Juvenile hormone connects larval nutrition with target of rapamycin signaling in the mosquito Aedes aegypti

Anautogenous mosquitoes require blood meals to promote egg development. If adequate nutrients are not obtained during larval development, the resulting "small" sized adult mosquitoes require multiple blood meals for egg development; markedly increasing host-vector contacts and the likelihood of disease transmission. Nutrient-sensitive target of rapamycin (TOR) signaling is a key signaling pathway that links elevated hemolymph amino acid levels derived from the blood meal to the expression of yolk protein precursors in the fat body. Here we report that the blood-meal-induced activation of the TOR-signaling pathway and subsequent egg maturation depends on the accumulation of adequate nutritional reserves during larval development. We have established well-nourished, "standard" mosquitoes and malnourished, "small" mosquitoes as models to address this nutrient sensitive pathway. This regulatory mechanism involves juvenile hormone (JH), which acts as a mediator of fat body competence, permitting the response to amino acids derived from the blood meal. We demonstrate that treatment with JH results in recovery of the TOR molecular machinery, Aedes aegypti cationic amino acid transporter 2 (AaiCAT2), TOR, and S6 kinase (S6K), in fat bodies of small mosquitoes, enabling them to complete their first gonotrophic cycle after a single blood meal. These findings establish a direct link between nutrient reserves and the establishment of TOR signaling in mosquitoes.     

A gain-of-function screen identifies wdb and lkb1 as lifespan-extending genes in Drosophila

The insulin/insulin-like growth factor (IGF) and the target of rapamycin (TOR) signaling pathways are known to regulate lifespan in diverse organisms. However, only a limited number of genes involved in these pathways have been examined regarding their effects on lifespan. Through a gain-of-function screen in Drosophila, we found that overexpression of the wdb gene encoding a regulatory subunit of PP2A, and overexpression of the lkb1 gene encoding a serine/threonine kinase, reduced organ size and extended lifespan. Overexpression of wdb also reduced the level of phosphorylated AKT, while overexpression of lkb1 increased the level of phosphorylated AMPK and decreased the level of phosphorylated S6K. Taken together, our results suggest that wdb- and lkb1-dependent lifespan extension is mediated by downregulation of S6K, a downstream component of the insulin/IGF and TOR signaling pathways.     

Regulation of neurogenesis and epidermal growth factor receptor signaling by the insulin receptor/target of rapamycin pathway in Drosophila

Determining how growth and differentiation are coordinated is key to understanding normal development, as well as disease states such as cancer, where that control is lost. We have previously shown that growth and neuronal differentiation are coordinated by the insulin receptor/target of rapamycin (TOR) kinase (InR/TOR) pathway. Here we show that the control of growth and differentiation diverge downstream of TOR. TOR regulates growth by controlling the activity of S6 kinase (S6K) and eIF4E. Loss of s6k delays differentiation, and is epistatic to the loss of tsc2, indicating that S6K acts downstream or in parallel to TOR in differentiation as in growth. However, loss of eIF4E inhibits growth but does not affect the timing of differentiation. We also show, for the first time in Drosophila, that there is crosstalk between the InR/TOR pathway and epidermal growth factor receptor (EGFR) signaling. InR/TOR signaling regulates the expression of several EGFR pathway components including pointedP2 (pntP2). In addition, reduction of EGFR signaling levels phenocopies inhibition of the InR/TOR pathway in the regulation of differentiation. Together these data suggest that InR/TOR signaling regulates the timing of differentiation through modulation of EGFR target genes in developing photoreceptors.     

Diet restriction‐induced healthy aging is mediated through the immune signaling component ZIP‐2 in Caenorhabditis elegans

Dietary restriction (DR) robustly delays the aging process in all animals tested so far. DR slows aging by negatively regulating the target of rapamycin (TOR) and S6 kinase (S6K) signaling pathway and thus inhibiting translation. Translation inhibition in C. elegans is known to activate the innate immune signal ZIP‐2. Here, we show that ZIP‐2 is activated in response to DR and in feeding‐defective eat‐2 mutants. Importantly, ZIP‐2 contributes to the improvements in longevity and healthy aging, including mitochondrial integrity and physical ability, mediated by DR in C. elegans. We further show that ZIP‐2 is activated upon inhibition of TOR/S6K signaling. However, DR‐mediated activation of ZIP‐2 does not require the TOR/S6K effector PHA‐4/FOXA. Furthermore, zip‐2 was not activated or required for longevity in daf‐2 mutants, which mimic a low nutrition status. Thus, DR appears to activate ZIP‐2 independently of PHA‐4/FOXA and DAF‐2. The link between DR, aging, and immune activation provides practical insight into the DR‐induced benefits on health span and longevity.     

