Yolk Protein Precursor Research Articles

A comparative study of vitellogenesis in Echinodermata: Lessons from the sea star

The provision of yolk precursor proteins to the oviparous egg is crucial for normal embryo development. In Echinodermata, a transferrin-like yolk component termed major yolk protein (MYP) is a major precursor protein in Echinoidea and Holothuroidea. In contrast, in Asteroidea a single vitellogenin (Vtg) was recently identified, but its role as primary yolk protein remains unclear. To resolve the apparent MYP-Vtg dichotomy in sea stars and to understand the dynamics of candidate yolk protein gene expression during the reproductive cycle, we investigated the molecular structures of sea star Vtg and MYP and quantified their transcript levels during oogenesis. By combining protein sequencing of the predominant proteins in ovulated eggs of Patiriella regularis with ovarian transcriptome sequencing and molecular cloning, we characterized two cDNAs encoding two bona fide Vtgs (PrVtg1 and PrVtg2) and a partial cDNA encoding MYP (PrMYP). PrMYP mRNA was found in low abundance in growing oocytes, possibly as maternal transcripts for translation after ovulation. In contrast, PrVtg transcripts, whose levels varied during the reproductive cycle, were not found in developing oocytes – rather, they were detected in ovarian follicle cells and pyloric caeca, indicating an extra-oocytic origin. Vtg accumulating in oocytes was stored in the form of cleaved products, which constituted the most abundant yolk polypeptides in ovulated sea star eggs; their levels decreased during early embryonic and larval development. Together, these traits are the hallmarks of a classical yolk protein – and hence, we contend that Vtg, and not MYP, is the main yolk protein in asteroids.

Mechanistic insights into induction of vitellogenin gene expression by estrogens in Sydney rock oysters, Saccostrea glomerata

Marine molluscs, such as oysters, respond to estrogenic compounds with the induction of the egg yolk protein precursor, vitellogenin (Vtg), availing a biomarker for estrogenic pollution. Despite this application, the precise molecular mechanism through which estrogens exert their action to induce molluscan vitellogenesis is unknown. As a first step to address this question, we cloned a gene encoding Vtg from the Sydney rock oyster Saccostrea glomerata (sgVtg). Using primers designed from a partial sgVtg cDNA sequence available in Genbank, a full-length sgVtg cDNA of 8498bp was obtained by 5′- and 3′-RACE. The open reading frame (ORF) of sgVtg was determined to be 7980bp, which is substantially longer than the orthologs of other oyster species. Its deduced protein sequence shares the highest homology at the N- and C-terminal regions with other molluscan Vtgs. The full-length genomic DNA sequence of sgVtg was obtained by genomic PCR and genome walking targeting the gene body and flanking regions, respectively. The genomic sequence spans 20kb and consists of 30 exons and 29 introns. Computer analysis identified three closely spaced half-estrogen responsive elements (EREs) in the promoter region and a 210-bp CpG island 62bp downstream of the transcription start site. Upregulation of sgVtg mRNA expression was observed in the ovaries following in vitro (explants) and in vivo (tank) exposure to 17β-estradiol (E2). Notably, treatment with an estrogen receptor (ER) antagonist in vitro abolished the upregulation, suggesting a requirement for an estrogen-dependent receptor for transcriptional activation. DNA methylation of the 5′ CpG island was analysed using bisulfite genomic sequencing of the in vivo exposed ovaries. The CpG island was found to be hypomethylated (with 0–3% methylcytosines) in both control and E2-exposed oysters. However, no significant differential methylation or any correlation between methylation and sgVtg expression levels was observed. Overall, the results support the possible involvement of an ERE-containing promoter and an estrogen-activated receptor in estrogen signalling in marine molluscs.

