Manipulating insulin signaling to enhance mosquito reproduction

<p>Abstract</p> <p>Backgrond</p> <p>In the mosquito <it>Aedes aegypti </it>the insulin/insulin growth factor I signaling (IIS) cascade is a key regulator of many physiological processes, including reproduction. Two important reproductive events, steroidogenesis in the ovary and yolk synthesis in the fat body, are regulated by the IIS cascade in mosquitoes. The signaling molecule phosphatase and tensin homolog (PTEN) is a key inhibitor of the IIS cascade that helps modulate the activity of the IIS cascade. In <it>Ae. aegypti</it>, six unique splice variants of AaegPTEN were previously identified, but the role of these splice variants, particularly AaegPTEN3 and 6, were unknown.</p> <p>Results</p> <p>Knockdown of AaegPTEN or its specific splice variant AaegPTEN6 (the splice variant thought to regulate reproduction in the ovary and fat body) using RNAi led to a 15–63% increase in egg production with no adverse effects on egg viability during the first reproductive cycle. Knockdown of AaegPTEN3, expressed predominantly in the head, had no effect on reproduction. We also characterized the protein expression patterns of these two splice variants during development and in various tissues during a reproductive cycle.</p> <p>Conclusion</p> <p>Previous studies in a range of organisms, including <it>Drosophila melanogaster </it>and <it>Caenorhabditis elegans</it>, have demonstrated that disruption of the IIS cascade leads to decreased reproduction or sterility. In this study we demonstrate that knockdown of the IIS inhibitor PTEN can actually increase reproduction in the mosquito, at least during the first reproductive cycle.</p

Viral Paratransgenesis in the Malaria Vector Anopheles gambiae

Paratransgenesis, the genetic manipulation of insect symbiotic microorganisms, is being considered as a potential method to control vector-borne diseases such as malaria. The feasibility of paratransgenic malaria control has been hampered by the lack of candidate symbiotic microorganisms for the major vector Anopheles gambiae. In other systems, densonucleosis viruses (DNVs) are attractive agents for viral paratransgenesis because they infect important vector insects, can be genetically manipulated and are transmitted to subsequent generations. However, An. gambiae has been shown to be refractory to DNV dissemination. We discovered, cloned and characterized the first known DNV (AgDNV) capable of infection and dissemination in An. gambiae. We developed a flexible AgDNV-based expression vector to express any gene of interest in An. gambiae using a two-plasmid helper-transducer system. To demonstrate proof-of-concept of the viral paratransgenesis strategy, we used this system to transduce expression of an exogenous gene (enhanced green fluorescent protein; EGFP) in An. gambiae mosquitoes. Wild-type and EGFP-transducing AgDNV virions were highly infectious to An. gambiae larvae, disseminated to and expressed EGFP in epidemiologically relevant adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations. These proof-of-principle data suggest that AgDNV could be used as part of a paratransgenic malaria control strategy by transduction of anti-Plasmodium peptides or insect-specific toxins in Anopheles mosquitoes. AgDNV will also be extremely valuable as an effective and easy-to-use laboratory tool for transient gene expression or RNAi in An. gambiae

Wolbachia in the flesh: symbiont intensities in germ-line and somatic tissues challenge the conventional view of Wolbachia transmission routes

Symbionts can substantially affect the evolution and ecology of their hosts. The investigation of the tissue-specific distribution of symbionts (tissue tropism) can provide important insight into host-symbiont interactions. Among other things, it can help to discern the importance of specific transmission routes and potential phenotypic effects. The intracellular bacterial symbiont Wolbachia has been described as the greatest ever panzootic, due to the wide array of arthropods that it infects. Being primarily vertically transmitted, it is expected that the transmission of Wolbachia would be enhanced by focusing infection in the reproductive tissues. In social insect hosts, this tropism would logically extend to reproductive rather than sterile castes, since the latter constitute a dead-end for vertically transmission. Here, we show that Wolbachia are not focused on reproductive tissues of eusocial insects, and that non-reproductive tissues of queens and workers of the ant Acromyrmex echinatior, harbour substantial infections. In particular, the comparatively high intensities of Wolbachia in the haemolymph, fat body, and faeces, suggest potential for horizontal transmission via parasitoids and the faecal-oral route, or a role for Wolbachia modulating the immune response of this host. It may be that somatic tissues and castes are not the evolutionary dead-end for Wolbachia that is commonly thought

