Vitellogenin Receptor Research Articles

Characterization of a Vitellogenin Receptor in the Bumblebee, Bombus lantschouensis (Hymenoptera, Apidae).

The vitellogenin receptor (VgR) belongs to the low-density lipoprotein receptor (LDLR) family, responsible for mediating the endocytosis of vitellogenin (Vg) into the ovaries to promote ovarian growth and oviposition. Here, we cloned and measured VgR gene expression characteristics in the bumblebee Bombus lantschouensis. RNA interference was used to validate VgR function. The results showed that the full length of the BLVgR cDNA was 5519 bp, which included a 5280 bp open reading frame encoding 1759 amino acids (AAs). Sequence alignment revealed that the protein contained 12 LDLa, 5 EGF, 2 EGF-CA and 10 LY domains. Phylogenetic analysis showed that BLVgR and the VgR of Bombus terrestris clustered together and the tree of bumblebees (Bombus) appeared as one clade next to honeybees (Apis). Transcript expression analysis showed that BLVgR was expressed in all tested tissues and showed the highest abundance in the ovaries. BLVgR expression was present in all developmental stages. However, the expression level in larvae was extremely low. In addition, the expression of BLVgR was significantly upregulated after egg laying in both workers and queens. In new emerging workers injected with 5 µg of VgR dsRNA, the expression level of BLVgR was 4-fold lower than that in the GFP dsRNA-injected group after 72 h. Furthermore, BLVgR silencing significantly reduced the number of eggs laid (3.67 ± 1.96 eggs) and delayed the first egg-laying time (16.31 ± 2.07 days) in worker microcolonies when compared to dsGFP (37.31 ± 4.09 eggs, 8.15 ± 0.22 days) and DEPC-treated water injected controls (16.42 ± 2.24 eggs, 10.00 ± 0.37 days). In conclusion, the BLVgR gene and its reproductive function were explored in the bumblebee B. lantschouensis. This gene plays an important role in egg laying time and egg number.

Molecular characterization of clathrin heavy chain (Chc) in Rhipicephalus haemaphysaloides and its effect on vitellogenin (Vg) expression via the clathrin-mediated endocytic pathway.

Clathrin plays an important role in arthropods, but its function in ticks has not been explored. Here, we describe the molecular characteristics of the clathrin heavy chain of the tick Rhipicephalus haemaphysaloides and its effects on yolk development. The open reading frame of the clathrin heavy chain (Chc) (Rh-Chc) gene consists of 5103 nucleotides encoding 670 amino acids, which is most closely related to that of Ixodes scapularis and relatively close to Homo sapiens and Drosophila melanogaster. Real-time qPCR revealed that Rh-Chc was expressed at all developmental stages and organs. After Rh-Chc is silenced, ticks did not feed and mortality rate was 100%. Moreover, Rh-Chc co-localized with Vitellogenin receptor (VgR) on oocyte membrane. Immunofluorescence showed that the expression of Vitellogenin (Vg) (Rh-Vg) was also closely related to Rh-Chc. Immunofluorescence showed that the expression of Vg was also closely related to Rh-Chc, Rh-Chc silencing slowed the development of oocytes in tick, and culture of ovary in vitro silenced Rh-Chc, the development of oocytes in ticks also slowed down. Overall, the results of this study indicated that Rh-Chc is a vital gene in the tick R. haemaphysaloides that plays an important role in its growth, development, and reproduction.

Changes in vitellogenin and estrogen receptor expression and 17β-estradiol concentration in male juvenile tilapia can be used to evaluate endocrine-disrupting chemicals

The effects of endocrine disruption with respect to potential human toxicities have been extensively evaluated to date. However, the standard testing methods used have not always taken the most pertinent approach. In this study, we used juvenile male tilapia (Oreochromis niloticus) as an animal model to test for endocrine disruption by chemicals. We measured 17β-estradiol (E2) concentrations, and the mRNA relative expression ratio (RER; treatment/control) of vitellogenin (vtg2) and estrogen receptors (ERs) to assess whether the effluent concentration of selected plasticizers disrupt E2 function in fish. We found that the vtg2 RER was significantly increased after exposure to 2.52 nM E2 for 5 days, 0.438 μM of bisphenol A (BPA) for 7 days, or 2.865 μM Cd2+ for 7 days. These data support vtg2 transcript level as a sensitive biomarker to evaluate contamination of water by endocrine disrupting chemicals (EDCs). However, vtg2 expression did not respond to fluctuations of E2 concentrations in the tilapia juveniles exposed to selected plasticizers. However, the RER of three types of ERs appeared to change dramatically upon exposure to plasticizers. ERα significantly increased, but ERβ2 decreased with 3.6 μM DEP exposure. Both ERα and ERβ2 decreased significantly after 1.44 μM DIBP exposure. We suggest that changes of vtg2 mRNA RER, E2 levels and ERs mRNA expression should be taken into consideration at the same time to determine if chemical contaminants in the water are endocrine disrupters.