Molecular Cloning and the Expression Profile of Vitellogenin in Relation to Tissue and Food Source inApolygus lucorum(Hemiptera: Miridae)

Vitellogenin proteins (Vgs) are precursors of the major egg yolk proteins in many oviparous vertebrates and invertebrates. Here we cloned the full-length cDNA of the Vg gene of Apolygus lucorum , an omnivorous mirid bug that feeds on various agricultural crops and small insects. The gene has 6,131 base pairs (bp) with an open reading frame (ORF) of 5,988 bp, encoding a protein with 1,996 amino acids, with molecular mass 218.3 kDa and isoelectric point 9.02. Phylogenetic analysis showed that Vg gene has a high similarity with that in Hemiptera insects and was most closely related to the rice leaf bug, Trigonotylus caelestialium . The expression of Vg gene was significantly higher in the fat body than in other tissues in females (ovary, hemolymph, gut, and malpighian tubule). Vg gene expression was extremely low in newly emerged adult females and increased with age. The effect of different food sources on the expression level of Vg in adult females was investigated after they were fed on green bean pods (Gb), H. armigera eggs (He), or a combination of green bean pods and H. armigera eggs (GbHe). The results showed that transcript level of Vg gene in females was significantly higher on the GbHe diet than on Gb or He. Our results demonstrate that A. lucorum can acquire nutrients from both plant and animal origin food sources, and the combined food sources significantly facilitated Vg synthesis.

Vitellogenesis and choriogenesis in fishes

In studies of sex discrimination in fish in the early 1900s, a specific antigen in the blood of gravid females was identified using immunological methods. At present, this specific antigen is known as vitellogenin, the major precursor of egg yolk protein that is synthesized in the female liver and is secreted into the blood to be incorporated into the egg. Recently, protein and gene analyses have revealed the presence of several vitellogenin variants. In addition, in the 1980s, choriogenin was identified as a novel precursor of egg envelope proteins that is secreted into the blood in response to stimulation by estrogen, similarly to vitellogenin. These two proteins not only play key roles in the process of oogenesis, but they are also used as effective biomarkers for assessing the impact of estrogen-like endocrine-disrupting chemicals (environmental hormones) in aquatic ecosystems.

Differential immune response of vitellogenin gene to Vibrio anguillarum in noble scallop Chlamys nobilis and its correlation with total carotenoid content.

Vitellogenin (Vg), an egg yolk precursor protein, not only functions as a source of nutrients and a nonpolar molecular carrier that combine and transfer lipids, proteins, vitamin and carotenoids to oocytes during the oogenesis. but also links with the immune defense in many oviparous animals. To investigate whether Vg plays a immune defensive role in noble scallop Chlamys nobilis, an acute Vibrio anguillarum infection experiment was conducted in orange and brown scallops with different carotenoids content. qRT-PCR result showed that Vg transcripts were significantly up-regulated after challenge with V. anguillarum in orange and brown shell scallops compared to the control group and Vg expression reached the highest spot at 6 h, indicated that Vg possessed an immune function in the noble scallop. Interestingly, a significantly positive correlation between Vg transcript levels and total carotenoids content in the ovary was observed, indicating that Vg gene expression was up regulated by carotenoids. The results suggest that Vg is a potent immune protector and carotenoid may linked with Vg plays an important role in host immune system against pathogens in noble scallop C. nobilis.

Age-dependent effects in fathead minnows from the anti-diabetic drug metformin

The anti-diabetic drug metformin is thought to be the pharmaceutical most deposited into the aquatic environment by mass at up to 6tons per year from individual WWTPs in urban areas. Recent studies have shown that exposure to 40ug/L of metformin increased the relative expression of the egg yolk precursor protein vitellogenin in adult male fathead minnows (Pimephales promelas) (FHM). For this study, the expression of several other genes involved in estrogen biosynthesis, clearance and downstream effects were assessed in FHM after treatment to three concentrations of metformin, to better understand the estrogenic effects of metformin on FHM. In contrast to the previous study, although upward trends were observed, metformin failed to significantly alter the expression of VTG, ERα, GnRH3, and CYP3A126 in adult male FHM. However, a concentration-dependent response to metformin was observed in younger 80–90day juvenile FHM. A 17.7-, 22-, and 22-fold increase in the relative expression of VTG mRNA in juvenile FHM exposed to 1, 10, and 100μg/L as compared to the control was observed. There was also a 3.3-, 4.7-, and 5.5-fold increase in GnRH3 in juvenile FHM exposed to 1, 10, and 100μg/L as compared to the control. Similarly, a 14-, 16-, and 24-fold increase in the relative expression of CYP3A126 mRNA was measured in juvenile FHM exposed to 1, 10 and 100μg/L metformin as compared to the control. These results indicate that juvenile FHM were more susceptible to the estrogenic effects of metformin during a 7-d exposure than older, sexually mature male FHM.

