The RNA interference pathway affects midgut infection- and escape barriers for Sindbis virus in <Emphasis Type="Italic">Aedes aegypti</Emphasis>

Background  

The RNA interference (RNAi) pathway acts as an innate antiviral immune response in Aedes aegypti, modulating arbovirus infection of mosquitoes. Sindbis virus (SINV; family: Togaviridae, genus: Alphavirus) is an arbovirus that infects Ae. aegypti in the laboratory. SINV strain TR339 encounters a midgut escape barrier (MEB) during infection of Ae. aegypti. The nature of this barrier is not well understood. To investigate the role of the midgut as the central organ determining vector competence for arboviruses, we generated transgenic mosquitoes in which the RNAi pathway was impaired in midgut tissue of bloodfed females. We used these mosquitoes to reveal effects of RNAi impairment in the midgut on SINV replication, midgut infection and dissemination efficiencies, and mosquito longevity.     

Efficiency of fungus‐impregnated black cloths combined with Imidacloprid for the control of adult Aedes aegypti (Diptera: Culicidae)

Entomopathogenic fungi are potential candidates for use in integrated vector management. However, efficient delivery systems for these fungi need to be investigated. It is known that adult mosquitoes are attracted to dark surfaces, and therefore, black cotton cloths impregnated with Metarhizium anisopliae alone or in combination with the insecticide imidacloprid (IMI) were tested under laboratory conditions. Black cloths impregnated with fungus were also tested in large‐cage trials under natural extradomicile conditions. Blood‐fed Rockefeller and wild‐type strain Aedes aegypti had higher levels of survival when compared with sucrose‐fed counterparts following exposure to fungus‐impregnated cloths. However, when blood‐fed A. aegypti were exposed to a combination of M. anisopliae + IMI, the survival rates were statistically equal to those of sucrose‐fed females. Large‐cage trials showed significant decreases in A. aegypti survival following a minimum 12 h exposure of the mosquitoes to fungus‐impregnated cloths. Increased exposure times results in further reductions in survival. The synergism between M. anisopliae and IMI resulted in reduced survival rates independent of feeding regime under laboratory conditions. Fungus‐impregnated cloths tested under simulated field conditions, considered to be unfavourable for fungal infection, resulted in significant reductions in adult A. aegypti survival. We are currently testing the combined use of fungi and insecticides against blood‐fed insects under simulated field conditions.

Significance and Impact of the Study

The use of fungus‐impregnated cotton cloths is a promising point source application method for the control of adult Aedes aegypti, and this strategy could be incorporated into an integrated vector management programme aiming to reduce the incidence of dengue fever.     

A dengue receptor as possible genetic marker of vector competence in <Emphasis Type="Italic">Aedes aegypti</Emphasis>

Background  

Vector competence refers to the intrinsic permissiveness of an arthropod vector for infection, replication and transmission of a virus. Notwithstanding studies of Quantitative Trait Loci (QTL) that influence the ability of Aedes aegypti midgut (MG) to become infected with dengue virus (DENV), no study to date has been undertaken to identify genetic markers of vector competence. Furthermore, it is known that mosquito populations differ greatly in their susceptibility to flaviviruses. Differences in vector competence may, at least in part, be due to the presence of specific midgut epithelial receptors and their identification would be a significant step forward in understanding the interaction of the virus with the mosquito. The first interaction of DENV with the insect is through proteins in the apical membrane of the midgut epithelium resulting in binding and receptor-mediated endocytosis of the virus, and this determines cell permissiveness to infection. The susceptibility of mosquitoes to infection may therefore depend on their specific virus receptors. To study this interaction in Ae. aegypti strains that differ in their vector competence for DENV, we investigated the DS3 strain (susceptible to DENV), the IBO-11 strain (refractory to infection) and the membrane escape barrier strain, DMEB, which is infected exclusively in the midgut epithelial cells.     

<Emphasis Type="Italic">Aedes aegypti</Emphasis>uses RNA interference in defense against Sindbis virus infection

Background  

RNA interference (RNAi) is an important anti-viral defense mechanism. The Aedes aegypti genome encodes RNAi component orthologs, however, most populations of this mosquito are readily infected by, and subsequently transmit flaviviruses and alphaviruses. The goal of this study was to use Ae. aegypti as a model system to determine how the mosquito's anti-viral RNAi pathway interacts with recombinant Sindbis virus (SINV; family Togaviridae, genus Alphavirus).     

Evidence of Experimental Vertical Transmission of Emerging Novel ECSA Genotype of Chikungunya Virus in Aedes aegypti

Background

Chikungunya virus (CHIKV) has emerged as one of the most important arboviruses of public health significance in the past decade. The virus is mainly maintained through human-mosquito-human cycle. Other routes of transmission and the mechanism of maintenance of the virus in nature are not clearly known. Vertical transmission may be a mechanism of sustaining the virus during inter-epidemic periods. Laboratory experiments were conducted to determine whether Aedes aegypti, a principal vector, is capable of vertically transmitting CHIKV or not.