Involvement of TRAF6 in regulating immune defense and ovarian development in Musca domestica

The tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is a key cytoplasm signaling adaptor that mediates signals activated by TNFR superfamily and the interleukin-1/Toll-like receptor (IL-1/TLR) superfamily. In the present research, a housefly Musca domestica TRAF6 (MdTRAF6) gene is identified and characterized, with a 51.7-kDa protein possessing a RING domain and a conserved C-terminal TRAF homology MATH domain encoded. MdTRAF6 is widely expressed in diverse tissues with high expression levels in gut and fat body, which is of the highest levels in adult in all growth stages. The expression of MdTRAF6 could be remarkably induced by bacterial challenge, and the silencing MdTRAF6 could alter the expressions of NF-κB-like genes (relish and dorsal) and antimicrobial peptide genes (cecropin, diptericin, attacin, muscin), thus leading elevated mortalities of larvae followed by bacterial infection. Inspiringly. MdTRAF6-depleted adult flies display higher mortality, lower fertility and reduced survival of offspring than the controls. Further investigation reveals that knockdown of MdTRAF6 disturbs the ovarian development and impaires the expressions of vitellogenin and vitellogenin receptor genes in the adult females. All these phenotypes show crucial roles of MdTRAF6 in innate immunity via positive regulation of the Toll pathway and negative regulation of the Imd pathway, and in reproduction by maintaining ovarian development.

Evidence for a transcellular route for vitellogenin transport in the telotrophic ovary of Podisus nigrispinus (Hemiptera: Pentatomidae)

Vitellogenin is the main yolk precursor protein in insect oocytes. It is synthesized in the fat body and released into the hemolymph. To reach the oocyte surface, vitellogenin must cross a single layer of follicular epithelium cells. The transport of vitellogenin across the follicular epithelium has been suggested to occur through the enlarged intercellular spaces (patency) by a paracellular route or by endocytosis by follicular cells and release onto oocyte surface in a transcelluar route. In this study, we investigated whether vitellogenin transport in the meroistic telotrophic ovary of Podisus nigrispinus (Hemiptera) occurs via a paracellular or transcellular route. Light and transmission electron microscopies showed that short cell–cell contacts with well-developed occluding septate junctions were present in follicular cells with patency. Immunofluorescence microscopy revealed the presence of vitellogenin receptors in the plasma membrane and of vitellogenin in the cytoplasm of follicular cells. Data suggest that cell–cell contacts serve as a barrier to large vitellogenin molecules and that this protein is transported via a transcellular route of receptor-mediated endocytosis.

Effects of 17β-estradiol injection and red-spectrum light on eyestalk hormones and vitellogenesis of the ornamental cleaner shrimp Lysmata amboinensis (De Man, 1888) (Decapoda: Caridea: Lysmatidae)

Abstract We analyzed the effects of 17β-estradiol (E2) injection and red light on the sexual maturation and vitellogenesis of the cleaner shrimp Lysmata amboinensis (De Man, 1888) by measuring the change of mRNA expression levels of eyestalk and vitellogenesis-related hormones induced by both factors. We examined the eyestalk crustacean hyperglycemic hormone (CHH) and vitellogenesis-inhibiting hormone (VIH) genes. The E2 injection did not affect changes of eyestalk hormones. The red light, however, caused a significant increase (P < 0.05) of CHH promoting maturity and a significant decrease of VIH, which suppressed vitellogenesis. The vitellogenin receptor (VTG-R) and mRNA expression (in situ hybridization) also caused the E2 injection and red-wavelength irradiation to induce synthesis of vitellogenin (VTG). Red-light-induced gonadal maturation, promoting hormone and VTG synthesis, but E2 did not change the eyestalk hormone. Red-light irradiation could thus be used as a novel, maturity-promoting alternative method in the culture of the species, which is different from the eyestalk-ablation method that has so far been used.

