Insulin-like peptides and the target of rapamycin pathway coordinately regulate blood digestion and egg maturation in the mosquito Aedes aegypti

Background

Mosquitoes are insects that vector many serious pathogens to humans and other vertebrates. Most mosquitoes must feed on the blood of a vertebrate host to produce eggs. In turn, multiple cycles of blood feeding promote frequent contacts with hosts and make mosquitoes ideal disease vectors. Both hormonal and nutritional factors are involved in regulating egg development in the mosquito, Aedes aegypti. However, the processes that regulate digestion of the blood meal remain unclear.

Methodology/Principal Findings

Here we report that insulin peptide 3 (ILP3) directly stimulated late phase trypsin-like gene expression in blood fed females. In vivo knockdown of the mosquito insulin receptor (MIR) by RNA interference (RNAi) delayed but did not fully inhibit trypsin-like gene expression in the midgut, ecdysteroid (ECD) production by ovaries, and vitellogenin (Vg) expression by the fat body. In contrast, in vivo treatment with double-stranded MIR RNA and rapamycin completely blocked egg production. In vitro experiments showed that amino acids did not simulate late phase trypsin-like gene expression in the midgut or ECD production by the ovaries. However, amino acids did enhance ILP3-mediated stimulation of trypsin-like gene expression and ECD production.

Conclusions/Significance

Overall, our results indicate that ILPs from the brain synchronize blood meal digestion and amino acid availability with ovarian ECD production to maximize Vg expression by the fat body. The activation of digestion by ILPs may also underlie the growth promoting effects of insulin and TOR signaling in other species.     

Juvenile hormone regulates vitellogenin gene expression through insulin-like peptide signaling pathway in the red flour beetle, Tribolium castaneum

Our recent studies identified juvenile hormone (JH) and nutrition as the two key signals that regulate vitellogenin (Vg) gene expression in the red flour beetle, Tribolium castaneum. Juvenile hormone regulation of Vg synthesis has been known for a long time in several insects, but the mechanism of JH action is not known. Experiments were conducted to determine the mechanism of action of these two signals in regulation of Vg gene expression. Injection of bovine insulin or FOXO double-stranded RNA into the previtellogenic, starved, or JH-deficient female adults increased Vg mRNA and protein levels, thereby implicating the pivotal role for insulin-like peptide signaling in the regulation of Vg gene expression and possible cross-talk between JH and insulin-like peptide signaling pathways. Reduction in JH synthesis or its action by RNAi-mediated silencing of genes coding for acid methyltransferase or methoprene-tolerant decreased expression of genes coding for insulin-like peptides (ILPs) and influenced FOXO subcellular localization, resulting in the down-regulation of Vg gene expression. Furthermore, JH application to previtellogenic female beetles induced the expression of genes coding for ILP2 and ILP3, and induced Vg gene expression. FOXO protein expressed in baculovirus system binds to FOXO response element present in the Vg gene promoter. These data suggest that JH functions through insulin-like peptide signaling pathway to regulate Vg gene expression.     

3-Oxoacyl-ACP Reductase from Schistosoma japonicum: Integrated In Silico-In Vitro Strategy for Discovering Antischistosomal Lead Compounds

Background

Schistosomiasis is a disease caused by parasitic worms and more than 200 million people are infected worldwide. The emergence of resistance to the most commonly used drug, praziquantel (PZQ), makes the development of novel drugs an urgent task. 3-oxoacyl-ACP reductase (OAR), a key enzyme involved in the fatty acid synthesis pathway, has been identified as a potential drug target against many pathogenic organisms. However, no research on Schistosoma japonicum OAR (SjOAR) has been reported. The characterization of the SjOAR protein will provide new strategies for screening antischistosomal drugs that target SjOAR.