Use of Vitellogenin as Biomarker Indicator in Sex Identification of Giant Grouper (Epinephelus lanceolatus)

Vitellogenin, the egg yolk precursor protein, has studied as biomarker for sex identification of giant grouper (Epinephelus lanceolatus). Vitellogenin was synthesized successfully in liver of male giant grouper by implanted with 2 mg/kg of Estradiol-17-s (E2). The levels of vitellogenin (vtg) in blood of E2-injected fish were comparable with those found in the blood of naturally male and female by observed with SDS-PAGE. With antigen from purified plasma E2-treated was produced polyclonal antibody of giant grouper after immunization process with rabbit for 72 days. Analyst by ELISA has demonstrated polyclonal antibody was developed within 2 weeks after primary immunization and greatest differences in absorbance at 1:8,000 dilutions. Validation by western blot, has verified vitellogenin as positive control (in female natural) and negative control (in male natural). It’s showed that the antigens vtg of giant grouper polyclonal antibody recognized the purified giant grouper vtg in natural female and treated estradiol male. The information on Vtg through this experiment could provide the basis of an immunological assay for sex identification and maturational status of the Giant grouper. Consequently, the aim of the present study was to establish a simple, efficient and non-lethal technique by using Vtg as biomarker indicator for rapid sex identification without killing or seriously injuring the sampled individuals.

Quantification of X. laevis vitellogenin by liquid chromatography tandem mass spectrometry

Over the last several decades, there has been an increase in public awareness and regulatory activity in regard to the presence of emerging contaminants in the environment that may have the potential to interact with the endocrine system of exposed wildlife. Alterations in vitellogenin (VTG), a high density yolk precursor protein, can indicate endocrine activity in oviparous species, including many fish and amphibians. While various methodologies and experiments have been performed to characterize baseline VTG concentrations among commonly studied fish species, fewer methodologies for accurately quantifying amphibian VTG are available. Since there is relatively little information available on background VTG levels in male and female frogs, the present investigation set out to quantify baseline levels of VTG in juvenile as well as adult male and female African clawed frogs (Xenopus laevis) using a newly developed liquid chromatography tandem mass spectrometry method. This new methodology for quantifying VTG in X. laevis frog blood plasma can be applied in mechanistic and toxicity studies with X. laevis to better characterize potential endocrine modes of action.

Spatiotemporal analysis of microRNA-8 reveals important role in mosquito reproductive processes

Female mosquitoes require a blood meal for reproduction, providing the underlying mechanism for the spread of many devastating vector-borne diseases in humans. Understanding the mechanisms that govern the major functions linked to the female mosquito's ability to utilize blood and develop eggs is of paramount importance. Reports have indicated that microRNAs (miRNAs) are differentially expressed in various tissues of the female mosquito upon the uptake of a blood meal. In our previous research, we have reported the importance of miRNAs in regulating mosquito blood digestion through the characterization of the conserved miRNA, miR-275, and the mosquito specific miRNA, miR-1174. Our most recent work has shown that the conserved miRNA, miR-8, targets the Wingless signaling pathway to regulate secretion of yolk protein precursors by the female mosquito fat body and accumulation into the developing ovaries. Here, we summarize the role of miRNAs in the female mosquito, particularly miR-8. We also discuss the recent advances in mosquito biology and how these genetic tools can enhance our understanding of miRNA function.

