Genomic disturbance of vitellogenin 2 (vtg2) leads to vitellin membrane deficiencies and significant mortalities at early stages of embryonic development in zebrafish (Danio rerio).

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

Molecular Characterization of Vitellogenin and Its Receptor in Spodoptera frugiperda (J. E. Smith, 1797), and Their Function in Reproduction of Female.

The fall armyworm Spodoptera frugiperda is a highly polyphagous invasive pest. The strong reproductive capacity is an important factor in the rapid colonization and expansion of S. frugiperda. Vitellogenin (Vg) and vitellogenin receptor (VgR) play important roles in insect reproduction. As the precursor of vitellin (Vn), Vg provides essential nutrition for embryonic development, and VgR mediates the uptake of Vg by oocytes. In this context, we cloned and characterized these two genes of S. frugiperda (SfVg and SfVgR) and evaluated their expression profiles in different developmental stages and tissues. The RNA interference experiment was used to investigate their function in vitellogenesis. The ORF values of SfVg and SfVgR were 5250 and 5445 bp, encoding 1749 and 1815 amino acid residues, respectively. The qRT-PCR results revealed that both SfVg and SfVgR were highly expressed in female adults; SfVg was specifically expressed in the fat body, whereas SfVgR was highly expressed in the ovary. In addition, the depletion of either SfVg or SfVgR hindered oocyte maturation and ovarian development, leading to a significant decrease in fecundity. The present study reveals the importance of SfVg and SfVgR in the vitellogenesis of S. frugiperda, laying a theoretical foundation for the development of pollution-free pest control strategies with SfVg and SfVgR as new targets.

The KNRL nuclear receptor controls hydrolase-mediated vitellin breakdown during embryogenesis in the brown planthopper, Nilaparvata lugens.

Vitellin (Vn) homeostasis is central to the fecundity of oviparous insects. Most studies have focused on the synthesis and transportation of Vn as a building block for developing eggs during vitellogenesis; however, less is known about how the utilization of this nutrient reserve affects embryonic development. Here, we show that the single ortholog of the knirps and knirps-like nuclear receptors, KNRL, negatively regulates Vn breakdown by suppressing the expression of hydrolase genes in the brown planthopper, Nilaparvata lugens. KNRL was highly expressed in the ovary of adult females, and knockdown of KNRL by RNA interference resulted in the acceleration of Vn breakdown and the inhibition of embryonic development. Transcriptome sequencing analysis revealed that numerous hydrolase genes, including cathepsins and trypsins were up-regulated after KNRL knockdown. At least eight of the nine significantly enriched Gene Ontology terms for the up-regulated genes were in proteolysis-related categories. The expression levels of five selected trypsin genes and the enzymatic activities of trypsin in the embryos were significantly increased after KNRL knockdown. Moreover, trypsin injection prolonged egg duration, delayed embryonic development, accelerated Vn breakdown and severely reduced egg hatchability, a pattern similar to that observed in KNRL-silenced N. lugens. These observations suggest that KNRL controls Vn breakdown in embryos via the transcriptional inhibition of hydrolases. Generally, this study provides a foundation for understanding how embryo nutrient reserves are mobilized during embryogenesis and identifies several genes and pathways that may prove valuable targets for pest control.

Ovary Proteome Analysis Reveals RH36 Regulates Reproduction via Vitellin Uptake Mediated by HSP70 Protein in Hard Ticks.

Ticks are blood-sucking vector arthropods, which play an important role in transmitting pathogens between humans and animals. RH36 is an immunomodulatory protein expressed in the salivary glands, but not other organs, of partially fed Rhipicephalus haemaphysaloides ticks, and it reaches its peak on the day of tick engorgement. RH36 gene silencing inhibited tick blood feeding and induced a significant decrease in tick oviposition, indicating that another function of immunosuppressor RH36 was regulating tick reproduction. Why did RH36 protein expressed uniquely in the salivary gland regulate tick reproduction? RH36 regulated positively the expression of vitellogenin in ovary, which indicated RH36 protein played an important role in the integration of nutrition and reproduction. According to proteomic analysis, heat shock protein 70 (HSP70) was significantly down-regulated in the immature ovary of post-engorged ticks. In addition, gene silencing of HSP70 not only inhibited tick blood-sucking and the expression of vitellogenin, but also increased tick death rate. These results suggested RH36 affected tick vitellogenin uptake and then regulated ovary cell maturation by modulating the expression of HSP70 protein, and finally controlled tick oviposition.