Methodology/Principal Findings

After cloning the SjOAR gene, recombinant SjOAR protein was purified and assayed for enzymatic activity. The tertiary structure of SjOAR was obtained by homology modeling and 27 inhibitor candidates were identified from 14,400 compounds through molecular docking based on the structure. All of these compounds were confirmed to be able to bind to the SjOAR protein by BIAcore analysis. Two compounds exhibited strong antischistosomal activity and inhibitory effects on the enzymatic activity of SjOAR. In contrast, these two compounds showed relatively low toxicity towards host cells.

Conclusions/Significance

The work presented here shows the feasibility of isolation of new antischistosomal compounds using a combination of virtual screening and experimental validation. Based on this strategy, we successfully identified 2 compounds that target SjOAR with strong antischistosomal activity but relatively low cytotoxicity to host cells.     

Identification and gene expression analyses of natriuretic peptide system in the ovary of goat (Capra hircus)

Natriuretic peptides (NPs) are involved in maintaining cardiovascular and fluid homeostasis, regulating reproductive processes and bone growth, and other numerous functions. To better understand the role of NPs in goat (Capra hircus), in the present study, full-length cDNAs of goat Nppa (natriuretic peptide precursor A), Nppb (natriuretic peptide precursor B) and Nppc (natriuretic peptide precursor C), respectively encoding ANP, BNP and CNP, were cloned from adult goat heart and ovary. The putative prepropeptide ANP (prepro-ANP) and prepro-CNP share a high amino acid sequence identity with other species. Real-time PCR showed that Nppa, Nppb and Nppc were widely expressed in adult goat tissues. The mRNA expression of Nppa and Nppb in the heart was extremely higher compared with other tissues. Nppc mRNA expression in the lung and uterus was also higher than in other tissues. The expression of Nppa, Nppb and Nppc genes was examined at different ovarian follicle stages using RT-PCR. The mRNAs of Nppa and Nppb were detected in secondary follicles as well as in COCs (cumulus–oocyte-complexes) and granulosa cells of antral follicles. However, the mRNA expression of Nppc was observed throughout ovarian follicle development, and it was especially higher in granulosa cells of antral follicles. In vitro, stimulating goat granulosa cells with FSH led to an increase in the expression of Nppc by dose- and time-dependent manners and a rapid decline was induced by LH stimulation, but the expression of Nppa and Nppb did not change after FSH or LH treatment. These results suggest that Nppc is a gonadotropin-induced gene in granulosa cells of goat ovary and CNP may be involved in the regulation of ovarian follicle development and oocyte maturation.     

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.     

The vitellogenin of Leucophaea maderae: Synthesis as a large phosphorylated precursor

Phosphorylation of vitellogenin (yolk protein precursor) and vitellin (major yolk protein) polypeptides of Leucophaea maderae was studied by [³²P]ortho phosphate labeling and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) autoradiography. The vitellogenin molecule was isolated from the hemolymph and fat body by antibody precipitation and high-performance liquid chromatography (HPLC), and shown to consist of at least five polypeptides (“subunits”) which had apparent molecular masses of 155, 112, 95, 92 and 54 kD. Labeling studies with ³²P showed that the covalently attached phosphorus was distributed in an uneven fashion among the five polypeptides. The two heavily-phosphorylated polypeptides, 112 and 54 kD, corresponded to the large and small, mature vitellin subunits. Quantitative SDS-PAGE analysis of long-term ³²P-labeled vitellin showed that these large and small “subunits” contained 55 and 30%, respectively, of the total radioactivity.When fat body was pulse-labeled with ³²P we found a heavily-phosphorylated intracellular 215 kD polypeptide which was precipitable with anti-vitellogenin. The synthesis of this intracellular precursorform of vitellogenin (pre-Vg) was under absolute juvenile hormone control. In vitro³²P pulse-chase experiments showed that pre-Vg was proteolytically processed within the fat body into some (or possibly all) of the mature vitellogenin subnits. Furthermore, peptide mapping confirmed that all of the phosphorylated vitellogenin subunits were derived from pre-Vg. Since previous studies have shown that phosphoserine residues account for essentially all of the covalently-attached phosphorus of the native vitellogenin molecule, we speculate that the asymmetric pattern of vitellogenin and vitellin subunit-phosphorylation is due to an uneven distribution of phosphoserine residues along the initial pre-Vg polypeptide chain. Finally, we conclude that phosphorylation of vitellogenin occurred post-translationally in the fat body endoplasmic reticulum because we could identify ³²P-labeled pre-Vg in purified microsomal vesicles but not in nascent vitellogenin polypeptide chains attached to vitellogenin polyribosomes.     