Immune-Relevant and Antioxidant Activities of Vitellogenin and Yolk Proteins in Fish.

Vitellogenin (Vtg), the major egg yolk precursor protein, is traditionally thought to provide protein- and lipid-rich nutrients for developing embryos and larvae. However, the roles of Vtg as well as its derived yolk proteins lipovitellin (Lv) and phosvitin (Pv) extend beyond nutritional functions. Accumulating data have demonstrated that Vtg, Lv and Pv participate in host innate immune defense with multifaceted functions. They can all act as multivalent pattern recognition receptors capable of identifying invading microbes. Vtg and Pv can also act as immune effectors capable of killing bacteria and virus. Moreover, Vtg and Lv are shown to possess phagocytosis-promoting activity as opsonins. In addition to these immune-relevant functions, Vtg and Pv are found to have antioxidant activity, which is able to protect the host from oxidant stress. These non-nutritional functions clearly deepen our understanding of the physiological roles of the molecules, and at the same time, provide a sound basis for potential application of the molecules in human health.

Genomic organization and transcriptional modulation in response to endocrine disrupting chemicals of three vitellogenin genes in the self-fertilizing fish Kryptolebias marmoratus

Vitellogenin (Vtg) is the precursor of egg yolk proteins, and its expression has been used as a reliable biomarker for estrogenic contamination in the aquatic environment. To examine the biomarker potential of the self-fertilizing killifish Kryptolebias marmoratus Vtgs (Km-Vtgs), full genomic DNAs of Km-Vtgs-Aa, Km-Vtgs-Ab, and Km-Vtgs-C were cloned, sequenced, and characterized. Three Vtg genes in K. marmoratus are tandemly placed in a 550kb section of the same chromosome. In silico analysis of promoter regions revealed that both the Km-Vtgs-Aa and Km-Vtgs-Ab genes had an estrogen response element (ERE), but the Km-Vtgs-C gene did not. However, all three Km-Vtgs genes had several ERE-half sites in their promoter regions. Phylogenetic analysis demonstrated that the three deduced amino acid residues were highly conserved with conventional Vtgs protein, forming distinctive clades within teleost Vtgs. Liver tissue showed the highest expression of Km-Vtg transcripts in all tested tissues (brain/pituitary, eye, gonad, intestine, skin, and muscle) in response to endocrine disrupting chemical (EDC)-exposed conditions. Km-Vtg transcripts were significantly increased in response to 17β-estradiol (E2), tamoxifen (TMX), 4-n-nonylphenol (NP), bisphenol A (BPA), and octylphenol (OP) over 24hr exposure. The Km-Vtg-A gene was highly expressed compared to the control in response to NP and OP. EDC-induced modulatory patterns of Km-Vtg gene expression were different depending on tissue, gender, and isoforms.

Efficiency of advanced wastewater treatment technologies for the reduction of hormonal activity in effluents and connected surface water bodies by means of vitellogenin analyses in rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta f. fario).

Endocrine effects in the aquatic environment are in the focus of scientists and media along with debates on the necessity of further steps in wastewater treatment. In the present study VTG responses were compared to evaluate upgrades at wastewater treatment plants (WWTPs). We investigated several advanced sewage treatment technologies at two WWTPs connected to the Schussen, a tributary of Lake Constance, for the reduction of hormonal activity: (1) a powdered activated charcoal filter at the WWTP Langwiese; and (2) a combination of ozonation, sand filter, and granulated activated carbon filter at the WWTP Eriskirch. Rainbow trout and brown trout were either directly exposed to the effluents in aquaria or cages, or in a bypass system flown through by surface water of the Schussen. As a reference, trout were kept in bypass aquaria at the Argen River, which is less influenced by micropollutants. As a biomarker for estrogenicity, we analyzed the yolk precursor protein vitellogenin in immature rainbow trout and brown trout and in trout larvae (100 days post-fertilization) prior to and after the upgrade with the new technologies. Trout of different ages and species were used to detect differences in their sensitivity. At both bypass stations, larvae of brown trout showed significantly higher vitellogenin levels prior to the upgrade compared to negative control levels. Female brown trout exposed at the bypass station downstream of the WWTP showed decreased vitellogenin levels after the upgrade. In 1-year-old immature trout directly exposed to the respective effluents, no significant effects of the upgrades on vitellogenin levels were found. In general, larger effects were observed in brown trout than in rainbow trout, indicating that they are more sensitive test organisms.

