleoptera: Coccinellidae), focusing on genes related to reproduction, such as vitellogenin (Vg) and its receptor (VgR). In the transcriptome of E. connexa, we found one VgR (EcVgR) and two Vg genes (EcVg1 and EcVg2). These genes have been validated by in silico analyses of functional domains and evolutionary relationships. Five-day-old females were injected with 500 ng/µL of a specific double-stranded RNA (dsRNA) (dsEcVg1, dsEcVg2, or dsEcVgR) for RNAi tests, while nonspecific dsRNA (dsGFP or dsAgCE8.1) was used as a control. Interestingly, dsEcVg2 was able to knockdown both Vg genes, while dsEcVg1 could silence only EcVg1. Additionally, the viability of the eggs was significantly reduced when both Vg genes were knocked down at the same time (after treatment with dsEcVg2 or "dsEcVg1+dsEcVg2"). Ultimately, malformed, nonviable eggs were produced when EcVgR was silenced. Interestingly, no dsRNA treatment had an impact on the quantity of eggs laid. Therefore, the feasibility of RNAi in E. connexa has been confirmed, suggesting that this coccinellid is an excellent Neotropical model for molecular research on natural enemies and for studying RNAi nontarget effects.

Genome-Wide Identification, Expression and Response to Estrogen of Vitellogenin Gene Family in Sichuan Bream (Sinibrama taeniatus).

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 17ß-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.

Changes in CpG Methylation of the Vitellogenin 1 Promoter in Adult Male Zebrafish after Exposure to 17α-Ethynylestradiol.

Numerous pharmaceutical and industrial chemicals are classified as endocrine-disrupting chemicals (EDCs) that interfere with hormonal homeostasis, leading to developmental disorders and other pathologies. The synthetic estrogen 17α-ethynylestradiol (EE2) is used in oral contraceptives and other hormone therapies. EE2 and other estrogens are inadvertently introduced into aquatic environments through municipal wastewater and agricultural effluents. Exposure of male fish to estrogens increases expression of the egg yolk precursor protein vitellogenin (Vtg), which is used as a molecular marker of exposure to estrogenic EDCs. The mechanisms behind Vtg induction are not fully known, and we hypothesized that it is regulated via DNA methylation. Adult zebrafish were exposed to either dimethyl sulfoxide or 20 ng/L EE2 for 14 days. Messenger RNA (mRNA) expression and DNA methylation were assessed in male zebrafish livers at 0, 0.25, 0.5, 1, 4, 7, and 14 days of exposure; and those of females were assessed at 13 days (n ≥ 4/group/time point). To test the persistence of any changes, we included a recovery group that received EE2 for 7 days and did not receive any for the following 7 days, in the total 14-day study. Methylation of DNA at the vtg1 promoter was assessed with targeted gene bisulfite sequencing in livers of adult male and female zebrafish. A significant increase in vtg1 mRNA was observed in the EE2-exposed male fish as early as 6 h. Interestingly, DNA methylation changes were observed at 4 days. Decreases in the overall methylation of the vtg1 promoter in exposed males resulted in levels comparable to those in female controls, suggesting feminization. Importantly, DNA methylation levels in males remained significantly impacted after 7 days post-EE2 removal, unlike mRNA levels. These data identify an epigenetic mark of feminization that may serve as an indicator of not only estrogenic exposure but also previous exposure to EE2. Environ Toxicol Chem 2024;43:1547-1556. © 2024 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Expression and function of the vitellogenin receptor in the hypopharyngeal glands of the honey bee Apis mellifera (Hymenoptera: Apidae) workers.

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.

The TCTP is essential for ovarian development and oviposition of Rhipicephalus haemaphysaloides.

Tick infestations transmit various infectious agents and result in significant socioeconomic consequences. Currently, the primary focus of tick control efforts is identifying potential targets for immune intervention. In a previous study, we identified a highly conserved protein abundant in tick haemolymph extracellular vesicles (EVs) known as translationally controlled tumour protein (TCTP). We have found that native TCTP is present in various tissues of the Rhipicephalus haemaphysaloides tick, including salivary glands, midgut, ovary, and fat body. Notably, TCTP is particularly abundant in the tick ovary and its levels increase progressively from the blood-feeding stage to engorgement. When the TCTP gene was knocked down by RNAi, there was a noticeable delay in ovarian development, and the reproductive performance, in terms of egg quantity and survival, was also hindered. Our investigations have revealed that the observed effects in ovary and eggs in dsRNA-treated ticks are not attributable to cell death mechanisms like apoptosis and autophagy but rather to the reduction in the expression of vitellogenin (Vg1, Vg2, and Vg3) and ferritin (ferritin 1 and ferritin 2) proteins crucial for ovarian development and embryo survival in ticks. Additionally, phylogenetic analysis and structural comparisons of RhTCTP and its orthologues across various tick species, vertebrate hosts, and humans have shown that TCTP is conserved in ticks but differs significantly between ticks and their hosts, particularly in the TCTP_1 and TCTP_2 domains. Overall, TCTP plays a vital role in tick reproductive development and presents itself as a potential target for tick control in both humans and animals.

Peroxisome biogenesis factor PEX14 is crucial for survival and fecundity of female brown planthopper, Nilaparvata lugens (Stål).

