Refractory Mosquitoes Research Articles

Transcriptome comparison of dengue-susceptible and -resistant field derived strains of Colombian Aedes aegypti using RNA-sequencing.

BACKGROUND Forty percent of the world’s population live in areas where they are at risk from dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Dengue viruses are transmitted primarily by the mosquito Aedes aegypti. In Cali, Colombia, approximately 30% of field collected Ae. aegypti are naturally refractory to all four dengue serotypes.OBJECTIVES Use RNA-sequencing to identify those genes that determine refractoriness in feral mosquitoes to dengue. This information can be used in gene editing strategies to reduce dengue transmission.METHODS We employed a full factorial design, analyzing differential gene expression across time (24, 36 and 48 h post bloodmeal), feeding treatment (blood or blood + dengue-2) and strain (susceptible or refractory). Sequences were aligned to the reference Ae. aegypti genome for identification, assembled to visualize transcript structure, and analyzed for dynamic gene expression changes. A variety of clustering techniques was used to identify the differentially expressed genes.FINDINGS We identified a subset of genes that likely assist dengue entry and replication in susceptible mosquitoes and contribute to vector competence.MAIN CONCLUSIONS The differential expression of specific genes by refractory and susceptible mosquitoes could determine the phenotype, and may be used to in gene editing strategies to reduce dengue transmission.

Effect of Oral Infection of Mayaro Virus on Fitness Correlates and Expression of Immune Related Genes in Aedes aegypti.

Mayaro virus is a mosquito-borne Alphavirus endemic to forests of tropical South America with a sylvatic cycle involving non-human primates and Haemagogus mosquitoes. Human infection with Mayaro virus causes a febrile illness and long-lasting arthralgia and cases are often associated with exposure to tropical forest habitats. Human movement between tropical forest habitats and urban settings may allow for imported cases and subsequent local transmission by domestic mosquito Aedes aegypti. The relative importance of Ae. aegypti as a vector of Mayaro virus may depend on the pathogenic effects of the virus on fitness correlates, especially those entomological parameters that relate to vectorial capacity. We performed mosquito infection studies and compared adult survival and fecundity of females from Brazilian and Floridian populations of Ae. aegypti following oral ingestion of uninfectious (control) and Mayaro virus infectious blood. Mayaro virus infected and refractory mosquitoes had similar or 30–50% lower fecundity than control (unexposed) mosquitoes, suggesting a reproductive cost to mounting an immune response or phenotypic expression of refractoriness. Survival of adult female mosquitoes and targeted gene expression in the Toll and IMD pathways were not altered by Mayaro virus infection. Adult lifespan and fecundity estimates were independent of measured viral titer in the bodies of mosquitoes. The lack of adverse effects of infection status on female survival suggests that Mayaro virus will not alter vectorial capacity mediated by changes in this parameter.

The mosquito melanization response requires hierarchical activation of non-catalytic clip domain serine protease homologs.

Serine protease cascades regulate important insect immune responses namely melanization and Toll pathway activation. An important component of these cascades are clip-domain serine protease homologs (cSPHs), which are non-catalytic, but essential for activating the enzyme prophenoloxidase (PPO) in the melanization response during septic infections. The activation of cSPHs requires their proteolytic cleavage, yet factors that control their activation and the complexity of their interactions within these cascades remain unclear. Here, we report the identification of CLIPA28 as a novel immune-related cSPH in the malaria vector Anopheles gambiae. Functional genetic analysis using RNA interference (RNAi) revealed that CLIPA28 is essential for the melanization of Plasmodium berghei parasites in refractory mosquitoes, and for mosquito resistance to fungal infections. We further show, using combined biochemical and genetic approaches, that CLIPA28 is member of a network of at least four cSPHs, whereby members are activated in a hierarchical manner following septic infections. Depletion of the complement-like protein TEP1 abolished the activation of this network after septic infections, whereas, depletion of the serine protease inhibitor 2 (SRPN2) triggered enhanced network activation, even in naïve mosquitoes, culminating in a dramatic reduction in cSPHs hemolymph levels, which paralleled that of PPO. Our data suggest that cSPHs are engaged in complex and multilayered interactions within serine protease cascades that regulate melanization, and identify TEP1 and SRPN2 as two master regulators of the cSPH network.