Target of rapamycin-dependent activation of S6 kinase is a central step in the transduction of nutritional signals during egg development in a mosquito

Female mosquitoes are effective disease vectors, because they take blood from vertebrate hosts to obtain nutrients for egg development. Amino acid signaling via the target of rapamycin (TOR) pathway has been identified as a key requirement for the activation of egg development after a blood meal. We report the characterization of the TOR kinase and one of its major downstream targets, S6 kinase, of the yellow fever mosquito Aedes aegypti during egg development in adult females. Both TOR and S6K mRNA are expressed at high levels in the ovaries and in lower levels in fat body and other tissues. After a blood meal, the subcellular localization of TOR shifts from the cytoplasm to the plasma membrane of fat body cells. By detecting phosphothreonine 388 of mosquito S6 kinase, we show that TOR activity strongly increases in fat body and ovaries after a blood meal in vivo. Furthermore, phosphorylation of S6 kinase increases in in vitro cultured fat bodies after stimulation with amino acids. This increase is sensitive to the TOR inhibitor rapamycin in a concentration-dependent manner but not to the phosphatidylinositol 3-kinase/phosphatidylinositol 3-kinase-related kinase inhibitor LY294002, the MAPK inhibitor PD98059, or the translational inhibitor cycloheximide. RNA interference-mediated reduction of S6 kinase strongly inhibits the amino acid-induced up-regulation of the major yolk protein vitellogenin in vitro and effectively disrupts egg development after a blood meal in vivo. Our data show that TOR-dependent activation of S6 kinase is a central step in the transduction of nutritional information during egg development in mosquitoes.     

S6 protein kinase activates Juvenile Hormone and vitellogenin production in the cockroach Blattella germanica

Nutritional status regulates different processes, such as growth and development, through TOR (target of rapamycin) and insulin receptor signalling pathways. The ribosomal S6 protein kinase (S6K) is a downstream element of both pathways. Using cDNA from the German cockroach Blattella germanica (L.), two S6K isoform sequences (BgS6K) are cloned. The long isoform differs from the short one by the insertion of a 22‐amino acid duplication, involving the key phosphorylation position Thr³⁹⁰. As a result of this, the long isoform presents a new, potentially regulatory phosphorylation site. RNA interference knockdown of both BgS6K isoforms induces an increase in the length of the last nymphal instar, together with a reduction in the mRNA levels of a number of enzymes of the Juvenile Hormone biosynthetic pathway in the corpora allata, vitellogenin mRNA levels in the fat body and basal oocyte length. Thus, BgS6K is important for nymphal development and is necessary for the full induction of Juvenile Hormone synthesis and vitellogenin production in adult females.     

Diacylglycerol lipase regulates lifespan and oxidative stress response by inversely modulating TOR signaling in Drosophila and C. elegans

Target of rapamycin (TOR) signaling is a nutrient‐sensing pathway controlling metabolism and lifespan. Although TOR signaling can be activated by a metabolite of diacylglycerol (DAG), phosphatidic acid (PA), the precise genetic mechanism through which DAG metabolism influences lifespan remains unknown. DAG is metabolized to either PA via the action of DAG kinase or 2‐arachidonoyl‐sn‐glycerol by diacylglycerol lipase (DAGL). Here, we report that in Drosophila and Caenorhabditis elegans, overexpression of diacylglycerol lipase (DAGL/inaE/dagl‐1) or knockdown of diacylglycerol kinase (DGK/rdgA/dgk‐5) extends lifespan and enhances response to oxidative stress. Phosphorylated S6 kinase (p‐S6K) levels are reduced following these manipulations, implying the involvement of TOR signaling. Conversely, DAGL/inaE/dagl‐1 mutants exhibit shortened lifespan, reduced tolerance to oxidative stress, and elevated levels of p‐S6K. Additional results from genetic interaction studies are consistent with the hypothesis that DAG metabolism interacts with TOR and S6K signaling to affect longevity and oxidative stress resistance. These findings highlight conserved metabolic and genetic pathways that regulate aging.     

