Using green fluorescent malaria parasites to screen for permissive vector mosquitoes.
ABSTRACT: BACKGROUND: The Plasmodium species that infect rodents, particularly Plasmodium berghei and Plasmodium yoelii, are useful to investigate host-parasite interactions. The mosquito species that act as vectors of human plasmodia in South East Asia, Africa and South America show different susceptibilities to infection by rodent Plasmodium species. P. berghei and P. yoelii infect both Anopheles gambiae and Anopheles stephensi, which are found mainly in Africa and Asia, respectively. However, it was reported that P. yoelii can infect the South American mosquito, Anopheles albimanus, while P. berghei cannot. METHODS: P. berghei lines that express the green fluorescent protein were used to screen for mosquitoes that are susceptible to infection by P. berghei. Live mosquitoes were examined and screened for the presence of a fluorescent signal in the abdomen. Infected mosquitoes were then examined by time-lapse microscopy to reveal the dynamic behaviour of sporozoites in haemolymph and extracted salivary glands. RESULTS: A single fluorescent oocyst can be detected in live mosquitoes and P. berghei can infect A. albimanus. As in other mosquitoes, P. berghei sporozoites can float through the haemolymph and invade A. albimanus salivary glands and they are infectious in mice after subcutaneous injection. CONCLUSION: Fluorescent Plasmodium parasites can be used to rapidly screen susceptible mosquitoes. These results open the way to develop a laboratory model in countries where importation of A. gambiae and A. stephensi is not allowed.
Project description:Malaria is endemic in the American continent and the Amazonian rainforest is the region with the highest risk of transmission. However, the lack of suitable experimental models to infect malaria vectors from the Americas has limited the progress to understand the biology of transmission in this region. Anopheles aquasalis, a major vector in coastal areas of South America, was found to be highly refractory to infection with two strains of Plasmodium falciparum (NF54 and 7G8) and with Plasmodium berghei (mouse malaria), even when the microbiota was eliminated with antibiotics and oxidative stress was reduced with uric acid. In contrast, An. aquasalis females treated with antibiotics and uric acid are susceptible to infection with a second murine parasite, Plasmodium yoelii nigeriensis N67 (PyN67). Anopheles albimanus, one of the main malaria vectors in Central America, Southern Mexico and the Caribbean, was more susceptible to infection with PyN67 than An. aquasalis, even in the absence of any pre-treatment, but was still less susceptible than Anopheles stephensi. Disruption of the complement-like system in An. albimanus significantly enhanced PyN67 infection, indicating that the mosquito immune system is mounting effective antiplasmodial responses. PyN67 has the ability to infect a broad range of anophelines and is an excellent model to study malaria transmission by South American vectors.
Project description:Mosquitoes infected with malaria parasites have demonstrated altered behaviour that may increase the probability of parasite transmission. Here, we examine the responses of the olfactory system in Plasmodium falciparum infected Anopheles gambiae, Plasmodium berghei infected Anopheles stephensi, and P. berghei infected An. gambiae. Infected and uninfected mosquitoes showed differential responses to compounds in human odour using electroantennography coupled with gas chromatography (GC-EAG), with 16 peaks triggering responses only in malaria-infected mosquitoes (at oocyst, sporozoite or both stages). A selection of key compounds were examined with EAG, and responses showed differences in the detection thresholds of infected and uninfected mosquitoes to compounds including lactic acid, tetradecanoic acid and benzothiazole, suggesting that the changes in sensitivity may be the reason for differential attraction and biting at the oocyst and sporozoite stages. Importantly, the different cross-species comparisons showed varying sensitivities to compounds, with P. falciparum infected An. gambiae differing from P. berghei infected An. stephensi, and P. berghei infected An. gambiae more similar to the P. berghei infected An. stephensi. These differences in sensitivity may reflect long-standing evolutionary relationships between specific Plasmodium and Anopheles species combinations. This highlights the importance of examining different species interactions in depth to fully understand the impact of malaria infection on mosquito olfactory behaviour.
Project description:These data were used to assess gene expression and dynamics between oocyst sporozoites and salivary gland sporozoites for both Plasmodium falciparum and Plasmodium yoelii. Overall design: Sporozoites (Plasmodium falciparum, Plasmodium yoelii) from either the oocyst or salivary gland of Anopheles stephensi mosquitoes were purified twice by an Accudenz discontinuous density gradient and their RNA was extracted using a Qiagen Rneasy Kit with added DNaseI module. All experiments were done in biological triplicate.