Induced changes in the proteomic profile of the phaeophyte Saccharina japonica upon colonization by the bryozoan Membranipora membranacea

The lacy crust bryozoan Membranipora membranacea frequently colonizes the late harvested blades of aquacultured Saccharina japonica. From proteomic profiles of S. japonica, 145 and 91 protein spots were detected from colonized and healthy tissues, respectively. Among them, 69 and 32 spots were significantly up- and downregulated, respectively, in expression level upon colonization. In M. membranacea colonized tissue, tripartite motif protein 2-like, microcompartments protein, carboxysome peptide shell peptide, trypsin precursor-like, transmembrane protein, two-component response regulator PilR, spermine/spermidine synthase, vanadium-dependent bromoperoxidase, peptide chain release factor 1, interaptin, 50S ribosomal protein L1P, plus agglutinin and leucine-rich repeat protein were upregulated, whereas protoporphyrinogen oxidase, PIH1 domain-containing protein 2, GTPase-activating protein alpha, cytoplasmic threonyl-tRNA synthetase, flavanone 3-hydroxylase, and eukaryotic translation initiation factor 3 proteins were downregulated. Moreover, DEAD/DEAH box helicase, glutamyl-tRNA reductase, and chaperone DnaJ protein were newly expressed in the colonized tissue. Most of the up- and downregulated proteins are known to be related to stress control, defense mechanisms, signal transduction, photosynthesis, protein metabolism, and the cytoskeleton.     

Cloning and functional characterization of a fatty acid transport protein (FATP) from the pheromone gland of a lichen moth, Eilema japonica, which secretes an alkenyl sex pheromone

Sex pheromones of moths are largely classified into two types based on the presence (Type I) or absence (Type II) of a terminal functional group. While Type-I sex pheromones are synthesized from common fatty acids in the pheromone gland (PG), Type-II sex pheromones are derived from hydrocarbons produced presumably in the oenocytes and transported to the PG via the hemolymph. Recently, a fatty acid transport protein (BmFATP) was identified from the PG of the silkworm Bombyx mori, which produces a Type-I sex pheromone (bombykol). BmFATP was shown to facilitate the uptake of extracellular fatty acids into PG cells for the synthesis of bombykol. To elucidate the presence and function of FATP in the PG of moths that produce Type-II sex pheromones, we explored fatp homologues expressed in the PG of a lichen moth, Eilema japonica, which secretes an alkenyl sex pheromone (Type II). A fatp homologue cloned from E. japonica (Ejfatp) was predominantly expressed in the PG, and its expression is upregulated shortly after eclosion. Functional expression of EjFATP in Escherichia coli enhanced the uptake of long chain fatty acids (C₁₈ and C₂₀), but not pheromone precursor hydrocarbons. To the best of our knowledge, this is the first report of the cloning and functional characterization of a FATP in the PG of a moth producing a Type-II sex pheromone. Although EjFATP is not likely to be involved in the uptake of pheromone precursors in E. japonica, the expression pattern of Ejfatp suggests a role for EjFATP in the PG not directly linked to pheromone biosynthesis.     