Arabidopsis TARGET OF RAPAMYCIN interacts with RAPTOR, which regulates the activity of S6 kinase in response to osmotic stress signals

TARGET OF RAPAMYCIN (TOR) kinase controls many cellular functions in eukaryotic cells in response to stress and nutrient availability and was shown to be essential for embryonic development in Arabidopsis thaliana. We demonstrated that Arabidopsis RAPTOR1 (a TOR regulatory protein) interacts with the HEAT repeats of TOR and that RAPTOR1 regulates the activity of S6 kinase (S6K) in response to osmotic stress. RAPTOR1 also interacts in vivo with Arabidopsis S6K1, a putative substrate for TOR. S6K1 fused to green fluorescent protein and immunoprecipitated from tobacco (Nicotiana tabacum) leaves after transient expression was active in phosphorylating the Arabidopsis ribosomal S6 protein. The catalytic domain of S6K1 could be phosphorylated by Arabidopsis 3-phosphoinositide-dependent protein kinase-1 (PDK1), indicating the involvement of PDK1 in the regulation of S6K. The S6K1 activity was sensitive to osmotic stress, while PDK1 activity was not affected. However, S6K1 sensitivity to osmotic stress was relieved by co-overexpression of RAPTOR1. Overall, these observations demonstrated the existence of a functional TOR kinase pathway in plants. However, Arabidopsis seedlings do not respond to normal physiological levels of rapamycin, which appears to be due its inability to bind to the Arabidopsis homolog of FKBP12, a protein that is essential for the binding of rapamycin with TOR. Replacement of the Arabidopsis FKBP12 with the human FKBP12 allowed rapamycin-dependent interaction with TOR. Since homozygous mutation in TOR is lethal, it suggests that this pathway is essential for integrating the stress signals into the growth regulation.     

马铃薯甲虫结节性硬化复合物基因LdTSC1和LdTSC2基因的克隆及功能分析

【目的】通过RNAi技术明确马铃薯甲虫TOR上游的关键信号集成节点及类胰岛素信号通道下游基因结节性硬化复合物TSC1和TSC2的功能。旨在为探明马铃薯甲虫类胰岛素信号转导提供更多理论支持。【方法】在NCBI(美国国家生物技术信息中心)获取马铃薯甲虫LdTSC1/2序列,分别利用多重序列比对和系统发育分析确定该基因的完整性和系统发育关系;采用喂食幼虫dsRNA的方法,观察该基因的调低对马铃薯甲虫幼虫生长发育、糖脂代谢的影响。【结果】克隆得到马铃薯甲虫TSC1编码蛋白的氨基酸序列与鞘翅目白蜡窄吉丁直系同源蛋白的氨基酸序列的自展一致度为100%,聚为一支;TSC2编码蛋白的氨基酸序列与鞘翅目白蜡窄吉丁和赤拟谷盗的同源蛋白氨基酸序列的自展一致度为100%,聚为一支。通过分别喂食2龄幼虫LdTSC1/2的dsRNA能有效降低靶标基因的表达量,幼虫出现体重减轻,化蛹率和羽化率显著下降,葡萄糖的吸收转化效率降低,海藻糖含量升高和甘油三酯均减少。【结论】下调2龄幼虫LdTSC1/2的表达量,导致试虫出现抑制了糖脂代谢、脂肪体减少、体重减轻以及发育延迟;结果表明LdTSC1/2调控了马铃薯甲虫幼虫的糖脂代谢过程,显著影响幼虫化蛹和蜕皮过程。     

Studies on the resting behaviour and host choice of Anopheles gambiae and An. arabiensis from Muleba,Tanzania

The relative efficacy of a mechanical (Prokopack) collection method vs. manual aspiration in the collection of resting mosquitoes was evaluated in northern Tanzania before and after an intervention using indoor residual spraying and longlasting insecticide‐treated nets. In smoke‐free houses mosquitoes were collected from the roof and walls, but in smoky houses mosquitoes were found predominantly on the walls. Anopheles gambiae (Diptera: Culicidae) constituted 97.7% of the 312 An. gambiae complex specimens identified before but only 19.3% of the 183 identified after the intervention. A single sampling with the Prokopack collected a third of the available insects. Anopheles gambiae completed its gonotrophic development indoors, whereas Anopheles arabiensis did so outdoors. In both species gonotrophic development took 2 days. Most unfed resting An. arabiensis collected outdoors were virgins, whereas the majority of engorged insects were parous (with well‐contracted sacs). Daily survival was estimated to be 80.0%. Only 9.4% of the engorged An. arabiensis collected outdoors and 47.1% of those collected indoors had fed on humans. Using the Prokopack sampler is more efficient than manual methods for the collection of resting mosquitoes. Malaria transmission may have been affected by a change in vector composition resulting from a change in feeding, rather than reduced survival. Monitoring the proportions of members of the An. gambiae complex may provide signals of an impending breakdown in control.