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Abstract The vitellogenin receptor (VgR) is highly expressed in the ovaries where it is responsible for vitellogenin (Vg) deposition during oogenesis in insects. Therefore, identifying the VgR of insect pests, and understanding the mechanism regulating its expression, could lead to the development of pest management strategies based on disrupting reproduction. We cloned and identified VgR in the cabbage beetle, Colaphellus bowringi , which is a serious pest of cruciferous vegetables in Asia. The regulation of VgR transcription by juvenile hormone (JH) was also investigated. The results show that C. bowringi VgR cDNA contains an open reading frame of 5310 bp encoding 1769 amino acid residues. Protein domain prediction indicates that C. bowringi VgR belongs to the LDLR gene superfamily, having the same group of structural domains that has been well characterized in other insects. VgR mRNA was highly expressed in the ovaries of reproductive female cabbage beetles. Knockdown of VgR reduced yolk deposition in the ovaries, increased the accumulation of Vg proteins in the hemolymph and decreased the transcription of Vg1 and Vg2 in the fat body. RNA interference and hormone challenge experiments showed that JH induced VgR transcription via the JH intracellular receptor methoprene-tolerant (Met) and the JH-responsive transcription factor Krüppel homolog 1 (Kr-h1). Our results suggest that there is a feedback loop between VgR transcription in the ovaries and Vg transcription in the fat body. JH acting through Met-Kr-h1 pathway induces the transcription of the VgR that is essential for Vg uptake and reproductive development. These findings not only reveal the potential JH signaling mechanism regulating VgR transcription, but may also contribute to the development of pest control strategies based on disrupting endocrine-regulated reproduction. Graphical abstract Unlabelled Image Highlights • Colaphellus bowringi VgR mRNA level was higher in the ovaries of reproductive females compared to diapause females. • Knockdown of VgR reduced yolk deposition in the ovaries and increased the accumulation of Vg proteins in the hemolymph. • There was a feedback loop between VgR transcription in the ovaries and Vg transcription in the fat body. • JH induced VgR transcription via the Met-Kr-h1 pathway. [ABSTRACT FROM AUTHOR]

Abstract Pesticides risk assessments have traditionally focused on the effects on standard parameters, such as mortality, reproduction and development. However, one of the first signs of adverse effects that occur in organisms exposed to stress conditions is an alteration in their genomic expression, which is specific to the type of stress, sensitive to very low contaminant concentrations and responsive in a few hours. The aim of the present study was to evaluate the single and binary mixture toxicity of commercial products of abamectin (Kraft® 36 EC) and difenoconazole (Score® 250 EC) to Folsomia candida. Laboratory toxicity tests were conducted to access the effects of these pesticides on springtail survival, reproduction and gene expression. The reproduction assays gave EC 50 and EC 10 values, respectively, of 6.3 and 1.4 mg a.s./kg dry soil for abamectin; 1.0 and 0.12 mg a.s./kg dry soil for Kraft® 36 EC; and 54 and 23 mg a.s./kg dry soil for Score® 250 EC. Technical difenoconazole did not have any effect at the concentrations tested. No significant differences in gene expression were found between the abamectin concentrations tested (EC 10 and EC 50) and the solvent control. Exposure to Kraft® 36 EC, however, significantly induced Cyp6 expression at the EC 50 level, while VgR was significantly downregulated at both the EC 10 and EC 50. Exposure to the simple pesticide mixture of Kraft® 36 EC + Score® 250 EC caused significant up regulation of ABC transporter, and significant down regulation of VgR relative to the controls. GABA receptor also showed significant down-regulation between the EC 10 and EC 50 mixture treatments. Results of the present study demonstrate that pesticide-induced gene expression effects precede and occur at lower concentrations than organism-level responses. Integrating "omic" endpoints in traditional bioassays may thus be a promising way forward in pesticide toxicity evaluations. Graphical abstract Image 1 Highlights • The use of gene expression to unravel pesticide effects was evaluated. • Gene expression and traditional parameters after single and mixture exposure were compared. • Gene expression parameters complement "traditional" test parameters. • Gene expression analyses has great potential to increase TKTD understanding of pesticide stress. [ABSTRACT FROM AUTHOR]