Molecular cloning, characterization and evolutionary analysis of vitellogenin in Chinese giant salamander Andrias davidianus

Abstract Vitellogenin (VTG) is the major precursor of yolk proteins and could transport many components into oocytes, and hence it was thought to be a potential marker of reproduction of oviparious animal. Andrias davidianus is a critically endangered amphibian species, of which the wild population decreased dramatically and the artificial breeding has been successfully established. In this study, a full-length VTG cDNA was cloned and characterized from A. davidianus (designated as AdVTG) using 5’ and 3’ rapid amplification cDNA ends and RT-PCR. The AdVTG cDNA is 5,418 base pair in length and it encodes 1,805 amino acid long protein with typical quadripartite domains and several conserved cleavage sites. The tissue expression pattern indicated that AdVTG is mainly expressed in liver in one-year old individual. The threedimensional model of AdVTG constructed by homology modelling revealed the presence of conserved N-terminal β-sheet and lipid-binding cavity. Moreover, the phylogenetic analysis of VTG in metazoa was carried out, which revealed that Anuraspecific gene duplication occurred after the divergence of Caudata. All these results would improve the understanding of the physiological roles of AdVTG in A. davidianus and contribute to elucidating the evolution of VTG in vertebrates.

A NONOVARY-SPECIFIC VITELLOGENIN RECEPTOR FROM THE ORIENTAL FRUIT FLY, Bactrocera dorsalis (HENDEL).

The yolk protein precursor, vitellogenin (Vg), is absorbed into growing oocytes via receptor-mediated endocytosis for embryonic development. In this study, a Vg receptor (VgR) cDNA of the oriental fruit fly (Bactrocera dorsalis Hendel) was cloned via RT-PCR and RACE (GenBank accession no. KR535603) and its expression analyzed. The BdVgR cDNA has a length of 6,585 bp encoding 1,923 amino acids. It has a conserved motif arrangement with other insect VgRs, and showed high identity to the B. cucurbitae VgR (91.4%). The expression of BdVgR mRNA and proteins was shown in both ovary and fat body. This is the first report on a nonovary-specific VgR from a nonsocial insect. In ovary, the expression of BdVgR mRNA and proteins was inconsistent, with the transcription, but not protein, level high on D0. In fat body, the expression levels of BdVgR mRNA and proteins were high on days 5 and 6. The function of BdVgR in the fat body is not clear. However, it may be involved in reuptake of yolk proteins from the hemolymph as an amino acid reservoir or as autocrine regulation of yolk protein expression.

Vitellogenin and vitellogenin receptor gene expression is associated with male and female parenting in a subsocial insect.

Complex social behaviour in Hymenoptera has been hypothesized to evolve by co-opting reproductive pathways (the ovarian ground plan hypothesis, OGPH) and gene networks (the reproductive ground plan hypothesis, RGPH). In support of these hypotheses, in eusocial Hymenoptera where there is reproductive division of labour, the yolk precursor protein vitellogenin (Vg) influences the expression of worker social behaviour. We suggest that co-opting genes involved in reproduction may occur more generally than just in the evolution of eusociality; i.e. underlie earlier stages of social evolution such as the evolution of parental care, given that reproduction and parental care rarely overlap. We therefore examined vitellogenin (vg) gene expression associated with parental care in the subsocial beetle Nicrophorus vespilloides. We found a significant reduction in the expression of vg and its receptor, vgr, in head tissue during active parental care, and confirmed that the receptor is expressed in the brains of both sexes. Ours is the first study to show that vgr is expressed in the brain of a non-eusocial insect. Given the association between behaviour and gene expression in both sexes, and the presence of vitellogenin receptors in the brain, we suggest that Vg was co-opted early in the evolution of sociality to have a regulatory function. This extends the association of Vg in parenting to subsocial species and outside of the Hymenoptera, and supports the hypothesis that the OGPH is general and that heterochrony in gene expression is important in the evolution of social behaviour and precedes subsequent evolutionary specialization of social roles.