Streamlined SMFA and mosquito dark-feeding regime significantly improve malaria transmission-blocking assay robustness and sensitivity

BackgroundThe development of malaria transmission-blocking strategies including the generation of malaria refractory mosquitoes to replace the wild populations through means of gene drives hold great promise. The standard membrane feeding assay (SMFA) that involves mosquito feeding on parasitized blood through an artificial membrane system is a vital tool for evaluating the efficacy of transmission-blocking interventions. However, despite the availability of several published protocols, the SMFA remains highly variable and broadly insensitive.MethodsThe SMFA protocol was optimized through coordinated culturing of Anopheles coluzzii mosquitoes and Plasmodium falciparum parasite coupled with placing mosquitoes under a strict dark regime before, during, and after the gametocyte feed.ResultsA detailed description of essential steps is provided toward synchronized generation of highly fit An. coluzzii mosquitoes and P. falciparum gametocytes in preparation for an SMFA. A dark-infection regime that emulates the natural vector-parasite interaction system is described, which results in a significant increase in the infection intensity and prevalence. Using this optimal SMFA pipeline, a series of putative transmission-blocking antimicrobial peptides (AMPs) were screened, confirming that melittin and magainin can interfere with P. falciparum development in the vector.ConclusionA robust SMFA protocol that enhances the evaluation of interventions targeting human malaria transmission in laboratory setting is reported. Melittin and magainin are identified as highly potent antiparasitic AMPs that can be used for the generation of refractory Anopheles gambiae mosquitoes.

The Composition of Midgut Bacteria in Aedes aegypti (Diptera: Culicidae) That Are Naturally Susceptible or Refractory to Dengue Viruses.

The composition, abundance, and diversity of midgut bacteria in mosquitoes can influence pathogen transmission. We used 16S rRNA microbiome profiling to survey midgut microbial diversity in pooled samples of laboratory colonized dengue-refractory, Cali-MIB, and dengue-susceptible, Cali-S Aedes aegypti (Linnaeus). The 16S rRNA sequences from the sugar-fed midguts of adult females clustered to 63 amplicon sequence variants (ASVs), primarily from Proteobacteria, Firmicutes, Flavobacteria, and Actinobacteria. An average of five ASVs dominated the midguts, and most ASVs were present in both Cali-MIB and Cali-S midguts. No differences in abundance were noted at any phylogenetic level (Phylum, Class, Order, Family, Genus) by analysis of composition of microbiome (w = 0). No community diversity metrics were significantly different between refractory and susceptible mosquitoes. These data suggest that phenotypic differences in the susceptibility to dengue virus between Cali-MIB and Cali-S are not likely due to major differences in midgut bacterial communities.

Metanetwork Transmission Model for Predicting a Malaria-Control Strategy.

Background: Mosquitoes are the primary vectors responsible for malaria transmission to humans, with numerous experiments having been conducted to aid in the control of malaria transmission. One of the main approaches aims to develop malaria parasite resistance within the mosquito population by introducing a resistance (R) allele. However, when considering this approach, some critical factors, such as the life of the mosquito, female mosquito fertility capacity, and human and mosquito mobility, have not been considered. Thus, an understanding of how mosquitoes and humans affect disease dynamics is needed to better inform malaria control policymaking.Methods: In this study, a method was proposed to create a metanetwork on the basis of the geographic maps of Gambia, and a model was constructed to simulate evolution within a mixed population, with factors such as birth, death, reproduction, biting, infection, incubation, recovery, and transmission between populations considered in the network metrics. First, the same number of refractory mosquitoes (RR genotype) was introduced into each population, and the prevalence of the R allele (the ratio of resistant alleles to all alleles) and malaria were examined. In addition, a series of simulations were performed to evaluate two different deployment strategies for the reduction of the prevalence of malaria. The R allele and malaria prevalence were calculated for both the strategies, with 10,000 refractory mosquitoes deployed into randomly selected populations or selection based on nodes with top-betweenness values. The 10,000 mosquitoes were deployed among 1, 5, 10, 20, or 40 populations.Results: The simulations in this paper showed that a higher RR genotype (resistant-resistant genes) ratio leads to a higher R allele prevalence and lowers malaria prevalence. Considering the cost of deployment, the simulation was performed with 10,000 refractory mosquitoes deployed among 1 or 5 populations, but this approach did not reduce the original malaria prevalence. Thus, instead, the 10,000 refractory mosquitoes were distributed among 10, 20, or 40 populations and were shown to effectively reduce the original malaria prevalence. Thus, deployment among a relatively small fraction of central nodes can offer an effective strategy to reduce malaria.Conclusion: The standard network centrality measure is suitable for planning the deployment of refractory mosquitoes.Importance: Malaria is an infectious disease that is caused by a plasmodial parasite, and some control strategies have focused on genetically modifying the mosquitoes. This work aims to create a model that takes into account mosquito development and malaria transmission among the population and how these factors influence disease dynamics so as to better inform malaria-control policymaking.