To evaluate potential involvement of clathrin in endocytosis of vitellogenin (Vtg) by teleost oocytes, cDNAs encoding clathrin heavy chain ( cltc ) were cloned from ovaries of cutthroat trout. Quantitative PCR revealed three types of cltc ( cltc-a1 , cltc-a2 , cltc-b ) to be expressed in 10 different tissues including the ovary. The cltc-a1 alone exhibited a significant decrease in ovarian expression during vitellogenesis; this was correlated with a corresponding decrease in transcripts encoding the major Vtg receptor (Vtgr). No development-related changes in ovarian cltc-a2 or cltc-b transcript levels were observed. In situ hybridization revealed a strong ctlc signal in pre-vitellogenic oocytes, but not in vitellogenic oocytes. Western blotting using a rabbit antiserum (a-Cltc) raised against a recombinant Cltc preparation detected a polypeptide band with an apparent mass of ~ 170 kDa in vitellogenic ovary extracts. Immunohistochemistry using a-Cltc revealed Cltc to be uniformly distributed throughout the ooplasm of perinucleolus stage oocytes, translocated to the periphery of lipid droplet stage oocytes, and localized to the oolemma during vitellogenesis. These patterns of cltc /Cltc distribution and abundance during oogenesis, which are identical to those previously reported for vtgr /Vtgr in this species, constitute the first empirical evidence that cltc-a1 /Cltc-a1 is involved in Vtg endocytosis via the Vtgr in teleost fish. [ABSTRACT FROM AUTHOR]

A complementary DNA (cDNA) that encodes the vitellogenin receptor (VgR) in the oriental river prawn, Macrobrachium nipponense , was cloned using expressed sequence tag analysis and a rapid amplification of cDNA ends approach. The coding region consists of 5920 base pairs (bp) that encode a 1902 amino acid protein, with a predicted molecular mass of 209 kDa. The coding region is flanked by a 45 bp 5ʹ-untranslated region (UTR) and a 166 bp 3ʹ-UTR. The deduced amino acid sequence of the M. nipponense VgR cDNA had typically conserved domains, such as an extracellular, lipoprotein-binding domain, epidermal growth factor-like and O -glycosylation domains, a transmembrane domain and a short C-terminal, cytosolic tail. Quantitative real-time PCR (qPCR) indicated that Mn-VgR is highly expressed in the female ovary. Expression analysis by qPCR demonstrated the larval and ovarian developmental stage-specific expression pattern. As the ovaries developed, the expression level of Mn-VgR gradually increased during the reproductive cycle (stage I), to reach a peak in stage III. Levels then dropped as a new development cycle was entered after reproduction molting. Eyestalk ablation led to a significant increase in the expression of Mn-VgR during the ovarian development stages ( P < 0.05), when compared with the eyestalk-intact group. The investigation revealed that eyestalk ablation initially affected Mn-VgR expression and then influenced vitellogenesis. In adult females, VgR RNA interference (RNAi) dramatically delayed the maturation of the ovary, in accordance with the gonad somatic index. In addition, Mn-VgR RNAi led to vitellin depletion in the oocytes and the accumulation of vitellin in the hepatopancreas. [ABSTRACT FROM AUTHOR]

Vitellogenin receptors (VgRs), members of the low-density lipoprotein receptor (LDLR) superfamily, are responsible for taking vitellogenin (Vg) into developing oocytes. Here the first full-length VgR cDNA from a hemipteran insect, the brown planthopper ( Nilaparvata lugens ), was cloned and sequenced. The complete mRNA sequence was 6174 bp in length with an open reading frame (ORF) of 5796 bp encoding 1931 amino acid residues. N. lugens VgR ( Nl VgR) contained two ligand-binding domains with five LDLR Class A cysteine-rich repeats in the first domain and eight in the second domain, which was similar to other insect VgRs. Nl VgR was specifically expressed in the ovary, and the mRNA level started to increase after adult female emergence, with a peak on day 7 in the adult stage, and then declined. Western blot analysis of Nl VgR protein revealed an ovary-specific expression pattern, which was consistent with NlVgR transcript detection. Injection with NlVgR double-stranded RNA (dsRNA) significantly disturbed NlVgR , which led to a decrease in Nl Vg protein content in the ovaries, an accumulation of Nl Vg protein in the hemolymph, the arrested development of ovaries, and the failure of insects to reproduce. Besides, Nl VgR expression was significantly upregulated after the topical application of juvenile hormone (JH) III. These results suggest that VgR is critical for Vg uptaking of oocytes and it plays an important role in insect fecundity. [ABSTRACT FROM AUTHOR]