Use of Immunofluorescence with Frozen Sections for the Detection of Vitellogenin Year-2 Class Natural Population Rainbow Trout-A Preliminary Investigation

Vitellogenin (VTG) is an egg yolk precursor protein produced in the liver in response to estrogen and is taken up by the oocytes of rainbow trout (Oncorhynchus mykiss) by receptor-mediated endocytosis. It is a useful biomarker which can be detected in blood plasma using an enzyme-linked-immunosorbent assay (ELISA) by using primary and secondary antibodies to detect the protein. This study utilized 12 juvenile male and female rainbow trout, to develop and test a new method as a hybrid of frozen section histology and ELISA to directly detect VTG within gonad tissue using immunofluorescence (IF). Frozen sections of gonads fluoresced bright yellow within Medium Stage IV cells, indicating the presence of VTG within the developing oocytes. Paraffin and frozen histology sections were also compared for efficacy and quality. The two methods produced similar results, however frozen sectioning was faster and the tissue was better preserved when viewed under the microscope. Paraffin provided excellent color contrast, but the tissue exhibited more damage when compared to the frozen sections. Use of frozen sections to examine morphology and IF to detect VTG in rainbow trout gonadal cells proved to be effective for analyzing VTG at the tissue level.

MicroRNA-8 targets the Wingless signaling pathway in the female mosquito fat body to regulate reproductive processes

Female mosquitoes require a blood meal for reproduction, and this blood meal provides the underlying mechanism for the spread of many important vector-borne diseases in humans. A deeper understanding of the molecular mechanisms linked to mosquito blood meal processes and reproductive events is of particular importance for devising innovative vector control strategies. We found that the conserved microRNA miR-8 is an essential regulator of mosquito reproductive events. Two strategies to inhibit miR-8 function in vivo were used for functional characterization: systemic antagomir depletion and spatiotemporal inhibition using the miRNA sponge transgenic method in combination with the yeast transcriptional activator gal4 protein/upstream activating sequence system. Depletion of miR-8 in the female mosquito results in defects related to egg development and deposition. We used a multialgorithm approach for miRNA target prediction in mosquito 3' UTRs and experimentally verified secreted wingless-interacting molecule (swim) as an authentic target of miR-8. Our findings demonstrate that miR-8 controls the activity of the long-range Wingless (Wg) signaling by regulating Swim expression in the female fat body. We discovered that the miR-8/Wg axis is critical for the proper secretion of lipophorin and vitellogenin by the fat body and subsequent accumulation of these yolk protein precursors by developing oocytes.

Genetic architecture of a hormonal response to gene knockdown in honey bees.

Variation in endocrine signaling is proposed to underlie the evolution and regulation of social life histories, but the genetic architecture of endocrine signaling is still poorly understood. An excellent example of a hormonally influenced set of social traits is found in the honey bee (Apis mellifera): a dynamic and mutually suppressive relationship between juvenile hormone (JH) and the yolk precursor protein vitellogenin (Vg) regulates behavioral maturation and foraging of workers. Several other traits cosegregate with these behavioral phenotypes, comprising the pollen hoarding syndrome (PHS) one of the best-described animal behavioral syndromes. Genotype differences in responsiveness of JH to Vg are a potential mechanistic basis for the PHS. Here, we reduced Vg expression via RNA interference in progeny from a backcross between 2 selected lines of honey bees that differ in JH responsiveness to Vg reduction and measured JH response and ovary size, which represents another key aspect of the PHS. Genetic mapping based on restriction site-associated DNA tag sequencing identified suggestive quantitative trait loci (QTL) for ovary size and JH responsiveness. We confirmed genetic effects on both traits near many QTL that had been identified previously for their effect on various PHS traits. Thus, our results support a role for endocrine control of complex traits at a genetic level. Furthermore, this first example of a genetic map of a hormonal response to gene knockdown in a social insect helps to refine the genetic understanding of complex behaviors and the physiology that may underlie behavioral control in general.