Neural and Endocrine Correlates of Early Life Abuse in Youth With Depression and Obesity

Depression and insulin resistance are becoming increasingly prevalent in younger populations. The origin and consequence of insulin resistance in depressed youth may, in part, be rooted in exposure to environmental stressors, such as early life abuse, that may lead to aberrant brain motivational networks mediating maladaptive food-seeking behaviors and insipient insulin resistance. In this paper, we aimed to investigate the impact of early life abuse on the development of insulin resistance in depressed and overweight youth aged 9 to 17 years. We hypothesized that youth with the greatest burden of early life abuse would have the highest levels of insulin resistance and corresponding aberrant reward network connectivities. To test this hypothesis, we evaluated sixty-nine depressed and overweight youth aged 9 to 17, using multimodal assessments of early life abuse, food-seeking behavior, and insulin resistance. Based on results of the Childhood Trauma Questionnaire (CTQ), we separated our study participants into two groups: 35 youth who reported high levels of the sum of emotional, physical, or sexual abuse and 34 youth who reported insignificant or no levels of any abuse. Results of an oral glucose tolerance test (OGTT) and resting state functional connectivity (RSFC), using the amygdala, insula, and nucleus accumbens (NAcc) as seed-based reward network regions of interest, were analyzed for group differences between high abuse and low abuse groups. High abuse youth exhibited differences from low abuse youth in amygdala-precuneus, NAcc-paracingulate gyrus, and NAcc-prefrontal cortex connectivities, that correlated with levels of abuse experienced. The more different their connectivity from of that of low abuse youth, the higher were their fasting glucose and glucose at OGTT endpoint. Importantly, level of abuse moderated the relation between reward network connectivity and OGTT glucose response. In contrast, low abuse youth showed hyperinsulinemia and more insulin resistance than high abuse youth, and their higher OGTT insulin areas under the curve correlated with more negative insula-precuneus connectivity. Our findings suggest distinct neural and endocrine profiles of youth with depression and obesity based on their histories of early life abuse

Comparisons of host mitochondrial, nuclear and endosymbiont bacterial genes reveal cryptic fig wasp species and the effects of Wolbachia on host mtDNA evolution and diversity

Background Figs and fig-pollinating wasp species usually display a highly specific one-to-one association. However, more and more studies have revealed that the "one-to-one" rule has been broken. Co-pollinators have been reported, but we do not yet know how they evolve. They may evolve from insect speciation induced or facilitated by Wolbachia which can manipulate host reproduction and induce reproductive isolation. In addition, Wolbachia can affect host mitochondrial DNA evolution, because of the linkage between Wolbachia and associated mitochondrial haplotypes, and thus confound host phylogeny based on mtDNA. Previous research has shown that fig wasps have the highest incidence of Wolbachia infection in all insect taxa, and Wolbachia may have great influence on fig wasp biology. Therefore, we look forward to understanding the influence of Wolbachia on mitochondrial DNA evolution and speciation in fig wasps. Results We surveyed 76 pollinator wasp specimens from nine Ficus microcarpa trees each growing at a different location in Hainan and Fujian Provinces, China. We found that all wasps were morphologically identified as Eupristina verticillata, but diverged into three clades with 4.22-5.28% mtDNA divergence and 2.29-20.72% nuclear gene divergence. We also found very strong concordance between E. verticillata clades and Wolbachia infection status, and the predicted effects of Wolbachia on both mtDNA diversity and evolution by decreasing mitochondrial haplotypes. Conclusions Our study reveals that the pollinating wasp E. verticillata on F. microcarpa has diverged into three cryptic species, and Wolbachia may have a role in this divergence. The results also indicate that Wolbachia strains infecting E. verticillata have likely resulted in selective sweeps on host mitochondrial DNA