Assessment of Gene Expression Biomarkers in the Chilean Pencil Catfish, Trichomycterus areolatus, from the Choapa River Basin, Coquimbo Chile.

The objective of this study was to describe changes in the gene expression in the Chilean catfish, Trichomycterus areolatus, based on their geographic location within the Choapa River. Genes of choice included those that are biomarkers of exposure to metals, oxidative stress, and endocrine disruption. Male and female T. areolatus were sampled from four sites in January 2015 differently impacted by human activities. In males, but not females, hepatic gene expression of heat shock protein (HSP70) and cytochrome P450 1A (CYP1A) were significantly elevated at the site adjacent to the small city of Salamanca, relative to the other sites. In females, hepatic HSP70, the aryl hydrocarbon receptor (AHR), and the estrogen responsive genes, vitellogenin (VTG) and estrogen receptor alpha (ERα), were significantly lower at the site located furthest downstream. A similar downstream pattern of lower expression levels also was found in ovarian tissue for the genes, HSP70 and ERα. Gill gene expression showed a unique pattern in females as levels of metallothionein were elevated at the site furthest downstream. While analytical chemistry of water samples provided limited evidence of agrichemical contamination, the gene expression data are consistent with an exposure to agrichemicals and metals. T. areolatus may be a valuable sentinel organism and its use as a bioindicator species in some rivers within Chile can provide considerable insight, particularly in situations analytical chemistry is limited by environmental constraints.

Molecular characterization and functional analysis of the vitellogenin receptor in the rice stem borer, Chilo suppressalis.

As a member of the low-density lipoprotein receptor (LDLR) superfamily, vitellogenin (Vg) receptor (VgR) is responsible for the uptake of Vg into developing oocytes and is a potential target for pest control. Here, a full-length VgR complementary DNA (named as CsVgR) was isolated and characterized in the rice stem borer, Chilo suppressalis. The composite CsVgR gene contained an open reading frame of 5,484 bp encoding a protein of 1,827 amino acid residues. Structural analysis revealed that CsVgR contained two ligand-binding domains (LBDs) with four Class A (LDLRA ) repeats in LBD1 and seven in LBD2, which was structurally different from most non-Lepidopteran insect VgRs having five repeats in LBD1 and eight in LBD2. The developmental expression analysis showed that CsVgR messenger RNA expression was first detectable in 3-day-old pupae, sharply increased in newly emerged female adults, and reached a peak in 2-day-old female adults. Consistent with most other insects VgRs, CsVgR was exclusively expressed in the ovary. Notably, injection of dsCsVgR into late pupae resulted in fewer follicles in the ovarioles as well as reduced fecundity, suggesting a critical role of CsVgR in female reproduction. These results may contribute to the development of RNA interference-mediated disruption of reproduction as a control strategy of C. suppressalis.

Accumulation and Depuration of Nonylphenol and Its Effect on the Expressions of Vitellogenin and Vitellogenin Receptor in Freshwater Prawn Macrobrachium rosenbergii

Accumulation of nonylphenol (NP) in hepatopancreas, gonad, eyestalk, and muscle of freshwater prawn Macrobrachium rosenbergii following 72h exposure to 100µg/L NP, and depuration of NP in these tissues at 0.5-192h post exposure were examined. We also examined the expressions of vitellogenin (Vg) and vitellogenin receptor (VgR) of prawn following 0-20days exposure to 0, 1, 10, and 100µg/L NP. NP accumulation in hepatopancreas and gonad with high concentration, and low concentration in muscle, but depurated faster in eyestalk and muscle. The expressions of vitellogenin (Vg) and vitellogenin receptor (VgR) increased directly with dose and time. In conclusion, NP accumulated significantly in gonad together with high Vg and VgR expressions, and depurated slow in hepatopancreas and gonad when prawns were removed back to control water. The induction of Vg and VgR under NP exposure might be a stress response in M. rosenbergii.