Identification of molecular markers linked to phenotypic parameters of reproduction is important and can be applied to domestication and selective breeding programs of the giant tiger shrimp ( Penaeus monodon ). In this study, the full-length cDNA and expression levels of P. monodon vitellogenin receptor ( PmVtgr ) during ovarian development in wild shrimp and association between its single nucleotide polymorphisms (SNPs) and reproduction-related parameters in domesticated shrimp were examined. PmVtgr was 5990 bp in length composing of an open reading frame (ORF) of 5832 bp deducing to a polypeptide of 1943 amino acids. PmVtgr was only expressed in ovaries but not in other tissues of female broodstock. The expression level of PmVtgr in premature ovaries of juveniles was significantly lower than in those of broodstock ( P < 0.05). In intact broodstock, PmVtgr mRNA significantly increased in stage IV (mature) relative to stage I (previtellogenic) ovaries ( P < 0.05). However, PmVtgr was comparably expressed at all stages of ovarian development in eyestalk-ablated shrimp ( P > 0.05). In addition, SNPs in the PmVtgr gene segment of 14-month-old P. monodon ( N = 64) were indirectly examined by PCR-SSCP. Significant relationships between different SSCP patterns of PmVtgr and important reproduction-related phenotypes (i.e. gonadosomatic index, GSI and ovarian weight) of P. monodon were found. Results were consistent when further tested against non-related 19-month-old shrimp ( N = 54, P < 0.05). Representative 14-month-old shrimp exhibiting different SSCP genotypes of PmVtgr were cloned and sequenced ( N = 10 for each SSCP pattern). SNP positions 126 (C → T), 226 (T → A), 441 (T → A), 477 (T → A), 499 (A → C), 500 (T → C), 519 (A → G), 540 (A → G), 545 (G → A), 614 (T → C) and 678 (A → C) of PmVtgr were significantly associated with GSI values of examined shrimp ( N = 40). [ABSTRACT FROM AUTHOR]

Abstract: We have identified the full-length cDNA encoding a vitellogenin receptor (VgR) from the African bont tick Amblyomma hebraeum Koch (1844). VgRs are members of the low-density lipoprotein receptor superfamily that promote the uptake of the yolk protein vitellogenin (Vg), from the haemolymph. The AhVgR (GenBank accession No. JX846592) is 5703 bp, and encodes an 1801 aa protein with a 196.5 kDa molecular mass following cleavage of a 22 aa signal peptide. Phylogenetic analysis indicates that AhVgR is highly similar to other tick VgRs. AhVgR is expressed in only the ovary of mated, engorged females, and is absent in all other female tissues and in both fed and unfed males. Unfed, adult females injected with a VgR-dsRNA probe to knock-down VgR expression experienced a significant delay in ovary development and started oviposition significantly later than controls. These results indicate that the expression of AhVgR is important for the uptake of Vg and subsequent maturation of the oocytes. [Copyright &y& Elsevier]

Abstract: A cDNA encoding a vitellogenin receptor with 8 ligand binding repeats (vtgr) was cloned from ovaries of the cutthroat trout, Oncorhynchus clarki. In situ hybridization and quantitative PCR analyses revealed that the main site of vtgr mRNA expression was the oocytes. Expression was strongly detected in perinucleous stage oocytes, gradually decreased as oocytes grew, and became hardly detectable in vitellogenic oocytes. A rabbit antibody (a-Vtgr) was raised against a recombinant Vtgr protein in order to immunologically detect and localize Vtgr within the ovarian follicles. Western blotting using a-Vtgr detected a bold band with an apparent mass of ~95–105kDa in an ovarian preparation that also bound Sakhalin taimen, Hucho perryi, vitellogenin in ligand blots. Immunohistochemistry using a-Vtgr revealed that the Vtgr was uniformly distributed throughout the ooplasm of perinucleolus stage oocytes, subsequently translocated to the periphery of lipid droplet stage oocytes, and became localized to the oolemma during vitellogenesis. We provide the first characterization of Vtgr at both the transcriptional and the translational levels in the cutthroat trout, and our results suggest that this receptor is involved in uptake of Vtg by oocytes of this species. [Copyright &y& Elsevier]

