印度疟疾媒介库态按蚊卵黄蛋白原基因的克隆与生物信息学分析（英文）

卵黄蛋白原（vitellogenin, Vg)是主要的卵黄蛋白前体, 在雌虫血餐之后在脂肪体内大量合成。卵黄蛋白原的调节元件已经被用于驱动蚊子（与寄生虫发生最大相互作用的场所）中抗寄生基因的组织特异性表达。不过, 迄今为止, 对在印度引起60%~70%疟疾发生的库态按蚊Anopheles culicifacies中的内源启动子尚未进行过分析。本研究通过PCR扩增了包括5′端上游调节区在内的库态按蚊A. culicifacies卵黄蛋白原基因, 并命名为AncuVg (GenBank登录号为JN113091)。它含有一个大约6.2 kb的开放阅读框, 编码2 052个氨基酸, 具有一个16个氨基酸残基的推断的信号肽。也含有一个N_Vitellogenin区和一个VWF型D区, 这两个区在其他昆虫卵黄蛋白原中也保守。估计多肽分子量为238.0 kDa, 含有4个共有的(RXXR/S)切割位点, C端附近有一个GL/ICG基序, 其后是9个半胱氨酸残基和1个位于GL/ICCG基序上游第18个氨基酸残基处的DGXR 基序。在推断的氨基酸序列上发现3个聚丝氨酸区, 其中2个位于氨基端, 1个位于羧基端。根据同义密码子相对使用概率值, 通过有效密码子数, 测定了蚊子卵黄蛋白原基因密码子的偏倚性程度。也预测了库态按蚊A. culicifacies Vg的三维结构。分析了AncuVg基因, 以理解Vg基因的转录调节。对Vg基因5′端上游区进行的系统发育分析表明, 它们聚类于蚊子的3大分枝。也用各种生物信息学工具分析分析了Vg的同源性和特征。     

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.     

COPI-mediated blood meal digestion in vector mosquitoes is independent of midgut ARF-GEF and ARF-GAP regulatory activities

We have previously shown that defects in COPI coatomer proteins cause 80% mortality in blood fed Aedes aegypti mosquitoes by 96 h post-feeding. In this study we show that similar deficiencies in COPII and clathrin mediated vesicle transport do not disrupt blood meal digestion and are not lethal, even though both COPII and clathrin functions are required for ovarian development. Since COPI vesicle transport is controlled in mammalian cells by upstream G proteins and associated regulatory factors, we investigated the function of the orthologous ADP-ribosylation factor 1 (ARF1) and ARF4 proteins in mosquito tissues. We found that both ARF1 and ARF4 function upstream of COPI vesicle transport in blood fed mosquitoes given that an ARF1/ARF4 double deficiency is required to phenocopy the feeding-induced mortality observed in COPI coatomer deficient mosquitoes. Small molecule inhibitors of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) are often transitory, and therefore, we investigated the role of five Ae. aegypti ARF-GEF and ARF-GAP proteins in blood meal digestion using RNA interference. Surprisingly, we found that ARF-GEF and ARF-GAP functions are not required for blood meal digestion, even though both vitellogenesis and ovarian development in Ae. aegypti are dependent on GBF1 (ARF-GEF) and GAP1/GAP2 (ARF-GAPs) proteins. Moreover, deficiencies in orthologous COPI regulating genes in Anopheles stephensi mosquitoes had similar phenotypes, indicating conserved functions in these two mosquito species. We propose that based on the need for rapid initiation of protein digestion and peritrophic membrane formation, COPI vesicle transport in midgut epithelial cells is not dependent on ARF-GEF and ARF-GAP regulatory proteins to mediate vesicle recycling within the first 48 h post-feeding.     

Lipophorin as a yolk protein precursor in the mosquito, Aedes aegypti

We examined expression of the lipophorin (Lp) gene, lipophorin (Lp) synthesis and secretion in the mosquito fat body, as well as dynamic changes in levels of this lipoprotein in the hemolymph and ovaries, during the first vitellogenic cycle of females of the yellow fever mosquito, Aedes aegypti. Lipophorin was purified by potassium bromide (KBr) density gradient ultracentrifugation and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Polyclonal antibodies were produced against individual Lp apoproteins, apolipoprotein-I (apoLp-I) and apolipoprotein-II (apoLp-II), with molecular weights of 240 and 75 kDa, respectively. We report here that in the mosquito A. aegypti, Lp was synthesized by the fat body, with a low level of the Lp gene expression and protein synthesis being maintained in pre- and postvitellogenic females. Following a blood meal, the Lp gene expression and protein synthesis were significantly upregulated. Our findings showed that the fat body levels of Lp mRNA and the rate of Lp secretion by this tissue reached their maximum at 18 h post-blood meal (PMB). 20-Hydroxyecdysone was responsible for an increase in the Lp gene expression and Lp protein synthesis in the mosquito fat body. Finally, the immunocytochemical localization of Lp showed that in vitellogenic female mosquitoes, this protein was accumulated by developing oocytes where it was deposited in yolk granules.     

Previtellogenic development and vitellogenin synthesis in the fat body of a mosquito: an ultrastructural and immunocytochemical study

We describe two phases, previtellogenic and vitellogenic, in the activity of the trophocytes in the fat body of the mosquito Aedes aegypti. The previtellogenic phase, leading to trophocyte competence to synthesize vitellogenin (Vg), occurred during the first 3 days after eclosion. This phase was characterized by enlargement and activation of the nucleoli, proliferation of ribosomes and rough endoplasmic reticulum (RER), development of Golgi complexes, and extensive invaginations of the plasma membrane. During the vitellogenic phase, initiated by a blood meal, Vg was first detected, by immunofluorescence, 1 hr after feeding. The intensity of the immunoreaction increased for the next 24 hr, was declining at 30 hr, and had disappeared by 48 hr. Vg synthesis was characterized ultrastructurally by the enlargement of the RER and the formation of dense secretion granules in Golgi complexes. These secretion granules were two to three times larger at the peak of Vg synthesis than at the beginning. The granules discharged their contents by exocytosis. Two electron microscopical immunocytochemical methods, immunoferritin and peroxidase-antiperoxidase, confirmed this pathway of Vg processing. For the first 12 hr after feeding. Vg synthetic organelles proliferated and the active nucleoli were multilobed; thereafter, while Vg synthesis continued, the nucleoli began to regress into compact bodies. Termination of Vg synthesis was marked by autophagical degradation of Vg synthetic and processing organelles.     

Short repeated elements in the upstream regulatory region of the SUC2 gene of Saccharomyces cerevisiae.

Expression of secreted invertase from the SUC2 gene is regulated by carbon catabolite repression. Previously, an upstream regulatory region that is required for derepression of secreted invertase was identified and shown to confer glucose-repressible expression to the heterologous promoter of a LEU2-lacZ fusion. In this paper we show that tandem copies of a 32-base pair (bp) sequence from the upstream regulatory region activate expression of the same LEU2-lacZ fusion. The level of expression increased with the number of copies of the element, but was independent of their orientation; the expression from constructions containing four copies of the sequence was only twofold lower than that when the entire SUC2 upstream regulatory region was present. This activation was not significantly glucose repressible. The 32-bp sequence includes a 7-bp motif with the consensus sequence (A/C)(A/G)GAAAT that is repeated at five sites within the upstream regulatory region. Genetic evidence supporting the functional significance of this repeated motif was obtained by pseudoreversion of a SUC2 deletion mutant lacking part of the upstream region, including two copies of the 7-bp element. In three of five pseudorevertants, the mutations that restored high-level SUC2 expression altered one of the remaining copies of the 7-bp element.