Graphene oxide mitigates endocrine disruption effects of bisphenol A on zebrafish at an early development stage

Development from embryos to larvae is an important life stage and impairments in this stage could affect the development and growth of fish. In this study, the endocrine disruption and developmental toxicities of bisphenol A (BPA) (50 and 500 μg/L) in the presence of graphene oxide (GO) (0.1 and 1 mg/L) were investigated on zebrafish embryos from 6 to 168 h post fertilization (hpf). BPA alone displayed significant endocrine disruption effects (increase in the estradiol (E2)/testosterone (T) ratio, vitellogenin (VTG) and estrogen receptor α (erα) in larvae), promoted embryos hatching and caused larvae malformation. There was a significant correlation between VTG level and erα expression, suggesting that erα played a vital role in VTG synthesis. However, all these adverse effects were alleviated distinctly in the presence of GO. GO formed a coating layer on the embryos chorion membrane, depressing absorption of BPA by the embryos. As a consequence, bioaccumulation of BPA in zebrafish co-exposed to GO and BPA decreased by >50% compared with the BPA single exposure group. Adsorption of BPA on GO might also make a partial contribution to the reduced accumulation of BPA in the larvae. The results demonstrated that GO could relieve the estrogenic and developmental effects of BPA on zebrafish in the early development stage.

Vitellogenin receptor and fatty acid profiles of individual lipid classes of oocytes from wild and captive-reared greater amberjack (Seriola dumerili) during the reproductive cycle

The greater amberjack Seriola dumerili (Risso, 1810) is a large migratory pelagic fish occurring in tropical and temperate waters with a great potential for the world aquaculture industry. Previous studies showed that wild-caught female greater amberjack reared in sea cages and handled during the reproductive season, underwent extensive ovarian atresia. This atresia, however, was not related to an insufficient liver transcription or oocyte uptake of vitellogenin (Vtg). In the present study, the structure of two greater amberjack vitellogenin receptors, namely Vtgr (Lr8-) and Lrp13, was characterized. Moreover, vtgr and lrp13 gene expression and the fatty acid profiles of specific phospholipids and neutral lipids were compared in the ovaries of wild and captive-reared greater amberjack during different phases of the reproductive cycle (i.e. early gametogenesis, advanced gametogenesis and spawning). Ovarian vtgr and lrp13 transcription was more active during early gametogenesis, suggesting that vitellogenin receptor transcripts were synthesized by previtellogenic oocytes and remained in the cellular mRNA pool until oocytes resumed meiosis and entered into secondary growth (i.e. vitellogenesis). Rearing of wild-caught greater amberjack in captivity together with handling during the reproductive season was associated with a reduced vtgr and lrp13 transcription and with a diminished capacity of oocytes in the early phase of gametogenesis (primary oocyte growth) to enter into vitellogenesis. During early gametogenesis, remarkable differences in the fatty acid composition were observed between wild and captive-reared individuals: all phospholipids of captive fish displayed dramatic increases of saturates (16:0 and 18:0) and decreases of arachidonic acid (ARA) and docosahexaenoic acid (DHA). The present study confirms the susceptibility of greater amberjack reproductive function to handling stress and suggests that the consequent extensive atresia of vitellogenic follicles originated during the primary oocytes growth when the capacity of oocytes to synthesize vitellogenin receptors was reduced. The study also suggests that this reduced capacity was associated with an altered oocyte phospholipid fatty acid composition during early gametogenesis.

Role of neuroparsin 1 in vitellogenesis in the mud crab, Scylla paramamosain

Neuroparsin (NP) is an important neuropeptide in invertebrates. It is well-known that NP displays multiple biological activities, including antidiuretic and inhibition of vitellogenesis in insects. However, the information about its effect in crustaceans is scarce. In this study, the sequence of Sp-NP1 was selected from the transcriptome database from the mud crab, Scylla paramamosain. Sequence analyses indicate that the Sp-NP1 amino acid (AA) sequences consist of a 27 AA signal peptide and a 74 AA mature peptide, which contains 12 cysteine residues. qRT-PCR analysis has revealed that the expressions of Sp-NP1 gene are high in the nervous tissues and extremely low in the ovary and hepatopancreas. In situ hybridization has shown that the positive signals are localized in cell cluster 6 of protocerebrum and cell clusters 10 and 11 of deutocerebrum. The presence of Sp-NP1 in the haemolymph has been detected in S. paramamosain through western blot, which indicates that Sp-NP1 serves as an endocrine factor in the regulation of physiological activities. In vitro experiments have further shown that the mRNA level of vitellogenin in the hepatopancreas notably decreases following administration of recombinant Sp-NP1, while the mRNA level of vitellogenin receptor and cyclin B in the ovary shows no significant differences. Collectively, Sp-NP1 possibly can inhibit the production of vitellogenin in the hepatopancreas and has no direct effect on the ovary in S. paramamosain.