Abstract: Many species of mites and ticks are of agricultural and medical importance. Much can be learned from the study of transcriptomes of acarines which can generate DNA-sequence information of potential target genes for the control of acarine pests. High throughput transcriptome sequencing can also yield sequences of genes critical during physiological processes poorly understood in acarines, i.e., the regulation of female reproduction in mites. The predatory mite, Phytoseiulus persimilis, was selected to conduct a transcriptome analysis using 454 pyrosequencing. The objective of this project was to obtain DNA-sequence information of expressed genes from P. persimilis with special interest in sequences corresponding to vitellogenin (Vg) and the vitellogenin receptor (VgR). These genes are critical to the understanding of vitellogenesis, and they will facilitate the study of the regulation of mite female reproduction. A total of 12,556 contiguous sequences (contigs) were assembled with an average size of 935bp. From these sequences, the putative translated peptides of 11 contigs were similar in amino acid sequences to other arthropod Vgs, while 6 were similar to VgRs. We selected some of these sequences to conduct stage-specific expression studies to further determine their function. [Copyright &y& Elsevier]

Abstract: We describe the first application of a non-radioactive ligand-blotting technique to the characterization of proteins interacting with nematode vitellins. Chromatographically purified vitellins from the free-living nematode Oscheius tipulae were labeled with fluorescein in vitro. Ligand-blotting assays with horseradish peroxidase-conjugated anti-fluorescein antibodies showed that labeled vitellins reacted specifically with a polypeptide of approximately 100 kDa, which we named P100. This polypeptide is a specific worm''s vitellin-binding protein that is present only in adult worms. Blots containing purified O. tipulae vitellin preparations showed no detectable signal in the 100 kDa region, ruling out any possibility of yolk polypeptides self-assembling under the conditions used in our assay. Experiments done in the presence of α-methyl mannoside ruled out the possibility of vitellins binding to P100 through mannose residues. Triton X-114 fractionation of whole worm extracts showed that P100 is either a membrane protein or has highly hydrophobic regions. [Copyright &y& Elsevier]

Abstract: Two members of the low density lipoprotein receptor (LDLR) family were identified as putative orthologs for a vitellogenin receptor (Amvgr) and a lipophorin receptor (Amlpr) in the Apis mellifera genome. Both receptor sequences have the structural motifs characteristic of LDLR family members and show a high degree of similarity with sequences of other insects. RT-PCR analysis of Amvgr and Amlpr expression detected the presence of both transcripts in different tissues of adult female (ovary, fat body, midgut, head and specifically hypopharyngeal gland), as well as in embryos. In the head RNA samples we found two variant forms of AmLpR: a full length one and a shorter one lacking 29 amino acids in the O-linked sugar domain. In ovaries the expression levels of the two honey bee LDLR members showed opposing trends: whereas Amvgr expression was upregulated as the ovaries became activated, Amlpr transcript levels gradually declined. In situ hybridization analysis performed on ovaries detected Amvgr mRNA exclusively in germ line cells and corroborated the qPCR results showing an increase in Amvgr gene expression concomitant with follicle growth. [Copyright &y& Elsevier]

Abstract: This is the first full-length message for a vitellogenin receptor (VgR) sequenced from ticks. VgRs, members of the low-density lipoprotein receptor (LDLR) superfamily, mediate the uptake of the yolk protein, vitellogenin (Vg), from the hemolymph. The VgR message from the American dog tick, Dermacentor variabilis (GenBank accession No. DQ103506.4) comprised 5673bp which coded for a 1798 aa deduced protein with a predicted 196.6kDa molecular mass. After removing the 20aa signal peptide, the 1778aa deduced mature protein had a predicted 196.6kDa molecular mass. BLAST comparisons showed the highest similarity to the VgR of the cockroach, Periplaneta americana. VgR message was expressed in mated female ovary but absent in female midgut and salivary glands or whole body mRNA from blood fed males, indicating that it is both sex and tissue specific. VgR transcript was absent in virgin (previtellogenic) females but present in ovaries of mated females following drop off. RNAi showed that unfed adult ticks injected with a VgR-dsRNA probe failed to lay eggs, develop brown eggs or fully express VgR transcript (Northern blots). In contrast, controls oviposited numerous normal brown eggs and showed strong expression of VgR transcripts. These results show that the expression of the VgR message is essential for Vg uptake and egg development in the American dog tick. [Copyright &y& Elsevier]