African tropical rainforest net carbon dioxide fluxes in the twentieth century

The African humid tropical biome constitutes the second largest rainforest region, significantly impacts global carbon cycling and climate, and has undergone major changes in functioning owing to climate and land-use change over the past century. We assess changes and trends in CO2 fluxes from 1901 to 2010 using nine land surface models forced with common driving data, and depict the inter-model variability as the uncertainty in fluxes. The biome is estimated to be a natural (no disturbance) net carbon sink (−0.02 kg C m−2 yr−1 or −0.04 Pg C yr−1, p < 0.05) with increasing strength fourfold in the second half of the century. The models were in close agreement on net CO2 flux at the beginning of the century (σ1901 = 0.02 kg C m−2 yr−1), but diverged exponentially throughout the century (σ2010 = 0.03 kg C m−2 yr−1). The increasing uncertainty is due to differences in sensitivity to increasing atmospheric CO2, but not increasing water stress, despite a decrease in precipitation and increase in air temperature. However, the largest uncertainties were associated with the most extreme drought events of the century. These results highlight the need to constrain modelled CO2 fluxes with increasing atmospheric CO2 concentrations and extreme climatic events, as the uncertainties will only amplify in the next century

Stable Isotope Analysis Can Potentially Identify Completely-Digested Bloodmeals in Mosquitoes

Background: Vertebrate bloodfeeding is a critical component of a mosquito’s ability to transmit pathogens that cause diseases such as malaria, dengue fever and viral encephalitis. Due to degradation by the digestive process, current methods to identify mosquito bloodmeal sources are only useful for approximately 36 hours post-feeding. A critical need exists for technologies to extend this window and gain a more complete picture of mosquito feeding behavior for epidemiological studies. Stable isotopes are useful for investigating organism feeding behavior because the isotopic ratio of an organism’s tissues reflects that of the material it ingests. Methodology/Principal Findings: Proof-of-principle data indicates that after bloodfeeding, Aedes albopictus mosquitoes acquire diagnostic Carbon and Nitrogen stable isotope profiles from their vertebrate hosts that can be accurately identified one week post-feeding, approximately 4 days after the entire bloodmeal has been digested. Total C/N ratio served as a biomarker marker for bloodfeeding (P,0.02), while dN was the most informative variable which could distinguish between unfed, chicken-fed and human-fed mosquitoes (P,0.01). By plotting C/N vs. dN, all feeding treatments could be identified in a double-blind analysis. Conclusions/Significance: These proof-of-principle experiments indicate that analysis of stable isotopes can be used to distinguish bloodfed from unfed mosquitoes, and also distinguish between different vertebrate bloodmeal sources eve

Wolbachia Infections in Anopheles gambiae Cells: Transcriptomic Characterization of a Novel Host-Symbiont Interaction

The endosymbiotic bacterium Wolbachia is being investigated as a potential control agent in several important vector insect species. Recent studies have shown that Wolbachia can protect the insect host against a wide variety of pathogens, resulting in reduced transmission of parasites and viruses. It has been proposed that compromised vector competence of Wolbachia-infected insects is due to up-regulation of the host innate immune system or metabolic competition. Anopheles mosquitoes, which transmit human malaria parasites, have never been found to harbor Wolbachia in nature. While transient somatic infections can be established in Anopheles, no stable artificially-transinfected Anopheles line has been developed despite numerous attempts. However, cultured Anopheles cells can be stably infected with multiple Wolbachia strains such as wAlbB from Aedes albopictus, wRi from Drosophila simulans and wMelPop from Drosophila melanogaster. Infected cell lines provide an amenable system to investigate Wolbachia-Anopheles interactions in the absence of an infected mosquito strain. We used Affymetrix GeneChip microarrays to investigate the effect of wAlbB and wRi infection on the transcriptome of cultured Anopheles Sua5B cells, and for a subset of genes used quantitative PCR to validate results in somatically-infected Anopheles mosquitoes. Wolbachia infection had a dramatic strain-specific effect on gene expression in this cell line, with almost 700 genes in total regulated representing a diverse array of functional classes. Very strikingly, infection resulted in a significant down-regulation of many immune, stress and detoxification-related transcripts. This is in stark contrast to the induction of immune genes observed in other insect hosts. We also identified genes that may be potentially involved in Wolbachia-induced reproductive and pathogenic phenotypes. Somatically-infected mosquitoes had similar responses to cultured cells. The data show that Wolbachia has a profound and unique effect on Anopheles gene expression in cultured cells, and has important implications for mechanistic understanding of Wolbachia-induced phenotypes and potential novel strategies to control malaria