Identification and Characterization of the Vitellogenin Receptor Gene and Its Role in Reproduction in the Alligatorweed Flea Beetle, Agasicles hygrophila.

Agasicles hygrophila is an effective biological control agent for the invasive weed Alternanthera philoxeroides, and because of this it has been introduced to many parts of the world where A. philoxeroides is a problem. Despite this, there are no reports at present about the reproduction of this important insect. Vitellogenin receptors (VgRs) belong to the superfamily of low-density lipoprotein receptors (LDLRs). One of the roles of VgRs is to regulate the absorption of yolk protein in insects. In this study, the full length vitellogenin receptor gene (AhVgR) from A. hygrophila was sequenced and found to encode a predicted protein of 1,642 amino acids. Sequence analysis of AhVgR revealed that it contains conserved structural motifs common to LDLR family members, and a phylogenetic analysis placed AhVgR as a separate group among the order Coleoptera. AhVgR was found to be specifically expressed in ovarian tissues, and it is first transcribed in the newly-emerged females. The expression patterns are consistent with VgR genes in other insects. RNA interference (RNAi)-mediated suppression of AhVgR gene expression in adult A. hygrophila females inhibited yolk protein deposition in the ovaries, shortened the ovariole, drastically reduced egg production, and ultimately led to a decrease in fecundity. In summary, our work shows that AhVgR is critical for transporting Vg into the oocytes and plays an important role in A. hygrophila reproduction.

Insulin signaling mediates previtellogenic development and enhances juvenile hormone-mediated vitellogenesis in a lepidopteran insect, Maruca vitrata

BackgroundInsulin/insulin-like growth peptide signaling (IIS) down-regulates hemolymph sugar level and facilitates larval growth in the soybean pod borer, Maruca vitrata. The objective of this study is to determine whether IIS of M. vitrata can mediate ovarian development of adult females.ResultsA pair of ovaries consists of 8 ovarioles, each of which is separated into distal germarium and proximal vitellarium in M. vitrata. In the germarium, oocyte development occurred with active mitotic activity which was visible by incorporating bromodeoxyribose uridine. Previtellogenic development and subsequent vitellogenesis began soon after adult emergence. They continued with increase of female age. Oocyte development was facilitated by up-regulation of vitellogenin (Vg) and Vg receptor (VgR) gene expression. Larval diets significantly influenced on ovarian development of M. vitrata because oocyte development varied with pupal size derived from larvae treated with different nutritional diets. Its ovarian development was dependent on endocrine signal(s) from the head because decapitation soon after adult emergence prevented oogenesis and subsequent vitellogenesis along with marked reduction of Vg and VgR expression. Topical application of juvenile hormone (JH) significantly recovered its ovarian development whereas farnesoic acid (a precursor of JH biosynthesis) or 20-hydroxyecdysone treatment did not. JH stimulated vitellogenesis and choriogenesis, but not previtellogenic development. In contrast, insulin injection to decapitated females stimulated oocyte differentiation and vitellogenesis along with increase of Vg and VgR expression. To further analyze the effect of insulin on ovarian development, expression of four IIS components (InR, FOXO, Akt, and TOR) genes was manipulated by RNA interference. Hemocoelic injection of gene-specific double stranded RNAs significantly reduced their target gene mRNA levels and interfered with ovarian development. An addition of insulin to JH treatment against decapitated females enhanced the gonadotropic effect of JH by stimulating oogenesis.ConclusionsIIS plays crucial role in mediating previtellogenic development of M. vitrata in response to nutrient signal. It also enhances the gonadotropic effect of JH II on vitellogenesis.