Schistosoma japonicum translationally controlled tumour protein,which is associated with the development of female worms,as a target for control of schistosomiasis

Schistosomiasis is a globally important helminthic disease of both humans and animals, and is the second most common parasitic disease after malaria. Although praziquantel is extensively used for treatment of parasitic diseases, drug resistance has been reported. Therefore, new drugs and effective vaccines are needed for continuous control of schistosomiasis. Eggs produced by schistosomes are responsible for the occurrence and spread of schistosomiasis. Revealing the reproductive mechanism of schistosomes will help to control this disease. In this study, the proteomic profiles of single-sex infected female worms and bisexual infected mature female worms of Schistosoma japonicum at 18, 21, 23 and 25 days p.i. were identified with isobaric tags for relative quantitation-coupled liquid chromatography–tandem mass spectrometry. Differentially expressed proteins were subsequently used for bioinformatic analysis. Six highly expressed differentially expressed proteins in mature female worms were selected and long-term interference with small interfering RNA (siRNA) was conducted to determine biological functions. SiRNA against S. japonicum translationally controlled tumour protein (SjTCTP) resulted in the most significant effect on the growth and development of MF worms. Sjtctp mRNA expression gradually increased over time with a high level of expression maintained at 25–42 days p.i., while levels were significantly higher in mature female worms than male and SF worms. The subsequent animal immune protection experiments showed that recombinant SjTCTP (rSjTCTP) reduced the number of adults by 44.7% (P < 0.01), average egg burden per gram of liver by 57.94% (P < 0.01), egg hatching rate by 47.57% (P < 0.01), and oviposition of individual females by 43.16%. rSjTCTP induced higher levels of serum IgG, IL-2, and IL-10 in mice. Collectively, these results show that SjTCTP is vital to reproduction of female worms and, thus, is a candidate antigen for immune protection.     

In vivo stimulation of vitellogenesis in Aedes aegypti with juvenile hormone,juvenile hormone analogue (ZR 515) and 20-hydroxyecdysone

Decapitated blood-fed Aedes aegypti mosquitoes do not undergo normal oöcyte maturation. Topical application of 1.25 ng JH analogue (ZR 515) or 250 ng JH-I restored ovarian development in 70–80% of the treated females. The rate of vitellogenin synthesis in these animals was 80% of normal blood-fed controls.When ligated abdomens were treated, 125 pg ZR 515 or 12.5 ng JH-I were sufficient to restore ovarian development in 80% of the animals. The rate of vitellogenin synthesis in these animals was 70% of normal blood-fed controls. On the other hand, injection of 1.25 μg 20-hydroxyecdysone was needed to restore ovarian development and vitellogenin synthesis in decapitated and abdominally ligated females.These experiments indicate that JH concentrations closer to the physiological norm than 20-hydroxyecdysone, can restore ovarian development and vitellogenin synthesis in vivo.     

Crustacean Vitellogenesis: Its Role in Oocyte Development

One of the major changes that occurs during the maturation ofoocytes is the accumulation of yolk protein, or vitellin (Vn).To better understand how this process is regulated, we characterizedthe Vn of the ridgeback shrimp, Sicyonia ingentis (Penaeoidea).This Vn is a 322 kDa molecule composed of three subunits. Usingpurified Vn, we developed an anti-Vn antiserum and used it tocharacterize vitellogenin by Western blot analysis. The antiserumwas also used in an ELISA to measure hemolymph levels of vitellogenin.Previous studies suggested the presence of vertebrate-type steroidsmight stimulate reproductive processes in decapod crustaceans.Treatment of sexually quiescent female shrimp with progesterone,hydroxyprogesterone, and estradiol did not increase hemolymphlevels of yolk protein precursor. The absence of a responseto these steroids may reflect the presence of other hormones(such as the gonad-inhibiting hormone) that prevent oocyte development.To examine the molecular basis for the regulation of vitellogenesis,ovarian and hepatopancreas expression cDNA libraries were screenedusing the anti-Vn antiserum. A 2.9 kilobase clone was isolatedfrom both cDNA libraries suggesting that both tissues are sitesof vitellogenin synthesis. These molecular tools should be usefulfor in vitro studies of vitellogenin synthesis.     