Peroxisomes are ubiquitous cellular organelles participating in a variety of critical metabolic reactions. PEX14 is an essential peroxin responsible for peroxisome biogenesis. In this study, we identified the human PEX14 homolog in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae). N. lugens PEX14 (NlPEX14) showed significant topological similarity to its human counterpart. It is expressed throughout all developmental stages, with the highest expression observed in adult insects. Down-regulation of NlPEX14 through injection of NlPEX14-specific double-strand RNA impaired nymphal development. Moreover, females subjected to dsNlPEX14 treatment exhibited a significantly reduced lifespan. Additionally, we found abnormal ovarian development and a significant decrease in the number of eggs laid in NlPEX14-downregulated females. Further experiments support that the shortening of lifespan and the decrease in female fecundity can be attributed, at least partially, to the accumulation of fatty acids and reduced expression of vitellogenin. Together, our study reveals an indispensable function of NlPEX14 for insect reproduction and establishes a causal connection between the phenotypes and peroxisome biogenesis, shedding light on the importance of peroxisomes in female fecundity.

Estetrol/drospirenone versus 17α-ethinylestradiol/drospirenone: An extended one generation test to evaluate the endocrine disruption potential on zebrafish (Danio rerio).

Combined oral contraceptives, comprising of both an oestrogen and a progestin component, are released in aquatic environments and potentially pose a risk to aquatic wildlife by their capacity to disrupt physiological mechanisms. In this study, the endocrine disruptive potential of two mixtures, 17α-ethinylestradiol (EE2), a synthetic oestrogen, or estetrol (E4), a natural oestrogen, with the progestin drospirenone (DRSP) have been characterised in three generations of zebrafish, according to an adapted Medaka Extended One Generation Reproduction Test. Zebrafish (Danio rerio) were exposed to a range of concentrations of EE2/DRSP and E4/DRSP (∼1×, ∼3×, ∼10× and ∼30× predicted environmental concentration, PEC). Survival, growth, hatching success, fecundity, fertilisation success, vitellogenin (VTG), gonad histopathology, sex differentiation, and transcriptional analysis of genes related to gonadal sex steroid hormones synthesis were assessed. In the F0 generation, exposure to EE2/DRSP at ∼10 and ∼30× PEC decreased fecundity and increased male VTG concentrations. The highest concentration of EE2/DRSP also affected VTG concentrations in female zebrafish and the expression of genes implicated in steroid hormones synthesis. In the F1 generation, sex determination was impaired in fish exposed to EE2/DRSP at concentrations as low as ∼3× PEC. Decreased fecundity and fertility, and abnormal gonadal histopathology were also observed. No effects were observed in the F2 generation. In contrast, E4/DRSP induced only minor histopathological changes and an increase in the proportion of males, at the highest concentration tested (∼30× PEC) in the F1 generation and had no effect on hatching success of F2 generation. Overall, this study suggests that the combination E4/DRSP has a more favourable environmental profile than EE2/DRSP.

Effects of crosstalk between steroid hormones mediated thyroid hormone in zebrafish exposed to 4-tert-octylphenol: Estrogenic and anti-androgenic effects.

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 17ß-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.

The role of multiple vitellogenins in early development of fishes.

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.

Structural modeling and gene expression analysis of phosvitinless vitellogenin (vgc) in the Indian freshwater murrel, Channa punctatus (Bloch, 1793).

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 E2 in blood induces expression of vgc gene. A rapid increase in the levels of E2 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.

Vitellogenin and its upstream gene TOR play essential roles in the reproduction of Dermanyssus gallinae.

In Dermanyssus gallinae, a hematophagous mite, the initiation of vitellogenesis induced by blood feeding is essential for its reproduction. However, the precise gene structures and physiological functions of Vg in D. gallinae and its upstream gene, Target of Rapamycin (TOR), have not been fully understood. This study revealed the presence of four homologous genes within D. gallinae, named Dg-Vg1, Dg-Vg1-like, Dg-Vg2, and Dg-Vg2-like, especially, Dg-Vg2-like was firstly identified in the mites. The expression levels of all these Vg genes were significantly higher in adult females than other stages. Following blood feeding, the expression levels of these genes increased significantly, followed by a subsequent decrease, aligning with egg production. Silencing Dg-Vgs by RNA interference (RNAi) led to decreased fecundity and egg hatching rates, as well as abnormal embryonic development, suggesting a vital role for Dg-Vgs in both egg formation and embryonic development. Furthermore, the knockdown of Dg-TOR significantly reduced the expression of Dg-Vgs and negatively impacted the reproductive capabilities of PRMs, indicating that TOR influences PRM reproduction by regulating the expression of Dg-Vgs. In summary, these findings demonstrated the crucial roles of Dg-Vgs and Dg-TOR in PRM reproduction, highlighting their potential as targets for pest control.

Molecular characterization of Vitellogenin-like1 gene in Sogatella furcifera (Hemiptera: Delphacidae), and its function on reproduction.

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.

Chlorothalonil as a potential endocrine disruptor in male zebrafish (Danio rerio): Impact on the hypothalamus-pituitary-gonad axis and sperm quality.

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.

Precise coordination between nutrient transporters ensures fertility in the malaria mosquito Anopheles gambiae.

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.

Sex-Biased Transcription Expression of Vitellogenins Reveals Fusion Gene and MicroRNA Regulation in the Sea Louse Caligus rogercresseyi.

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.

Yolk proteins of the schistosomiasis vector snail Biomphalaria glabrata revealed by multi-omics analysis.

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.

Juvenile hormone induces reproduction via miR-1175-3p in the red imported fire ant, Solenopsis invicta.

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.

Male vitellogenin regulates gametogenesis through a testis-enriched big protein in Chrysopa pallens.

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.

Thyroid-gonadal hormonal interplay in zebrafish exposed to sodium perchlorate: Implications for reproductive health.

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.

Transcriptional Response of Vitellogenin Gene in Flatfish to Environmental Pollutants from Two Regions of the Gulf of Mexico.

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.