Reduced Glutamine Synthetase Activity Alters the Fecundity of Female Bactrocera dorsalis (Hendel)

Glutamine synthetase (GS) is a key enzyme in glutamine synthesis and is associated with multiple physiological processes in insects, such as embryonic development, heat shock response, and fecundity regulation. However, little is known about the influence of GS on female fecundity in the oriental fruit fly, Bactrocera dorsalis. Based on the cloning of BdGSs, mitochondrial BdGSm and cytoplasmic BdGSc, we determined their expressions in the tissues of adult B. dorsalis. BdGSm was highly expressed in the fat body, while BdGSc was highly expressed in the head and midgut. Gene silencing by RNA interference against two BdGSs isoforms suppressed target gene expression at the transcriptional level, leading to a reduced ovarian size and lower egg production. The specific inhibitor L-methionine S-sulfoximine suppressed enzyme activity, but only the gene expression of BdGSm was suppressed. A similar phenotype of delayed ovarian development occurred in the inhibitor bioassay. Significantly lower expression of vitellogenin and vitellogenin receptor was observed when GS enzyme activity was suppressed. These data illustrate the effects of two GS genes on adult fecundity by regulating vitellogenin synthesis in different ways.

Vitellogenin Receptor as a Target for Tick Control: A Mini-Review.

While much effort has been put into understanding vitellogenesis in insects and other organisms, much less is known of this process in ticks. There are several steps that facilitate yolk formation in developing oocytes of which the vitellogenin receptor (VgR) is a key component. The tick VgR binds vitellogenin (Vg) circulating in the hemolymph to initiate receptor-mediated endocytosis and its transformation into vitellin (Vn). The conversion of Vg into Vn, the final form of the yolk protein, occurs inside oocytes of the female tick ovary. Vn is critical to tick embryos since it serves as the nutritional source for their development, survival, and reproduction. Recent studies also suggest that pathogenic microbes, i.e., Babesia spp., that rely on ticks for propagation and dissemination likely “hitchhike” onto Vg molecules as they enter developing oocytes through the VgR. Suppressing VgR messenger RNA synthesis via RNA interference (RNAi) completely blocked Babesia spp. transmission into developing tick oocytes, thereby inhibiting vertical transmission of these pathogenic microbes from female to eggs. To date, VgRs from only four tick species, Dermacentor variabilis, Rhipicephalus microplus, Amblyomma hebraeum, and Haemaphysalis longicornis, have been fully sequenced and characterized. In contrast, many more VgRs have been described in various insect species. VgR is a critical component in egg formation and maturation that can serve as a precise target for tick control. However, additional research will help identify unique residues within the receptor that are specific to ticks or other arthropod disease vectors while avoiding cross-reactivity with non-target species. Detailed knowledge of the molecular structure and functional role of tick VgRs will enable development of novel vaccines to control ticks and tick-borne diseases.

A de novo transcriptome assembly approach elucidates the dynamics of ovarian maturation in the swordfish (Xiphias gladius)

The Mediterranean swordfish (Xiphias gladius) has been recently classified as overfished and in 2016, the International Commission for the Conservation of the Atlantic Tunas (ICCAT) established a multi-annual management plan to recover this stock. To successfully achieve this goal, knowledge about swordfish biology is needed. To date, few studies on swordfish have been performed and none of them has provided useful insights into the reproductive biology at molecular level. Here we set to characterise the molecular dynamics underlying ovarian maturation by employing a de novo transcriptome assembly approach. Differential gene expression analysis in mature and immature ovaries identified a number of differentially expressed genes associated with biological processes driving ovarian maturation. Focusing on ovarian steroidogenesis and vitellogenin uptake, we depict the molecular dynamics characterizing these processes while a phylogenetic analysis let us identify a candidate vitellogenin receptor. This is the first swordfish transcriptome assembly and these findings provide in-depth understanding of molecular processes describing ovarian maturation. Moreover, the establishment of a publicly available database containing information on the swordfish transcriptome aims to boost research on this species with the long-term of developing more comprehensive and successful stock management plans.

Intracellular localization of vitellogenin receptor mRNA and protein during oogenesis of a parthenogenetic tick, Haemaphysalis longicornis