Proteome-wide analysis of Anopheles culicifacies mosquito midgut: new insights into the mechanism of refractoriness

BackgroundMidgut invasion, a major bottleneck for malaria parasites transmission is considered as a potential target for vector-parasite interaction studies. New intervention strategies are required to explore the midgut proteins and their potential role in refractoriness for malaria control in Anopheles mosquitoes. To better understand the midgut functional proteins of An. culicifacies susceptible and refractory species, proteomic approaches coupled with bioinformatics analysis is an effective means in order to understand the mechanism of refractoriness. In the present study, an integrated in solution- in gel trypsin digestion approach, along with Isobaric tag for relative and absolute quantitation (iTRAQ)–Liquid chromatography/Mass spectrometry (LC/MS/MS) and data mining were performed to identify the proteomic profile and differentially expressed proteins in Anopheles culicifacies susceptible species A and refractory species B.ResultsShot gun proteomics approaches led to the identification of 80 proteins in An. culicifacies susceptible species A and 92 in refractory species B and catalogue was prepared. iTRAQ based proteomic analysis identified 48 differentially expressed proteins from total 130 proteins. Of these, 41 were downregulated and 7 were upregulated in refractory species B in comparison to susceptible species A. We report that the altered midgut proteins identified in naturally refractory mosquitoes are involved in oxidative phosphorylation, antioxidant and proteolysis process that may suggest their role in parasite growth inhibition. Furthermore, real time polymerase chain reaction (PCR) analysis of few proteins indicated higher expression of iTRAQ upregulated protein in refractory species than susceptible species.ConclusionThis study elucidates the first proteome of the midguts of An. culicifacies sibling species that attempts to analyze unique proteogenomic interactions to provide insights for better understanding of the mechanism of refractoriness. Functional implications of these upregulated proteins in refractory species may reflect the phenotypic characteristics of the mosquitoes and will improve our understandings of blood meal digestion process, parasite vector interactions and proteomes of other vectors of human diseases for development of novel vector control strategies.

Vector competence and innate immune responses to dengue virus infection in selected laboratory and field-collected Stegomyia aegypti (= Aedes aegypti).

Control of dengue virus (DenV) transmission, primarily based on strategies to reduce populations of the principle vector Stegomya aegypti (= Aedes aegypti) (Diptera: Culicidae), is difficult to sustain over time. Other potential strategies aim to manipulate characteristics such as vector competence (VC), the innate capacity of the vector to transmit the virus. Previous studies have identified genetic factors, including differential expression of apoptosis-related genes, associated with the refractory and susceptible phenotypes in selected strains of S. aegypti from Cali, Colombia. The present study was designed to evaluate the variability of VC in selected strains against different DenV serotypes and to determine whether field-collected mosquitoes respond similarly to selected laboratory strains in terms of enhanced or reduced expression of apoptosis-related genes. Vector competence differed between strains, but did not differ in response to different DenV serotypes. Differences in VC were observed among mosquitoes collected from different localities in Cali. The overexpression of the pro-apoptosis genes, caspase 16 and Aedronc, was conserved in field-collected refractory mosquitoes and the selected laboratory refractory strain. The results suggest that the apoptosis response is conserved among all refractory mosquitoes to inhibit the development of all DenV serotypes.

Mitigating Future Avian Malaria Threats to Hawaiian Forest Birds from Climate Change.