Investigation of three enzymes and their roles in the embryonic development of parthenogenetic Haemaphysalis longicornis.

BACKGROUND: The tick Haemaphysalis longicornis exhibits two separate reproductive populations: bisexual and parthenogenetic, which have diploid and triploid karyotypes, respectively. The parthenogenetic population can undergo engorgement without copulation and produce viable female-only offspring with a longer incubation period than the bisexual population. Three enzymes, cathepsin B, cathepsin D and acid phosphatase, were found to be involved in vitellin degradation during the embryonic development of bisexual H. longicornis. However, the expression and activity profiles of these enzymes during the embryonic development of parthenogenetic ticks remain unknown. In the present study, the transcriptional expression profile, enzyme activity and roles in embryogenesis of the three enzymes during the embryonic development of parthenogenetic H. longicornis were investigated. METHODS: Quantitative real-time polymerase chain reaction (qPCR) and fluorescence detection were used to analyze the dynamic changes in the three enzymes during embryogenesis. The roles of the three enzymes during embryogenesis were also explored using RNA interference (RNAi). RESULTS: The three enzymes were all expressed during embryonic development in parthenogenetic H. longicornis. The expression of cathepsin B was highest on day 15, whereas that of cathepsin D was highest on day 3 and the peak of acid phosphatase expression occurred on day 9. The activity of cathepsin B was highest on day 3 and lowest on day 5, then gradually increased and remained stable. Cathepsin D activity was highest on day 1 and showed a gradually decreasing trend, whereas acid phosphatase showed the opposite trend and reached a peak on day 23. RNA interference experiments in engorged female ticks revealed that there was no significant difference in the number of eggs laid, but the hatching rate of the eggs was significantly decreased. CONCLUSION: The three enzymes all play important roles in embryonic development of H. longicornis, but the expression patterns and changes in the activity of the enzymes in the bisexual and parthenogenetic populations are different. The results will help a better understanding of the similarities and differences underlying embryonic development in the bisexual and parthenogenetic populations and contribute to the future exploration of the development of the parthenogenetic population of H. longicornis.

Interaction of nuclear ERs and GPER in vitellogenesis in zebrafish.

Estrogens exert their biological functions through the estrogen receptors (ERs). In zebrafish, three nuclear estrogen receptors (nERs) named ERα, ERß1 and ERß2 and one membrane-bound G protein-coupled estrogen receptor (GPER) are identified. Vitellogenin (Vtg) is predominantly expressed in liver and strongly response to the stimulation of estrogen. It has been proposed that all three nERs are functionally involved in vitellogenesis and ERα may act as the major mediator in teleost. However, the role of GPER and its interaction with nERs in this process are not yet defined in teleost species. In the present study, we provide genetic evidence for the functional significance of ERα that the expression of Vtg genes (vtg1, vtg2, vtg3) and their response to estradiol stimulation were significantly decreased in esr1 mutant zebrafish. Activation of ERß1 and ERß2 induced Vtg expression through ERα. Moreover, the involvement of GPER in vitellogenesis and its interaction with nERs in zebrafish were firstly proposed in this work. Activation of GPER induced Vtg genes expression while inhibition of GPER significantly attenuated the estrogenic effect on Vtg. Both treatments altered the expression levels of nERs, suggesting GPER acts interactively with nERs. Collectively, the involvement of both nERs and GPER in regulation of vitellogenesis is demonstrated. ERα is the central factor, acting interactively with ERß1, ERß2 and GPER, and GPER regulates vitellogenesis directly and interactively with nERs.

Short-term induction of vitellogenesis in the immature male yellowfin seabream ( Acanthopagrus latus) exposed to bisphenol A and 17 ß-estradiol.