BackgroundVitellogenin (Vg), a key molecule for oocyte development synthesized in the fat body during blood-feeding, is released into the hemolymph and then taken into the oocytes via Vg receptor (VgR) in ticks. Previously, we showed that VgR mRNA is expressed in the ovary at the adult stage of parthenogenetic Haemaphysalis longicornis ticks and its expression increases after blood-feeding. However, intracellular localization of VgR mRNA and protein at each developmental stage of oocytes during oogenesis remains largely unclear.MethodsmRNA and protein expression profiles of H. longicornis VgR (HlVgR) in the oocytes from the unfed to oviposition periods were analyzed by real-time PCR, in situ hybridization, and immunostaining. To elucidate the timing of the onset of Vg uptake, RNA interference (RNAi)-mediated gene silencing of HlVgR was performed.ResultsIn situ hybridization revealed that HlVgR mRNA was detected in the cytoplasm of stage I-III oocytes, and weaker positive signals for HlVgR mRNA were found in the cell periphery of stage IV and V oocytes. Likewise, HlVgR protein was detected by immunostaining in the cytoplasm of stage I-III oocytes and in the cell periphery of stage IV and V oocytes. Each developmental stage of the oocytes showed distinct patterns of mRNA and protein expression of HlVgR. Moreover, RNAi of HlVgR caused delayed or arrested development in the oocytes. The ovaries of control ticks showed all developmental stages of oocytes, whereas stage I-III oocytes were found in the ovaries of HlVgR-RNAi ticks at 5 days after engorgement.ConclusionsThese results suggest that active uptake of Vg is required for development from stage III to stage IV during oogenesis. Our data clearly revealed an apparent shift in the intracellular localization of VgR for both mRNA and protein level in oocytes during oogenesis.

Gene expression profiling of ovary identified eggshell proteins regulated by 20-hydroxyecdysone in Bactrocera dorsalis

The oriental fruit fly, Bactrocera dorsalis, is one of the most destructive pests worldwide. The frequent use of chemical insecticides has led B. dorsalis to develop resistance to many insecticides in recent decades. New high-throughput-sequenced transcriptomes, as well as genomes, have revealed a large number of reference genes for functional target identification. Here, we performed digital gene expression profiling of ovary and testis of B. dorsalis adults. Various genes were identified to be highly expressed in B. dorsalis ovary. The genes encoding components of eggshell, vitelline membrane proteins (Vmps) and chorion-related proteins, were identified to be tissue-specifically expressed in ovary. Five cytochrome P450 genes were also identified to be highly expressed in ovary. Three of them were ecdysone synthesis pathway genes indicating the ovary as a potential synthesis site of female. The up-regulated expression of Vmps by exogenous 20-hydroxyecdysone implied the hormonal regulation of eggshell formation during ovarian development. Many other genes with potential functions in ovarian development were also identified, including vitellogenin receptor, insulin receptor, NASP protein, and odorant binding protein. These findings should promote our understanding of the regulation of vitellogenesis and eggshell formation and enable exploration of potentially novel pest control targets.

Transcriptomic information from the ovaries of red swamp crayfish (Procambarus clarkii) provides new insights into development of ovaries and embryos

The red swamp crayfish Procambarus clarkii is a commercially important aquaculture species in inland China. Currently, the major constraint on the crayfish industry is the shortage of high-quality juveniles. Understanding the mechanisms that regulate ovarian development and maturation in female P. clarkii is crucial for the resolution of this problem. Through RNA-Seq technology, we first obtained the deep-coverage transcriptome sequencing data from the ovary of P. clarkii. A total of 31, 080, 133 clean reads were retrieved after removing the adapters in reads and filtering out low-quality data. All reads were assembled into 69, 261 unigenes with N50 of 1, 129 bp, of which 24, 014 unigenes matched sequences in the GenBank non-redundant database. From this transcriptome data, we obtained the full-length of one fragment that was previously identified to be differently expressed between precocious and normally grown P. clarkii ovaries. This gene encodes a peptide which shared the highest identity with RDH11 from other species, thus it was named PcRDH11 (P. clarkii retinol dehydrogenase 11). qRT-PCR analysis showed that PcRDH11 was highly transcribed in the ovary at the late vitellogenic stage. In situ hybridization exhibited specific expression of PcRDH11 in follicular cells around the early oocytes, but in the late vitellogenic stage, it moved to the ooplasm. Knockdown of PcRDH11 by injection of double-stranded RNA (dsRNA) resulted in decreased transcription of vitellogenin (Vg) and vitellogenin receptor (VgR). Treatment of early embryos with dsPcRDH11 caused arrest of the embryonic development and death of embryos. These results suggest that PcRDH11 may play an important role in ovarian development and embryogenesis. Our data provide insights into the molecular regulatory mechanisms of ovarian development in P. clarkii and other crustaceans, and will be helpful for the development of new techniques for artificial regulation of the reproductive process in crayfish aquaculture.