A Crayfish Insulin-like-binding Protein: ANOTHER PIECE IN THE ANDROGENIC GLAND INSULIN-LIKE HORMONE PUZZLE IS REVEALED*

Across the animal kingdom, the involvement of insulin-like peptide (ILP) signaling in sex-related differentiation processes is attracting increasing attention. Recently, a gender-specific ILP was identified as the androgenic sex hormone in Crustacea. However, moieties modulating the actions of this androgenic insulin-like growth factor were yet to be revealed. Through molecular screening of an androgenic gland (AG) cDNA library prepared from the crayfish Cherax quadricarinatus, we have identified a novel insulin-like growth factor-binding protein (IGFBP) termed Cq-IGFBP. Based on bioinformatics analyses, the deduced Cq-IGFBP was shown to share high sequence homology with IGFBP family members from both invertebrates and vertebrates. The protein also includes a sequence determinant proven crucial for ligand binding, which according to three-dimensional modeling is assigned to the exposed outer surface of the protein. Recombinant Cq-IGFBP (rCq-IGFBP) protein was produced and, using a “pulldown” methodology, was shown to specifically interact with the insulin-like AG hormone of the crayfish (Cq-IAG). Particularly, using both mass spectral analysis and an immunological tool, rCq-IGFBP was shown to bind the Cq-IAG prohormone. Furthermore, a peptide corresponding to residues 23–38 of the Cq-IAG A-chain was found sufficient for in vitro recognition by rCq-IGFBP. Cq-IGFBP is the first IGFBP family member shown to specifically interact with a gender-specific ILP. Unlike their ILP ligands, IGFBPs are highly conserved across evolution, from ancient arthropods, like crustaceans, to humans. Such conservation places ILP signaling at the center of sex-related phenomena in early animal development.     

Fermentation and purification of lacZ-fused single chain insulin precursor for (B30-homoserine) human insulin

In order to produce the single chain precursor of a novel human insulin analogue, (B³⁰-homoserine) insulin, the fermentative behaviors ofEscherichia coli JM103 were studied, which harbors pKBA plasmid carrying a hybrid gene in which the gene for a single chain precursor was fused withlacZ gene undertac promoter. The maximal induction of gene expression was achieved when more than 0.05 mM of isopropyl-β-D-thiogalactopyranoside (IPTG) was supplemented to fermentation medium after 4 h cultivation ofE. coli, and followed by longer than 2-h fermentation. The hybrid protein of the single chain insulin precursor was isolated from cytoplasmic inclusion bodies by dissolving in 8M urea solution, and purified through DEAE-Sephacel and Sephadex G-200 column chromatographies with a recovery of 35%. The finally purified hybrid protein showed a single band on sodium dodecyl sulfate-polyacrylamide gel.     

Genetic variations in the pre-feeding and pre-oviposition periods among four pure lines in the brown planthopper, <Emphasis Type="Italic">Nilaparvata lugens</Emphasis> (Hemiptera: Auchenorrhyncha:Delphacidae)

In the brown planthopper, Nilaparvata lugens Stål, traits related to ovarian development were compared among four pure lines predominantly expressing the designated characteristics: blackish short-winged (BS), yellowish brown short-winged (YS), blackish long-winged (BL), and yellowish brown long-winged (YL). The pre-oviposition period was longer in the following order: BS < YS < BL = YL line. The pre-feeding period, namely, the time of non-feeding after adult emergence, which was estimated from dry weight loss during starvation, was also longer in the same order: BS < YS < BL = YL. Vitellogenin, the precursor of yolk protein, first appeared in the hemolymph about 1½ days later after the initiation of feeding in respective lines. One-day’s starvation did not have any effect on the timing of vitellogenin’s appearance in the long-winged lines (BL, YL), but delayed it by 1 day in the short-winged lines (BS, YS). Further, we showed that the synthesis of vitellogenin mRNA was induced in the adults even before feeding by being topically applied with juvenile hormone III (JH III). These results suggest that feeding first triggers the increase of JH III titer, which activates vitellogenin synthesis, and enhances ovarian development. Thus, genetic differences in the pre-feeding periods cause the differences in the timings of physiological events relating to ovarian development among the four lines.     