Wolbachia Stimulates Immune Gene Expression and Inhibits Plasmodium Development in Anopheles gambiae

The over-replicating wMelPop strain of the endosymbiont Wolbachia pipientis has recently been shown to be capable of inducing immune upregulation and inhibition of pathogen transmission in Aedes aegypti mosquitoes. In order to examine whether comparable effects would be seen in the malaria vector Anopheles gambiae, transient somatic infections of wMelPop were created by intrathoracic inoculation. Upregulation of six selected immune genes was observed compared to controls, at least two of which (LRIM1 and TEP1) influence the development of malaria parasites. A stably infected An. gambiae cell line also showed increased expression of malaria-related immune genes. Highly significant reductions in Plasmodium infection intensity were observed in the wMelPop-infected cohort, and using gene knockdown, evidence for the role of TEP1 in this phenotype was obtained. Comparing the levels of upregulation in somatic and stably inherited wMelPop infections in Ae. aegypti revealed that levels of upregulation were lower in the somatic infections than in the stably transinfected line; inhibition of development of Brugia filarial nematodes was nevertheless observed in the somatic wMelPop infected females. Thus we consider that the effects observed in An. gambiae are also likely to be more pronounced if stably inherited wMelPop transinfections can be created, and that somatic infections of Wolbachia provide a useful model for examining effects on pathogen development or dissemination. The data are discussed with respect to the comparative effects on malaria vectorial capacity of life shortening and direct inhibition of Plasmodium development that can be produced by Wolbachia

Wolbachia infections that reduce immature insect survival: Predicted impacts on population replacement

<p>Abstract</p> <p>Background</p> <p>The evolutionary success of <it>Wolbachia </it>bacteria, infections of which are widespread in invertebrates, is largely attributed to an ability to manipulate host reproduction without imposing substantial fitness costs. Here, we describe a stage-structured model with deterministic immature lifestages and a stochastic adult female lifestage. Simulations were conducted to better understand <it>Wolbachia </it>invasions into uninfected host populations. The model includes conventional <it>Wolbachia </it>parameters (the level of cytoplasmic incompatibility, maternal inheritance, the relative fecundity of infected females, and the initial <it>Wolbachia </it>infection frequency) and a new parameter termed relative larval viability (<it>RLV</it>), which is the survival of infected larvae relative to uninfected larvae.</p> <p>Results</p> <p>The results predict the <it>RLV </it>parameter to be the most important determinant for <it>Wolbachia </it>invasion and establishment. Specifically, the fitness of infected immature hosts must be close to equal to that of uninfected hosts before population replacement can occur. Furthermore, minute decreases in <it>RLV </it>inhibit the invasion of <it>Wolbachia </it>despite high levels of cytoplasmic incompatibility, maternal inheritance, and low adult fitness costs.</p> <p>Conclusions</p> <p>The model described here takes a novel approach to understanding the spread of <it>Wolbachia </it>through a population with explicit dynamics. By combining a stochastic female adult lifestage and deterministic immature/adult male lifestages, the model predicts that even those <it>Wolbachia </it>infections that cause minor decreases in immature survival are unlikely to invade and spread within the host population. The results are discussed in relation to recent theoretical and empirical studies of natural population replacement events and proposed applied research, which would use <it>Wolbachia </it>as a tool to manipulate insect populations.</p