Genotype effect on lifespan following vitellogenin knockdown

Honey bee workers display remarkable flexibility in the aging process. This plasticity is closely tied to behavioral maturation. Workers who initiate foraging behavior at earlier ages have shorter lifespans, and much of the variation in total lifespan can be explained by differences in pre-foraging lifespan. Vitellogenin (Vg), a yolk precursor protein, influences worker lifespan both as a regulator of behavioral maturation and through anti-oxidant and immune functions. Experimental reduction of Vg mRNA, and thus Vg protein levels, in wild-type bees results in precocious foraging behavior, decreased lifespan, and increased susceptibility to oxidative damage. We sought to separate the effects of Vg on lifespan due to behavioral maturation from those due to immune and antioxidant function using two selected strains of honey bees that differ in their phenotypic responsiveness to Vg gene knockdown. Surprisingly, we found that lifespans lengthen in the strain described as behaviorally and hormonally insensitive to Vg reduction. We then performed targeted gene expression analyses on genes hypothesized to mediate aging and lifespan: the insulin-like peptides (Ilp1 and 2) and manganese superoxide dismutase (mnSOD). The two honey bee Ilps are the most upstream components in the insulin-signaling pathway, which influences lifespan in Drosophila melanogaster and other organisms, while manganese superoxide dismutase encodes an enzyme with antioxidant functions in animals. We found expression differences in the llps in fat body related to behavior (llp1 and 2) and genetic background (Ilp2), but did not find strain by treatment effects. Expression of mnSOD was also affected by behavior and genetic background. Additionally, we observed a differential response to Vg knockdown in fat body expression of mnSOD, suggesting that antioxidant pathways may partially explain the strain-specific lifespan responses to Vg knockdown.

Molecular characterization and developmental expression of vitellogenin in the oriental river prawn Macrobrachium nipponense and the effects of RNA interference and eyestalk ablation on ovarian maturation

Vitellogenin (Vg) is the precursor of yolk protein, which functions as a nutritive resource that is important for embryonic growth and gonad development. In this study, the cDNA encoding the Vg gene from the oriental river prawn Macrobrachium nipponense was cloned using expressed sequence tag (EST) analysis and the rapid amplification of cDNA ends (RACE) approach. The transcript encoded 2536 amino acids with an estimated molecular mass of 286.810kDa. Quantitative real-time PCR indicated high expression of Mn-Vg in the female ovary, hemocytes, and hepatopancreas. As ovaries developed, the expression level of Mn-Vg increased in both the hepatopancreas and ovary. In the hepatopancreas, the expression level rose more slowly at the early stage of vitellogenesis and reached the peak more rapidly compared to the expression pattern in ovary. The observed changes in Mn-Vg expression level at different development stages suggest the role of nutrient source in embryonic and larval development. Eyestalk ablation caused the Mn-Vg expression level to increase significantly compared to eyestalk-intact groups during the ovary development stages. Ablation accelerated ovary maturation by removing hormone inhibition of Mn-Vg in the hepatopancreas and ovary. In adult females, Mn-Vg dsRNA injection resulted in decreased expression of Mn-Vg in both the hepatopancreas and ovary, and two injection treatment dramatically delayed ovary maturation. Vg RNA interference down-regulated the vitellogenin receptor (VgR) expression level in the ovary, which illustrates the close relationship between Vg and VgR in the process of vitellogenesis.