Bisphenol A (BPA) is a known environmental endocrine-disrupting chemical that is widely used in plastics manufacturing. BPA enters in the aquatic environment mainly through urban and industrial sewage effluents, thereby posing a potential threat to organisms living in these ecosystems. This study was conducted to investigate the effect of BPA on VTG production with direct (sodium dodecyl sulfate-polyarylamide gel electrophoresis) and indirect (alkali-labile phosphate (ALP), total plasma calcium and protein) methods in immature male yellowfin seabream ( Acanthopagrus latus) as a marine fish model. Fish were randomly distributed into seven groups that were administered 1, 10, 50, and 100 µg g-1 week-1 of BPA and 2 µg g-1week-1 of 17ß-estradiol (E2) over a period of 2 weeks. Solvent controls received olive oil, whereas controls were not injected. The fish were sampled on days 0, 7, and 14, and their blood plasma and liver were obtained. The results showed that the hepatosomatic index of all treated fish was elevated in comparison with controls. Direct and indirect indicators showed that fish VTG protein was induced by BPA and E2 exposure. The protein was found to have two bands with molecular weights around 210 and 190 KDa. ALP, total plasma calcium and protein levels were increased in dose- and time-dependent manners. The results of this study demonstrated that short-term exposure of yellowfin seabream to BPA induced adverse effects in the reproductive system of hermaphrodite fish.

Purification and Characterization of Vitellin from the Egg of the Suminoe Oyster Crassostrea ariakensis and Cross-Reactivity of Anti-vitellin Antibody with Other Marine Invertebrate Egg Proteins.

A polyclonal antibody specific to an egg protein of Suminoe oyster Crassostrea ariakensis was previously developed in our laboratory to assess the reproductive life cycle of the oyster. The present study was undertaken to investigate vitellin of C. ariakensis (CAVt). Vitellin is an essential component of egg proteins in marine invertebrates as it provides energy and nutrients to the embryo and larvae. CAVt was purified from eggs of the oyster using ammonium sulfate precipitation followed by affinity chromatography with Concanavalin A-agarose. Native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate PAGE showed that CAVt is a high molecular weight [532 kiloDaltons (kDa)] protein, with multiple subunits. Similar to other vitellin proteins, it is a phospholipoglycoprotein composed of phospholipids (12.06%), carbohydrates (mannose, 10.08% or glucose, 9.84%), and alkali-labile phosphates (4.16%). Affinity chromatography, enzyme-linked immunosorbent aasay (ELISA) and western blot analysis revealed that CAVt is only present in the ovary, and two subunits of CAVt (72 and 35 kDa) are believed to be incorporated from the hemolymph into the oocyte. The antibody specific to CAVt (anti-CAVt), raised in rabbit, strongly cross reacted with the egg proteins of oyster species and scallops, suggesting that the antigenic epitopes are highly conserved among species. Our results suggest that the anti-CAVt antibody can be used to develop a tool similar to ELISA or western blotting for investigation of the effect of microorganisms on reproduction as well as the effect of chemicals on the endocrine system in C. ariakensis.

Regulating gonad inhibition and vitellogenin/vitellin induction in Penaeus monodon using mature GIH fusion protein and polyclonal antisera.

Gonad inhibiting hormone (GIH), type II class of the CHH family neuropeptides, is released by the neurohaemal XO-SG complex of the eyestalk. The inhibitory function of GIH has a pivotal role in gonad development and reproduction. In this study, we report the expression and production of a thioredoxin-fused mature GIH protein (mf-PmGIH) of Penaeus monodon in a bacterial system and its use as antigen to raise polyclonal antiserum (anti-mf-PmGIH). The mature GIH gene of 237bp that codes for 79 amino acids, was cloned into the Escherichia coli thioredoxin gene fusion expression system. The expression vector construct (mf-PmGIH+pEt32a+) upon induction produced 32.16kDa mature GIH fusion protein (mf-PmGIH)·The purified fusion protein was used as exogenous GIH and as antigen to raise polyclonal antisera. The fusion protein when injected into juvenile shrimp significantly reduced vitellogenin/vitellin levels by 31.55% within 72h in comparison to the controls showing the gonad inhibiting property. Vitellogenin/vitellin levels were significantly induced by 74.10% within 6h when polyclonal antiserum (anti-mf-PmGIH - 1:500) was injected in P. monodon. Anti-mf-PmGIH immunolocalized GIH producing neurosecretory cells in the eyestalk of P. monodon. The present manuscript reports an innovative means of gonad inhibition and vitellogenin/vitellin induction with thioredoxin fused GIH and antisera developed.

Cry1Ab-expressing rice did not influence expression of fecundity-related genes in the wolf spider Pardosa pseudoannulata.