Identification and expression analyses of BAMBI mediated by FSH in swine luteinizing granulosa cells

Transforming growth factor-β and related growth factors are essential regulators for the development of follicles. Bone morphogenic protein (BMP) and activin membrane-bound inhibitor (BAMBI) was reported as a key factor participating in the transforming growth factor-β signal pathway. To investigate the role of BAMBI in porcine granulosa cells, the full length of the BAMBI was cloned from porcine ovarian cDNA. The results of bioinformatics analyses showed that the signaling peptide was located in between positions 20 and 21. The results of online prediction on phosphorylation sites indicate that the sites of Ser, Thr, and Tyr are 9, 1, and 1, respectively. In addition, BAMBI was highly homologous in rodent and livestock. Real-time quantitative polymerase chain reaction (qPCR) indicated that BAMBI was widely expressed in porcine tissues. Immunofluorescence showed that BAMBI was located in both nucleus and cytoplasm. Stimulating the granulosa cells with FSH in vitro could alter BAMBI expression level in a time-dependent manner. Moreover, the expression level declined after treatment with FSH. These results indicated that BAMBI is an FSH-repressed gene in porcine luteinizing granulosa cells and it may be involved in the regulation of ovarian follicle development and oocyte maturation.     

Haemolymph vitellogenin,juvenile hormone,and oöcyte growth in the adult cockroach Nauphoeta cinerea during first pre-oviposition period

In the cockroach Nauphoeta cinerea the incorporation of a protein of low solubility into the oöcytes begins at day 5 of its adult life. An immunologically identical protein appears in the haemolymph two days earlier. The concentration of this protein, i.e. ‘vitellogenin’ in the haemolymph increases up to the onset of yolk incorporation into the oöcytes. During ovarian development no correlation could be detected between vitellogenin titre and several other parameters (ovary dry weight, length of the basal oöcytes, haemolymph protein concentration, body weight and age when ovulation occurred). In young females vitellogenin titre depends on the age, i.e. the volume of the corpora allata and hence on the presence and the titre of JH. During the period of egg maturation the total haemolymph protein concentration generally tends to drop while materials not precipitable by trichloracetic acid circulate at higher concentration after ecdysis and before ovulation.Early decapitation prevents vitellogenin synthesis and oöcyte growth, but when JH is applied to decapitated females, the normal vitellogenin titre is re-established, ovarian development, however, cannot be fully resumed. A dose-response curve shows that serial application of the hormone is much more effective than single large doses. Farnesylmethylester, a JH mimic, is about a hundred times less active, but more persistent than JH. Copulation seems to enhance the synthesis and release of endogenous JH, while food and water uptake are necessary to guarantee and optimal ovarian development. JH and high vitellogenin titre never restore ovarian development in females deprived of food and/or water or in those decapitated shortly after ecdysis.     

Secretory Expression of Insulin Precursor in Pichia pastoris and Simple Procedure for Producing Recombinant Human Insulin

Abstract

In this work, Pichia pastoris was applied to produce human insulin by a simple procedure. The synthesized insulin precursor (ILP) gene was inserted into pPIC9K to obtain secretary expression plasmid pPIC9K/ILP. Pichia pastoris GS115 was transformed by pPIC9K/ILP and the high expresser was screened. In a 16 L fermentor, the insulin precursor production was 3.6 g/L. Insulin precursor, purified by one-step chromatography, was converted into human insulin by transpeptidation. The yield of the processing procedure from insulin precursor to insulin reached up to 70%. In vivo assay showed that the biological activity of the produced recombinant human insulin was 28.8 U/mg.