Absract: A cDNA encoding the vitellogenin receptor (VgR) of the cockroach, Leucophaea maderae (Lm) was cloned and sequenced. The coding region consisted of 5454bp flanked by a 67bp 5′-untranslated region (UTR) and a 168bp 3′-UTR, which encoded a 1792-residues mature protein with a predicted molecular mass of 199.84kDa. The deduced amino acid sequence of the LmVgR cDNA revealed two ligand-binding domains with 5 repeats in the first domain and 8 in the second domain similar to those of other insect VgRs. Northern hybridization analysis revealed the presence of a ∼7.3kb transcript that was specifically expressed only in the ovarian tissues. The developmental expression profile demonstrated that LmVgR mRNA exists throughout the ovarian development, and that the transcriptional level is especially high in the previtellogenic periods. The Immunoblot analysis detected an ovary-specific VgR protein with a M r of ∼215kDa under reducing conditions. The VgR protein continues to increase until day 3 of adult emergence, declines thereafter slightly until day 6, then rises again and reaches to its maximum on day 9 during the vitellogenic periods. Compared to other insect VgRs, the LmVgR primary protein structure shows an overall amino acid identity between 33% and 65% to their corresponding gene products. The similarity was high when compared with other cockroach VgRs described from Periplaneta americana (55%) and Blattella germanica (65%). A proposed phylogenetic (neighbor-joining) relationship suggests that LmVgR and B. germanica VgR have closer ancestry than from P. americana VgR. The cytoplasmic tail of LmVgR contains a leucine-isoleucine internalization signal similar to other insect counterparts unlike the NPXY motif of other LDLR family members. The cockroaches VgRs harbor, in addition, a putative NPTF motif for internalization. The sequence reported in this paper has been submitted to GenBank (accession number: AB255883). [Copyright &y& Elsevier]

Recent studies of vitellogenesis engendered a novel model of teleost yolk formation in which multiple yolk precursors, vitellogenins (Vtgs), and their receptors (Vtgrs) interact to ensure proper yolk composition for embryonic development and larval growth. As a step toward verification of this concept, we examined the role of one candidate Vtgr, termed low-density lipoprotein receptor relative with eight ligand-binding repeat (Lr8), in the medaka, a representative teleost and established laboratory model. A homozygous lr8 knock out (lr8 -KO) medaka was produced to perform reverse-genetic functional analyses. In ovaries of wild type (WT) medaka, Western blotting detected a putative Lr8 protein band at ~130 kDa, while immunohistochemistry detected the putative Lr8 signal at the periphery of the oocyte underneath the zona radiata. These signals disappeared in ovaries of the lr8 -KO group. Offspring of lr8 -KO medaka exhibited decreased survival rate compared to WT fish, but KO of lr8 was not 100% lethal. There was no significant difference in total yolk protein content or size of eggs between WT and lr8- KO fish. However, LC-MS/MS analyses revealed a remarkable decrease in the relative abundance of yolk proteins derived from VtgAb in lr8 -KO eggs, in conjunction with a compensatory increase in proteins derived from VtgAa1. These findings strongly support the conclusion that Lr8 is an important receptor for VtgAb in medaka. The disruption of proper yolk composition by lr8 -KO is possibly one cause of the low offspring survival. [Display omitted] • Knockout of the lr8 gene confirmed that Lr8 is a receptor for vitellogenin uptake into medaka oocytes. • Knockout of lr8 disrupted yolk composition and diminished egg quality evidenced by reduced larval survival • Compensation for lr8 knockout via increased VtgAa1 contributions to yolk was evident [ABSTRACT FROM AUTHOR]

The vitellogenin receptor (VgR) plays a critical role in egg development by mediating endocytosis of the major yolk protein precursor vitellogenin (Vg). Therefore, identifying the VgR of beneficial insects and its characterization could lead to the development of novel egg production strategies to enhance their commercial values. Here, we present the cloning, expression, and functional characterization of the VgR from an economically important eri silkworm, Samia ricini. The complete mRNA sequence was 6002 bp with an ORF of 5484 bp, encoding a protein of 1827 amino acids. Sequence analyses revealed that the Sr VgR contained all of the conservative structural motifs characteristics of LDLR family members. The SrVgR was specifically expressed in the ovary, and the mRNA level increased steadily in pupal stages, reached its peak on day 9, and then declined to a bare minimum in adults. RNA interference (RNAi) clearly reduced the VgR transcript levels, disrupted the ovarian development resulting in malformed ovarioles and abnormal development of eggs. Taken together, these data provide conclusive evidence for the essential roles of VgR in insect reproduction. Unlabelled Image • Cloned and characterized the vitellogenin receptor from eri silkworm, Samia ricini • RNA interference (RNAi) of VgR gene drastically disrupted the ovarian development • Clearly demonstrated an essential role of VgR in the egg development of eri silkworm • SrVgR can be used as a marker gene for improving the yield potential in silkworm breeds in terms of fecundity [ABSTRACT FROM AUTHOR]