Methodology/Principal Findings

Female Ae. aegypti were orally infected with a novel ECSA genotype of CHIKV in the 2^nd gonotrophic cycle. On day 10 post infection, a non-infectious blood meal was provided to obtain another cycle of eggs. Larvae and adults developed from the eggs obtained following both infectious and non-infectious blood meal were tested for the presence of CHIKV specific RNA through real time RT-PCR. The results revealed that the larvae and adults developed from eggs derived from the infectious blood meal (2^nd gonotrophic cycle) were negative for CHIKV RNA. However, the larvae and adults developed after subsequent non-infectious blood meal (3^rd gonotrophic cycle) were positive with minimum filial infection rates of 28.2 (1∶35.5) and 20.2 (1∶49.5) respectively.

Conclusion/Significance

This study is the first to confirm experimental vertical transmission of emerging novel ECSA genotype of CHIKV in Ae. aegypti from India, indicating the possibilities of occurrence of this phenomenon in nature. This evidence may have important consequence for survival of CHIKV during adverse climatic conditions and inter-epidemic periods.     

<Emphasis Type="Italic">Gdt</Emphasis>2 regulates the transition of <Emphasis Type="Italic">Dictyostelium</Emphasis>cells from growth to differentiation

Background  

Dictyostelium life cycle consists of two distinct phases – growth and development. The control of growth-differentiation transition in Dictyostelium is not completely understood, and only few genes involved in this process are known.     

High-level gene expression in Aedes albopictus cells using a baculovirus Hr3 enhancer and IE1 transactivator

Background  

Aedes aegypti is the key vector of both the Yellow Fever and Dengue Fever viruses throughout many parts of the world. Low and variable transgene expression levels due to position effect and position effect variegation are problematic to efforts to create transgenic laboratory strains refractory to these viruses. Transformation efficiencies are also less than optimal, likely due to failure to detect expression from all integrated transgenes and potentially due to limited expression of the transposase required for transgene integration.     

Dynamics of infection and competition between two strains of <Emphasis Type="Italic">Trypanosoma brucei brucei</Emphasis>in the tsetse fly observed using fluorescent markers

Background  

Genetic exchange occurs between Trypanosoma brucei strains during the complex developmental cycle in the tsetse vector, probably within the salivary glands. Successful mating will depend on the dynamics of co-infection with multiple strains, particularly if intraspecific competition occurs. We have previously used T. brucei expressing green fluorescent protein to study parasite development in the vector, enabling even one trypanosome to be visualized. Here we have used two different trypanosome strains transfected with either green or red fluorescent proteins to study the dynamics of co-infection directly in the tsetse fly.     

Towards a semen proteome of the dengue vector mosquito: protein identification and potential functions

Background

No commercially licensed vaccine or treatment is available for dengue fever, a potentially lethal infection that impacts millions of lives annually. New tools that target mosquito control may reduce vector populations and break the cycle of dengue transmission. Male mosquito seminal fluid proteins (Sfps) are one such target since these proteins, in aggregate, modulate the reproduction and feeding patterns of the dengue vector, Aedes aegypti. As an initial step in identifying new targets for dengue vector control, we sought to identify the suite of proteins that comprise the Ae. aegypti ejaculate and determine which are transferred to females during mating.

Methodology and Principal Findings

Using a stable-isotope labeling method coupled with proteomics to distinguish male- and female-derived proteins, we identified Sfps and sperm proteins transferred from males to females. Sfps were distinguished from sperm proteins by comparing the transferred proteins to sperm-enriched samples derived from testes and seminal vesicles. We identified 93 male-derived Sfps and 52 predicted sperm proteins that are transferred to females during mating. The Sfp protein classes we detected suggest roles in protein activation/inactivation, sperm utilization, and ecdysteroidogenesis. We also discovered that several predicted membrane-bound and intracellular proteins are transferred to females in the seminal fluids, supporting the hypothesis that Ae. aegypti Sfps are released from the accessory gland cells through apocrine secretion, as occurs in mammals. Many of the Ae. aegypti predicted sperm proteins were homologous to Drosophila melanogaster sperm proteins, suggesting conservation of their sperm-related function across Diptera.

Conclusion and Significance

This is the first study to directly identify Sfps transferred from male Ae. aegypti to females. Our data lay the groundwork for future functional analyses to identify individual seminal proteins that may trigger female post-mating changes (e.g., in feeding patterns and egg production). Therefore, identification of these proteins may lead to new approaches for manipulating the reproductive output and vectorial capacity of Ae. aegypti.     