The impact of Bacillus thuringiensis (Bt) toxin proteins on non-target predatory arthropods is not well understood at the cellular and molecular levels. Here, we investigated the potential effects of Cry1Ab expressing rice on fecundity of the wolf spider, Pardosa pseudoannulata, and some of the underlying molecular mechanisms. The results indicated that brown planthoppers (BPHs) reared on Cry1Ab-expressing rice accumulated the Cry toxin and that reproductive parameters (pre-oviposition period, post-oviposition stage, number of eggs, and egg hatching rate) of the spiders that consumed BPHs reared on Bt rice were not different from those that consumed BPHs reared on the non-Bt control rice. The accumulated Cry1Ab did not influence several vitellin (Vt) parameters, including stored energy and amino acid composition, during one generation. We considered the possibility that the Cry toxins exert their influence on beneficial predators via more subtle effects detectable at the molecular level in terms of gene expression. This led us to transcriptome analysis to detect differentially expressed genes in the ovaries of spiders exposed to dietary Cry1Ab and their counterpart control spiders. Eight genes, associated with vitellogenesis, vitellogenin receptor activity, and vitellin membrane formation were not differentially expressed between ovaries from the treated and control spiders, confirmed by qPCR analysis. We infer that dietary Cry1Ab expressing rice does not influence fecundity, nor expression levels of Vt-associated genes in P. pseudoannulata.

Identification of new markers for the Schistosoma mansoni vitelline lineage.

Schistosomes cause significant morbidity and mortality in millions of the world's poorest people. While parasite egg-induced inflammation is the primary driver of host pathology, relatively little is known at the molecular level about the organ systems that participate in schistosome egg production (i.e., testes, ovaries and vitellaria). Here we use transcriptional profiling and in situ hybridization to characterise the vitellarium of Schistosoma mansoni. We uncovered several previously uncharacterised vitellaria-specific factors and defined molecular markers for various stages in the vitellocyte differentiation process. These data provide the framework for future in-depth molecular studies exploring the biology of this important parasite organ.

Juvenile Hormone Activates the Transcription of Cell-division-cycle 6 (Cdc6) for Polyploidy-dependent Insect Vitellogenesis and Oogenesis.

Although juvenile hormone (JH) is known to prevent insect larval metamorphosis and stimulate adult reproduction, the molecular mechanisms of JH action in insect reproduction remain largely unknown. Earlier, we reported that the JH-receptor complex, composed of methoprene-tolerant and steroid receptor co-activator, acts on mini-chromosome maintenance (Mcm) genes Mcm4 and Mcm7 to promote DNA replication and polyploidy for the massive vitellogenin (Vg) synthesis required for egg production in the migratory locust (Guo, W., Wu, Z., Song, J., Jiang, F., Wang, Z., Deng, S., Walker, V. K., and Zhou, S. (2014) PLoS Genet. 10, e1004702). In this study we have investigated the involvement of cell-division-cycle 6 (Cdc6) in JH-dependent vitellogenesis and oogenesis, as Cdc6 is essential for the formation of prereplication complex. We demonstrate here that Cdc6 is expressed in response to JH and methoprene-tolerant, and Cdc6 transcription is directly regulated by the JH-receptor complex. Knockdown of Cdc6 inhibits polyploidization of fat body and follicle cells, resulting in the substantial reduction of Vg expression in the fat body as well as severely impaired oocyte maturation and ovarian growth. Our data indicate the involvement of Cdc6 in JH pathway and a pivotal role of Cdc6 in JH-mediated polyploidization, vitellogenesis, and oogenesis.

Very high-density lipoprotein and vitellin as carriers of novel biliverdins IXα with a farnesyl side-chain presumably derived from heme A in Spodoptera littoralis.

Bilins in complex with specific proteins play key roles in many forms of life. Biliproteins have also been isolated from insects; however, structural details are rare and possible functions largely unknown. Recently, we identified a high-molecular weight biliprotein from a moth, Cerura vinula, as an arylphorin-type hexameric storage protein linked to a novel farnesyl biliverdin IXα; its unusual structure suggests formation by cleavage of mitochondrial heme A. In the present study of another moth, Spodoptera littoralis, we isolated two different biliproteins. These proteins were identified as a very high-density lipoprotein (VHDL) and as vitellin, respectively, by mass spectrometric sequencing. Both proteins are associated with three different farnesyl biliverdins IXα: the one bilin isolated from C. vinula and two new structurally closely related bilins, supposed to be intermediates of heme A degradation. The different bilin composition of the two biliproteins suggests that the presumed oxidations at the farnesyl side-chain take place mainly during egg development. The egg bilins are supposedly transferred from hemolymph VHDL to vitellin in the female. Both biliproteins show strong induced circular dichroism activity compatible with a predominance of the M-conformation of the bilins. This conformation is opposite to that of the arylphorin-type biliprotein from C. vinula. Electron microscopy of the VHDL-type biliprotein from S. littoralis provided a preliminary view of its structure as a homodimer and confirmed the biochemically determined molecular mass of ∼350 kDa. Further, images of S. littoralis hexamerins revealed a 2 × 3 construction identical to that known from the hexamerin from C. vinula.