Avian malaria, transmitted by Culex quinquefasciatus mosquitoes in the Hawaiian Islands, has been a primary contributor to population range limitations, declines, and extinctions for many endemic Hawaiian honeycreepers. Avian malaria is strongly influenced by climate; therefore, predicted future changes are expected to expand transmission into higher elevations and intensify and lengthen existing transmission periods at lower elevations, leading to further population declines and potential extinction of highly susceptible honeycreepers in mid- and high-elevation forests. Based on future climate changes and resulting malaria risk, we evaluated the viability of alternative conservation strategies to preserve endemic Hawaiian birds at mid and high elevations through the 21st century. We linked an epidemiological model with three alternative climatic projections from the Coupled Model Intercomparison Project to predict future malaria risk and bird population dynamics for the coming century. Based on climate change predictions, proposed strategies included mosquito population suppression using modified males, release of genetically modified refractory mosquitoes, competition from other introduced mosquitoes that are not competent vectors, evolved malaria-tolerance in native honeycreepers, feral pig control to reduce mosquito larval habitats, and predator control to improve bird demographics. Transmission rates of malaria are predicted to be higher than currently observed and are likely to have larger impacts in high-elevation forests where current low rates of transmission create a refuge for highly-susceptible birds. As a result, several current and proposed conservation strategies will be insufficient to maintain existing forest bird populations. We concluded that mitigating malaria transmission at high elevations should be a primary conservation goal. Conservation strategies that maintain highly susceptible species like Iiwi (Drepanis coccinea) will likely benefit other threatened and endangered Hawai’i species, especially in high-elevation forests. Our results showed that mosquito control strategies offer potential long-term benefits to high elevation Hawaiian honeycreepers. However, combined strategies will likely be needed to preserve endemic birds at mid elevations. Given the delay required to research, develop, evaluate, and improve several of these currently untested conservation strategies we suggest that planning should begin expeditiously.

Molecular Analysis of Pfs47-Mediated Plasmodium Evasion of Mosquito Immunity

Malaria is a life-threatening disease caused by Plasmodium falciparum parasites that is transmitted through the bites of infected anopheline mosquitoes. P. falciparum dispersal from Africa, as a result of human migration, required adaptation of the parasite to several different indigenous anopheline species. The mosquito immune system can greatly limit infection and P. falciparum evolved a strategy to evade these responses that is mediated by the Pfs47 gene. Pfs47 is a polymorphic gene with signatures of diversifying selection and a strong geographic genetic structure at a continental level. Here, we investigated the role of single four amino acid differences between the Pfs47 gene from African (GB4 and NF54) and a New World (7G8) strains that differ drastically in their ability to evade the immune system of A. gambiae L35 refractory mosquitoes. Wild type NF54 and GB4 parasites can survive in this mosquito strain, while 7G8 parasites are eliminated. Our studies indicate that replacement in any of these four single amino acids in Pfs47 from the NF54 strain by those present in 7G8, completely disrupts the ability of NF54 parasites to hide from the mosquito immune system. One of these amino acid replacements had the opposite effect on A. albimanus mosquitoes, and enhanced infection. We conclude that malaria transmission involves a complex interplay between the genetic background of the parasite and the mosquito and that Pfs47 can be critical in this interaction as it mediates Plasmodium immune evasion through molecular interactions that need to be precise in some parasite/vector combinations.

Variation in susceptibility of African Plasmodium falciparum malaria parasites to TEP1 mediated killing in Anopheles gambiae mosquitoes.

Anopheles gambiae s.s. mosquitoes are efficient vectors for Plasmodium falciparum, although variation exists in their susceptibility to infection. This variation depends partly on the thioester-containing protein 1 (TEP1) and TEP depletion results in significantly elevated numbers of oocysts in susceptible and resistant mosquitoes. Polymorphism in the Plasmodium gene coding for the surface protein Pfs47 modulates resistance of some parasite laboratory strains to TEP1-mediated killing. Here, we examined resistance of P. falciparum isolates of African origin (NF54, NF165 and NF166) to TEP1-mediated killing in a susceptible Ngousso and a refractory L3–5 strain of A. gambiae. All parasite clones successfully developed in susceptible mosquitoes with limited evidence for an impact of TEP1 on transmission efficiency. In contrast, NF166 and NF165 oocyst densities were strongly reduced in refractory mosquitoes and TEP1 silencing significantly increased oocyst densities. Our results reveal differences between African P. falciparum strains in their capacity to evade TEP1-mediated killing in resistant mosquitoes. There was no significant correlation between Pfs47 genotype and resistance of a given P. falciparum isolate for TEP1 killing. These data suggest that polymorphisms in this locus are not the sole mediators of immune evasion of African malaria parasites.