Evolutionary lability of a complex life cycle in the aphid genus <Emphasis Type="Italic">Brachycaudus</Emphasis>

Background  

Most aphid species complete their life cycle on the same set of host-plant species, but some (heteroecious species) alternate between different hosts, migrating from primary (woody) to secondary (herbaceous) host plants. The evolutionary processes behind the evolution of this complex life cycle have often been debated. One widely accepted scenario is that heteroecy evolved from monoecy on woody host plants. Several shifts towards monoecy on herbaceous plants have subsequently occurred and resulted in the radiation of aphids. Host alternation would have persisted in some cases due to developmental constraints preventing aphids from shifting their entire life cycle to herbaceous hosts (which are thought to be more favourable). According to this scenario, if aphids lose their primary host during evolution they should not regain it. The genus Brachycaudus includes species with all the types of life cycle (monoecy on woody plants, heteroecy, monoecy on herbs). We used this genus to test hypotheses concerning the evolution of life cycles in aphids.     

Meiosis-specific gene discovery in plants: RNA-Seq applied to isolated <Emphasis Type="Italic">Arabidopsis</Emphasis> male meiocytes

Background  

Meiosis is a critical process in the reproduction and life cycle of flowering plants in which homologous chromosomes pair, synapse, recombine and segregate. Understanding meiosis will not only advance our knowledge of the mechanisms of genetic recombination, but also has substantial applications in crop improvement. Despite the tremendous progress in the past decade in other model organisms (e.g., Saccharomyces cerevisiae and Drosophila melanogaster), the global identification of meiotic genes in flowering plants has remained a challenge due to the lack of efficient methods to collect pure meiocytes for analyzing the temporal and spatial gene expression patterns during meiosis, and for the sensitive identification and quantitation of novel genes.     

Parity and Longevity of Aedes aegypti According to Temperatures in Controlled Conditions and Consequences on Dengue Transmission Risks

Background

In Guadeloupe, Aedes aegypti mosquitoes are the only vectors of dengue and chikungunya viruses. For both diseases, vector control is the only tool for preventing epidemics since no vaccine or specific treatment is available. However, to efficiently implement control of mosquitoes vectors, a reliable estimation of the transmission risks is necessary. To become infective an Ae. aegypti female must ingest the virus during a blood meal and will not be able to transmit the virus during another blood-meal until the extrinsic incubation period is completed. Consequently the aged females will carry more infectious risks. The objectives of the present study were to estimate under controlled conditions the expectation of infective life for females and thus the transmission risks in relation with their reproductive cycle and parity status.

Methodology/Principal Findings

Larvae of Ae. aegypti were collected in central Guadeloupe and breed under laboratory conditions until adult emergence. The experiments were performed at constant temperatures (± 1.5°C) of 24°C, 27°C and 30°C on adults females from first generation (F1). Females were kept and fed individually and records of blood-feeding, egg-laying and survival were done daily. Some females were dissected at different physiological stages to observe the ovaries development. The data were analyzed to follow the evolution of parity rates, the number of gonotrophic cycles, the fecundity and to study the mean expectation of life and the mean expectation of infective life for Ae. aegypti females according to temperatures. The expectation of life varies with the parity rates and according to the temperatures, with durations from about 10 days at low parity rates at the higher temperature to an optimal duration of about 35 days when 70% of females are parous at 27°C. Infective life expectancy was found highly variable in the lower parous rates and again the optimal durations were found when more than 50% of females are parous for the mean temperatures of 27°C and 30°C.

Conclusion

Parity rates can be determined for field collected females and could be a good proxy of the expectation of infective life according to temperatures. However, for the same parity rates, the estimation of infective life expectation is very different between Ae. aegypti and Anopheles gambiae mosquitoes. Correlation of field parity rates with transmission risks requires absolutely to be based on Ae. aegypti models, since available Anopheles sp. models underestimate greatly the females longevity.     

Bacteriocyte dynamics during development of a holometabolous insect,the carpenter ant <Emphasis Type="Italic">Camponotus floridanus</Emphasis>

Background  

The carpenter ant Camponotus floridanus harbors obligate intracellular mutualistic bacteria (Blochmannia floridanus) in specialized cells, the bacteriocytes, intercalated in their midgut tissue. The diffuse distribution of bacteriocytes over the midgut tissue is in contrast to many other insects carrying endosymbionts in specialized tissues which are often connected to the midgut but form a distinct organ, the bacteriome. C. floridanus is a holometabolous insect which undergoes a complete metamorphosis. During pupal stages a complete restructuring of the inner organs including the digestive tract takes place. So far, nothing was known about maintenance of endosymbionts during this life stage of a holometabolous insect. It was shown previously that the number of Blochmannia increases strongly during metamorphosis. This implicates an important function of Blochmannia in this developmental phase during which the animals are metabolically very active but do not have access to external food resources. Previous experiments have shown a nutritional contribution of the bacteria to host metabolism by production of essential amino acids and urease-mediated nitrogen recycling. In adult hosts the symbiosis appears to degenerate with increasing age of the animals.     