The Role of DmCatD, a Cathepsin D-Like Peptidase, and Acid Phosphatase in the Process of Follicular Atresia in Dipetalogaster maxima (Hemiptera: Reduviidae), a Vector of Chagas' Disease.

In this work, we have investigated the involvement of DmCatD, a cathepsin D-like peptidase, and acid phosphatase in the process of follicular atresia of Dipetalogaster maxima, a hematophagous insect vector of Chagas' disease. For the studies, fat bodies, ovaries and hemolymph were sampled from anautogenous females at representative days of the reproductive cycle: pre-vitellogenesis, vitellogenesis as well as early and late atresia. Real time PCR (qPCR) and western blot assays showed that DmCatD was expressed in fat bodies and ovaries at all reproductive stages, being the expression of its active form significantly higher at the atretic stages. In hemolymph samples, only the immunoreactive band compatible with pro-DmCatD was observed by western blot. Acid phosphatase activity in ovarian tissues significantly increased during follicular atresia in comparison to pre-vitellogenesis and vitellogenesis. A further enzyme characterization with inhibitors showed that the high levels of acid phosphatase activity in atretic ovaries corresponded mainly to a tyrosine phosphatase. Immunofluorescence assays demonstrated that DmCatD and tyrosine phosphatase were associated with yolk bodies in vitellogenic follicles, while in atretic stages they displayed a different cellular distribution. DmCatD and tyrosine phosphatase partially co-localized with vitellin. Moreover, their interaction was supported by FRET analysis. In vitro assays using homogenates of atretic ovaries as the enzyme source and enzyme inhibitors demonstrated that DmCatD, together with a tyrosine phosphatase, were necessary to promote the degradation of vitellin. Taken together, the results strongly suggested that both acid hydrolases play a central role in early vitellin proteolysis during the process of follicular atresia.

CALCIUM-INDUCED LIPID PEROXIDATION IS MEDIATED BY RHODNIUS HEME-BINDING PROTEIN (RHBP) AND PREVENTED BY VITELLIN.

Lipid peroxidation is promoted by the quasi-lipoxygenase (QL) activity of heme proteins and enhanced by the presence of free calcium. Unlike mammalian plasma, the hemolymph of Rhodnius prolixus, a vector of Chagas disease, contains both a free heme-binding protein (RHBP) and circulating lipoproteins. RHBP binds and prevents the heme groups of the proteins from participating in lipid peroxidation reactions. Herein, we show that despite being bound to RHBP, heme groups promote lipid peroxidation through a calcium-dependent QL reaction. This reaction is readily inhibited by the presence of ethylene glycol tetraacetic acid (EGTA), the antioxidant butylated hydroxytoluene or micromolar levels of the main yolk phosphoprotein vitellin (Vt). The inhibition of lipid peroxidation is eliminated by the in vitro dephosphorylation of Vt, indicating that this reaction depends on the interaction of free calcium ions with negatively charged phosphoamino acids. Our results demonstrate that calcium chelation mediated by phosphoproteins occurs via an antioxidant mechanism that protects living organisms from lipid peroxidation.

Vitellogenesis of diphyllobothriidean cestodes (Platyhelminthes).

The recently erected cestode order Diphyllobothriidea is unique among all tapeworm orders in that its species infect all major groups of tetrapods, including man. In the present paper, the vitellogenesis of representatives of all three currently recognized families of this order was evaluated, based on ultrastructural (transmission electron microscopy) and cytochemical (detection of glycogen) observations. Vitelline follicles of all taxa studied, i.e. Cephalochlamys namaquensis from clawed frogs (Xenopus), Duthiersia expansa from monitors (Varanus) and Schistocephalus solidus that matures in fish-eating birds, contain vitelline cells at various stages of development and interstitial cells. Developing vitellocytes are characterized by the presence of mitochondria, granular endoplasmic reticulum and Golgi complexes involved in the synthesis of shell globules and formation of shell globule clusters. Mature vitellocytes contain lipids and glycogen in different proportions. The most significant differences among the three diphyllobothriidean families were found in the presence or absence of lamellar bodies. Variations of vitelline clusters morphology and types of lipid droplets are described and discussed in relation to the presumed evolutionary history of diphyllobothriideans, which belong to the most basal cestode groups.