A Bayesian Hierarchical Model for Estimation of Abundance and Spatial Density of Aedes aegypti.

Strategies to minimize dengue transmission commonly rely on vector control, which aims to maintain Ae. aegypti density below a theoretical threshold. Mosquito abundance is traditionally estimated from mark-release-recapture (MRR) experiments, which lack proper analysis regarding accurate vector spatial distribution and population density. Recently proposed strategies to control vector-borne diseases involve replacing the susceptible wild population by genetically modified individuals’ refractory to the infection by the pathogen. Accurate measurements of mosquito abundance in time and space are required to optimize the success of such interventions. In this paper, we present a hierarchical probabilistic model for the estimation of population abundance and spatial distribution from typical mosquito MRR experiments, with direct application to the planning of these new control strategies. We perform a Bayesian analysis using the model and data from two MRR experiments performed in a neighborhood of Rio de Janeiro, Brazil, during both low- and high-dengue transmission seasons. The hierarchical model indicates that mosquito spatial distribution is clustered during the winter (0.99 mosquitoes/premise 95% CI: 0.80–1.23) and more homogeneous during the high abundance period (5.2 mosquitoes/premise 95% CI: 4.3–5.9). The hierarchical model also performed better than the commonly used Fisher-Ford’s method, when using simulated data. The proposed model provides a formal treatment of the sources of uncertainty associated with the estimation of mosquito abundance imposed by the sampling design. Our approach is useful in strategies such as population suppression or the displacement of wild vector populations by refractory Wolbachia-infected mosquitoes, since the invasion dynamics have been shown to follow threshold conditions dictated by mosquito abundance. The presence of spatially distributed abundance hotspots is also formally addressed under this modeling framework and its knowledge deemed crucial to predict the fate of transmission control strategies based on the replacement of vector populations.

Fighting Arbovirus Transmission: Natural and Engineered Control of Vector Competence in Aedes Mosquitoes.

Control of aedine mosquito vectors, either by mosquito population reduction or replacement with refractory mosquitoes, may play an essential role in the fight against arboviral diseases. In this review, we will focus on the development and application of biological approaches, both natural or engineered, to limit mosquito vector competence for arboviruses. The study of mosquito antiviral immunity has led to the identification of a number of host response mechanisms and proteins that are required to control arbovirus replication in mosquitoes, though more factors influencing vector competence are likely to be discovered. We will discuss key aspects of these pathways as targets either for selection of naturally resistant mosquito populations or for mosquito genetic manipulation. Moreover, we will consider the use of endosymbiotic bacteria such as Wolbachia, which in some cases have proven to be remarkably efficient in disrupting arbovirus transmission by mosquitoes, but also the use of naturally occurring insect-specific viruses that may interfere with arboviruses in mosquito vectors. Finally, we will discuss the use of paratransgenesis as well as entomopathogenic fungi, which are also proposed strategies to control vector competence.

Exsheathment and midgut invasion of nocturnally subperiodic Brugia malayi microfilariae in a refractory vector, Aedes aegypti (Thailand strain).

Exsheathment and midgut invasion of nocturnally subperiodic Brugia malayi microfilariae were analyzed using light and scanning electron microscopy in a refractory vector, Aedes aegypti (Thailand strain). Results showed that exsheathed microfilariae represented only approximately 1% of the total microfilaria midguts dissected at 5-min post-infected blood meal (PIBM). The percentage of exsheathed microfilariae found in midguts progressively increased to about 20, 60, 80, 90, and 100% at 1-, 2-5-, 6-12-, 18-36-, and 48-h PIBM, respectively. Importantly, all the microfilariae penetrating the mosquito midguts were exsheathed. Midgut invasion by the exsheathed microfilariae was observed between 2- and 48-h PIBM. SEM analysis revealed sheathed microfilariae surrounded by small particles and maceration of the microfilarial sheath in the midguts, suggesting that the midguts of the refractory mosquitoes might have protein(s) and/or enzyme(s) and/or factor(s) that induce and/or accelerate exsheathment. The microfilariae penetrated the internal face of the peritrophic matrix (PM) by their anterior part and then the midgut epithelium, before entering the hemocoel suggesting that PM was not a barrier against the microfilariae migrating towards the midgut. Melanized microfilariae were discovered in the hemocoel examined at 96-h PIBM suggesting that the refractory mosquitoes used melanization reactions against this parasite. This study provided evidence that A. aegypti (Thailand strain) has refractory mechanisms against B. malayi in both midgut and hemocoel.