Comparison of ESTs from juvenile and adult phases of the giant unicellular green alga <Emphasis Type="Italic">Acetabularia acetabulum</Emphasis>

Background  

Acetabularia acetabulum is a giant unicellular green alga whose size and complex life cycle make it an attractive model for understanding morphogenesis and subcellular compartmentalization. The life cycle of this marine unicell is composed of several developmental phases. Juvenile and adult phases are temporally sequential but physiologically and morphologically distinct. To identify genes specific to juvenile and adult phases, we created two subtracted cDNA libraries, one adult-specific and one juvenile-specific, and analyzed 941 randomly chosen ESTs from them.     

LmxMPK4, a mitogen-activated protein (MAP) kinase homologue essential for promastigotes and amastigotes of <Emphasis Type="Italic">Leishmania mexicana</Emphasis>

Background  

Leishmania parasites undergo profound morphological and biochemical changes while passing through their life cycle. Protein kinases have been shown to be involved in the differentiation from the extracellular flagellated promastigotes to the intracellular "non-flagellated" amastigotes and vice versa. Moreover, these enzymes are likely involved in the regulation of the proliferation of the different life stages.     

Ecological modeling of Aedes aegypti (L.) pupal production in rural Kamphaeng Phet, Thailand

Background

Aedes aegypti (L.) is the primary vector of dengue, the most important arboviral infection globally. Until an effective vaccine is licensed and rigorously administered, Ae. aegypti control remains the principal tool in preventing and curtailing dengue transmission. Accurate predictions of vector populations are required to assess control methods and develop effective population reduction strategies. Ae. aegypti develops primarily in artificial water holding containers. Release recapture studies indicate that most adult Ae. aegypti do not disperse over long distances. We expect, therefore, that containers in an area of high development site density are more likely to be oviposition sites and to be more frequently used as oviposition sites than containers that are relatively isolated from other development sites. After accounting for individual container characteristics, containers more frequently used as oviposition sites are likely to produce adult mosquitoes consistently and at a higher rate. To this point, most studies of Ae. aegypti populations ignore the spatial density of larval development sites.

Methodology

Pupal surveys were carried out from 2004 to 2007 in rural Kamphaeng Phet, Thailand. In total, 84,840 samples of water holding containers were used to estimate model parameters. Regression modeling was used to assess the effect of larval development site density, access to piped water, and seasonal variation on container productivity. A varying-coefficients model was employed to account for the large differences in productivity between container types. A two-part modeling structure, called a hurdle model, accounts for the large number of zeroes and overdispersion present in pupal population counts.

Findings

The number of suitable larval development sites and their density in the environment were the primary determinants of the distribution and abundance of Ae. aegypti pupae. The productivity of most container types increased significantly as habitat density increased. An ecological approach, accounting for development site density, is appropriate for predicting Ae. aegypti population levels and developing efficient vector control programs.     

<Emphasis Type="Italic">Drosophila mitoferrin</Emphasis>is essential for male fertility: evidence for a role of mitochondrial iron metabolism during spermatogenesis

Background  

Mammals and Drosophila melanogaster share some striking similarities in spermatogenesis. Mitochondria in spermatids undergo dramatic morphological changes and syncytial spermatids are stripped from their cytoplasm and then individually wrapped by single membranes in an individualization process. In mammalian and fruit fly testis, components of the mitochondrial iron metabolism are expressed, but so far their function during spermatogenesis is unknown. Here we investigate the role of Drosophila mitoferrin (dmfrn), which is a mitochondrial carrier protein with an established role in the mitochondrial iron metabolism, during spermatogenesis.     

Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway

Βackground  

The methylotrophic yeast Pichia pastoris has become an important host organism for recombinant protein production and is able to use methanol as a sole carbon source. The methanol utilization pathway describes all the catalytic reactions, which happen during methanol metabolism. Despite the importance of certain key enzymes in this pathway, so far very little is known about possible effects of overexpressing either of these key enzymes on the overall energetic behavior, the productivity and the substrate uptake rate in P. pastoris strains.     

Maintenance metabolism and carbon fluxes in <Emphasis Type="Italic">Bacillus</Emphasis> species

Background  

Selection of an appropriate host organism is crucial for the economic success of biotechnological processes. A generally important selection criterion is a low maintenance energy metabolism to reduce non-productive consumption of substrate. We here investigated, whether various bacilli that are closely related to Bacillus subtilis are potential riboflavin production hosts with low maintenance metabolism.