Purification of vitellin and dynamics of vitellogenesis in the parthenogenetic tick Haemaphysalis longicornis (Acari: Ixodidae).

Vitellin (Vt) was purified from eggs of parthenogenetic bush tick Haemaphysalis longicornis by gel filtration and ion exchange chromatography. Our results revealed that only one single Vt existed in parthenogenetic bush tick, and the purified Vt was proved to be a hemoglycolipoprotein consisting of nine polypeptides with molecular weights of 203, 147, 126, 82, 74, 70, 61, 47 and 31 kDa, respectively. Polyclonal antibody and monoclonal antibody against Vt were produced using the purified Vt. The change in vitellogenin (Vg) and Vt levels over time of the parthenogenetic H. longicornis was established, and the Vg content in haemolymph and Vt in ovary at different feeding or engorgement statuses was also determined using a double antibody sandwich enzyme-linked immunosorbent assay. The Vg level in haemolymph was distinctly increased on the day of engorgement (1.785 mg/mL) and continued to increase until 2nd day post-engorgement (5.611 mg/mL). There was a slight decrease in Vg level after 4 days of engorgement, and a second peak was observed on day 2 post-oviposition (10.774 mg/mL). Subsequently, Vg content continuously decreased and reached a low level on the 10th day post-oviposition. The Vt content in ovary continuously increased once the female reached its critical weight (0.024 mg per female), and reached the maximum level on day 2 post-oviposition (1.942 mg per female). Afterwards, Vt content rapidly decreased.

TBT effects on the development of intersex (ovotestis) in female fresh water prawn Macrobrachium rosenbergii.

The impact of tributyltin (TBT) on the female gonad and the endocrine system in Macrobrachium rosenbergii was studied. Prawns were exposed to environmentally realistic concentrations of 10, 100, and 1000 ng/L of TBT for 6 months. Dose dependent effects were noticed in TBT exposed prawns. At 1000 ng/L TBT caused ovotestis formation (formation of male germ cells in ovary). Presence immature oocytes, fusion of developing oocytes, increase in interstitial connective tissues, and its modification into tubular like structure and abundance of spermatogonia in the ovary of TBT treated prawns. The control prawn ovary showed normal architecture of cellular organelles such as mature oocytes with type 2 yolk globules, lipid droplets, normal appearance of yolk envelop, and uniformly arranged microvilli. On the other hand, type 1 yolk globules, reduced size of microvilli, spermatogonial cells in ovary, spermatogonia with centrally located nucleus, and chromatin distribution throughout the nucleoplasm were present in the TBT treated group. Immunofluorescence staining indicated a reduction in vitellin content in ovary of TBT treated prawn. Moreover, TBT had inhibited the vitellogenesis by causing hormonal imbalance in M. rosenbergii. Thus, the present investigation demonstrates that TBT substantially affects sexual differentiation and gonadal development in M. rosenbergii.

Transcriptome analysis of female reproductive tissues of Anastrepha obliqua and molecular evolution of eggshell proteins in the fraterculus group.

The investigation of cDNA libraries has been an important tool for the identification of new genes in nonmodel species such as the fruit flies from the Anastrepha fraterculus group. In the present study, we constructed a cDNA library from the female reproductive tissues of Anastrepha obliqua aiming to identify genes with high evolutionary rates. We sequenced 2304 clones obtained from the female reproductive tissues of A. obliqua flies. The expressed sequence tags generated a total of 816 unigenes which were classified into different protein classes. Among these,we identified chorionic and vitelline protein genes as being among the most highly expressed. We used unigene sequences to amplify a set of chorionic and vitelline genes, involved in the formation of the eggshell,in species of the fraterculus group. Four chorionic genes and two vitelline genes showed evidence of positive selection along the Anastrepha and/or Tephritidae lineage. The signal of selection detected for Vm26Aa was possibly generated by a gene duplication event. The rapid evolutionary rates indicate that these genes could serve as important markers in population and evolutionary studies, not only for species of this group, but possibly also for other Diptera.