Comparative Expression Profiles of Midgut Genes in Dengue Virus Refractory and Susceptible Aedes aegypti across Critical Period for Virus Infection

Background Aedes aegypti is the primary mosquito vector for dengue virus (DENV) worldwide. Infectivity of dengue virus varies among natural populations of this mosquito. How A. aegypti responds to DENV infection relative to which genes and associated pathways contribute to its differential susceptibility as a vector is not well defined.Methods/Principal FindingsHere, we used custom cDNA microarrays to identify groups of genes that were differentially expressed in midgut tissues between susceptible and refractory strains in a highly time specific manner. While genes involved in protein processing in the endoplasmic reticulum, mRNA surveillance, and the proteasome were significantly up-regulated in the susceptible strain, several metabolic processes including glycolysis, glycan biosynthesis and Wnt pathway were active in the refractory strain. In addition, several key signaling genes were expressed as common responsive genes in both susceptible and refractory mosquitoes that may be necessary for signal transduction to trigger the appropriate host response to the viral infection. These are coordinately expressed in the form of tight gene networks and expression clusters that may be necessary to differentially contribute to the progression of dengue infection between the two strains.ConclusionsOur data show that highly correlated differential expression of responsive genes throughout the post infection period in A. aegypti midgut tissues is necessary for a coordinated transcriptional response of the mosquito genes to host or defend the viral infection.

Global Cross-Talk of Genes of the Mosquito Aedes aegypti in Response to Dengue Virus Infection

BackgroundThe mosquito Aedes aegypti is the primary vector of dengue virus (DENV) infection in humans, and DENV is the most important arbovirus across most of the subtropics and tropics worldwide. The early time periods after infection with DENV define critical cellular processes that determine ultimate success or failure of the virus to establish infection in the mosquito.Methods and ResultsTo identify genes involved in these processes, we performed genome-wide transcriptome profiling between susceptible and refractory A. aegypti strains at two critical early periods after challenging them with DENV. Genes that responded coordinately to DENV infection in the susceptible strain were largely clustered in one specific expression module, whereas in the refractory strain they were distributed in four distinct modules. The susceptible response module in the global transcriptional network showed significant biased representation with genes related to energy metabolism and DNA replication, whereas the refractory response modules showed biased representation across different metabolism pathway genes including cytochrome P450 and DDT [1,1,1-Trichloro-2,2-bis(4-chlorophenyl) ethane] degradation genes, and genes associated with cell growth and death. A common core set of coordinately expressed genes was observed in both the susceptible and refractory mosquitoes and included genes related to the Wnt (Wnt: wingless [wg] and integration 1 [int1] pathway), MAPK (Mitogen-activated protein kinase), mTOR (mammalian target of rapamycin) and JAK-STAT (Janus Kinase - Signal Transducer and Activator of Transcription) pathways.ConclusionsOur data revealed extensive transcriptional networks of mosquito genes that are expressed in modular manners in response to DENV infection, and indicated that successfully defending against viral infection requires more elaborate gene networks than hosting the virus. These likely play important roles in the global-cross talk among the mosquito host factors during the critical early DENV infection periods that trigger the appropriate host action in susceptible vs. refractory mosquitoes.

Parasite Killing in Malaria Non-Vector Mosquito Anopheles culicifacies Species B: Implication of Nitric Oxide Synthase Upregulation

Background Anopheles culicifacies, the main vector of human malaria in rural India, is a complex of five sibling species. Despite being phylogenetically related, a naturally selected subgroup species B of this sibling species complex is found to be a poor vector of malaria. We have attempted to understand the differences between vector and non-vector Anopheles culicifacies mosquitoes in terms of transcriptionally activated nitric oxide synthase (AcNOS) physiologies to elucidate the mechanism of refractoriness. Identification of the differences between genes and gene products that may impart refractory phenotype can facilitate development of novel malaria transmission blocking strategies. Methodology/Principal Findings We conducted a study on phylogenetically related susceptible (species A) and refractory (species B) sibling species of An. culicifacies mosquitoes to characterize biochemical and molecular differences in AcNOS gene and gene elements and their ability to inhibit oocyst growth. We demonstrate that in species B, AcNOS specific activity and nitrite/nitrates in mid-guts and haemolymph were higher as compared to species A after invasion of the mid-gut by P. vivax at the beginning and during the course of blood feeding. Semiquantitative RT-PCR and real time PCR data of AcNOS concluded that this gene is more abundantly expressed in midgut of species B than in species A and is transcriptionally upregulated post blood meals. Dietary feeding of L-NAME along with blood meals significantly inhibited midgut AcNOS activity leading to an increase in oocyst production in An. culicifacies species B. Conclusions/Significance We hypothesize that upregulation of mosquito innate cytotoxicity due to NOS in refractory strain to Plasmodium vivax infection may contribute to natural refractoriness in An. culicifacies mosquito population. This innate capacity of refractory mosquitoes could represent the ancestral function of the mosquito immune system against the parasite and could be utilized to understand the molecular basis of refractoriness in planning effective vector control strategies.

Induction of reaper ortholog mx in mosquito midgut cells following baculovirus infection

Many vertebrate and insect viruses possess antiapoptotic genes that are required for their infectivity. This led to the hypothesis that apoptosis is an innate immunoresponse important for limiting virus infections. The role of apoptosis may be especially important in insect antiviral defense because of the lack of adaptive immunity. However, the cellular mechanism that elicits apoptosis in response to viral infection in insects has not been determined. Using an in vivo infection system with the mosquito baculovirus CuniNPV (Culex nigripalpus nucleopolyhedrovirus), we demonstrated that michelob_x (mx), the mosquito ortholog of Drosophila proapoptotic gene reaper, is specifically induced in larval midgut cells following viral infection. Interestingly, the dynamics of mx induction corresponds with the outcome of the infection. In the permissive mosquito C. quinquefasciatus, a slow induction of mx failed to induce prompt apoptosis, and the infected cells eventually undergo necrosis with heavy loads of encapsulated viruses. In contrast, in the refractory mosquito Aedes aegypti, a rapid induction of mx within 30 min p.i. is followed by apoptosis within 2-6 h p.i., suggesting a possible role for apoptosis in limiting viral infection. When the execution of apoptosis was delayed by caspase inhibitors, viral gene expression became detectable in the A. aegypti larvae.

Expression Profile of Prophenoloxidase-Encoding (<I>acppo6</I>) Gene of Plasmodium vivax-Refractory Strain of Anopheles culicifacies

Anopheles culicifacies is the main vector for transmission of Plasmodium vivax malaria in the Indian subcontinent. A strain of An. culicifacies isolated from its natural niche displayed complete refractoriness to P. vivax by melanotic encapsulation of ookinetes. Prophenoloxidases are key components of the phenoloxidase cascade that leads to recognition and melanization of invading organisms. We isolated and cloned prophenoloxidase-encoding acppo6 gene of An. culicifacies and analyzed its expression profile under various regimens of immune challenge. The acppo6 was differentially expressed during various stages of larval development. The acppo6 transcription was also up-regulated in response to bacteria and Plasmodium vinckei petteri challenge. The transcript levels of the acppo6 gene were higher in naive adult refractory female mosquitoes as compared with female susceptible mosquitoes. Furthermore, the induction of acppo6 in the susceptible strain upon Plasmodium infection was negligible as compared with that of the refractory strain. The observation is suggestive of the role of acppo6 in effectuating a melanotic response in Plasmodium-incompetent naturally occurring refractory An. culicifacies strain.

Differential Gene Expression from Midguts of Refractory and Susceptible Lines of the Mosquito,Aedes aegypti, Infected with Dengue-2 Virus

Suppressive subtractive hybridization was used to evaluate the differential expression of midgut genes of feral populations of Aedes aegypti (Diptera: Culicidae) from Colombia that are naturally refractory or susceptible to Dengue-2 virus infection. A total of 165 differentially expressed sequence tags (ESTs) were identified in the subtracted libraries. The analysis showed a higher number of differentially expressed genes in the susceptible Ae. aegypti individuals than the refractory mosquitoes. The functional annotation of ESTs revealed a broad response in the susceptible library that included immune molecules, metabolic molecules and transcription factors. In the refractory strain, there was the presence of a trypsin inhibitor gene, which could play a role in the infection. These results serve as a template for more detailed studies aiming to characterize the genetic components of refractoriness, which in turn can be used to devise new approaches to combat transmission of dengue fever.