Project description:BACKGROUND: Functional screens based on dsRNA-mediated gene silencing identified several Anopheles gambiae genes that limit Plasmodium berghei infection. However, some of the genes identified in these screens have no effect on the human malaria parasite Plasmodium falciparum; raising the question of whether different mosquito effector genes mediate anti-parasitic responses to different Plasmodium species. RESULTS: Four new An. gambiae (G3) genes were identified that, when silenced, have a different effect on P. berghei (Anka 2.34) and P. falciparum (3D7) infections. Orthologs of these genes, as well as LRIM1 and CTL4, were also silenced in An. stephensi (Nijmegen Sda500) females infected with P. yoelii (17XNL). For five of the six genes tested, silencing had the same effect on infection in the P. falciparum-An. gambiae and P. yoelii-An. stephensi parasite-vector combinations. Although silencing LRIM1 or CTL4 has no effect in An. stephensi females infected with P. yoelii, when An. gambiae is infected with the same parasite, silencing these genes has a dramatic effect. In An. gambiae (G3), TEP1, LRIM1 or LRIM2 silencing reverts lysis and melanization of P. yoelii, while CTL4 silencing enhances melanization. CONCLUSION: There is a broad spectrum of compatibility, the extent to which the mosquito immune system limits infection, between different Plasmodium strains and particular mosquito strains that is mediated by TEP1/LRIM1 activation. The interactions between highly compatible animal models of malaria, such as P. yoelii (17XNL)-An. stephensi (Nijmegen Sda500), is more similar to that of P. falciparum (3D7)-An. gambiae (G3).
Project description:BACKGROUND:The native gut microbiota of Anopheles mosquitoes is known to play a key role in the physiological function of its host. Interestingly, this microbiota can also influence the development of Plasmodium in its host mosquitoes. In recent years, much interest has been shown in the employment of gut symbionts derived from vectors in the control of vector-borne disease transmission. In this study, the midgut microbial diversity has been characterized among laboratory-reared adult Anopheles stephensi mosquitoes, from the colony created by rearing progeny of wild-caught mosquitoes (obtained from three different locations in southern India) for multiple generations, using 16S ribosomal RNA (rRNA) gene sequencing approach. Further, the influence of native midgut microbiota of mosquitoes on the development of rodent malaria parasite Plasmodium berghei in its host has been studied. METHODS:The microbial diversity associated with the midgut of An. stephensi mosquitoes was studied by sequencing V3 region of 16S ribosomal RNA (rRNA) gene. The influence of native midgut microbiota of An. stephensi mosquitoes on the susceptibility of the mosquitoes to rodent malaria parasite P. berghei was studied by comparing the intensity and prevalence of P. berghei infection among the antibiotic treated and untreated cohorts of mosquitoes. RESULTS:The analysis of bacterial diversity from the midguts of An. stephensi showed Proteobacteria as the most dominant population among the three laboratory-reared strains of An. stephensi studied. Major genera identified among these mosquito strains were Acinetobacter, Pseudomonas, Prevotella, Corynebacterium, Veillonella, and Bacillus. The mosquito infectivity studies carried out to determine the implication of total midgut microbiota on P. berghei infection showed that mosquitoes whose native microbiota cleared with antibiotics had increased susceptibility to P. berghei infection compared to the antibiotic untreated mosquitoes with its natural native microbiota. CONCLUSIONS:The use of microbial symbiont to reduce the competence of vectors involved in disease transmission has gained much importance in recent years as an emerging alternative approach towards disease control. In this context, the present study was aimed to identify the midgut microbiota composition of An. stephensi, and its effect on the development of P. berghei. Interestingly, the analysis of midgut microbiota from An. stephensi revealed the presence of genus Veillonella in Anopheles species for the first time. Importantly, the study also revealed the negative influence of total midgut microbiota on the development of P. berghei in three laboratory strains of An. stephensi, emphasizing the importance of understanding the gut microbiota in malaria vectors, and its relationship with parasite development in designing strategies to control malaria transmission.
Project description:BACKGROUND:Sugar-feeding provides energy for mosquitoes. Facilitated glucose transporters (GLUTs) are responsible for the uptake of glucose in animals. However, knowledge of GLUTs function in Anopheles spp. is limited. METHODS:Phylogenetic analysis of GLUTs in Anopheles stephensi was performed by the maximum likelihood and Bayesian inference methods. The spatial and temporal expression patterns of four Asteglut genes were analyzed by qPCR. The function of Asteglut1 was examined using a dsRNA-mediated RNA interference method. Transcriptome analysis was used to investigate the global influence of Asteglut1 on mosquito physiology. RESULTS:We identified 4 glut genes, Asteglut1, Asteglutx, Asteglut3 and Asteglut4 in An. stephensi. Asteglut1, Asteglut3 and Asteglut4 were mainly expressed in the midgut. Plasmodium berghei infection differentially regulated the expression of Asteglut genes with significant downregulation of Asteglut1 and Asteglut4, while upregulation of Asteglutx. Only knocking-down Asteglut1 facilitated Plasmodium berghei infection in An. stephensi. This might be due to the accumulation of glucose prior to blood-feeding in dsAsteglut1-treated mosquitoes. Our transcriptome analysis revealed that knockdown of Asteglut1 differentially regulated expression of genes associated with multiple functional clusters, especially those related to detoxification and immunity. The dysregulation of multiple pathways might contribute to the increased P. berghei infection. CONCLUSIONS:Our study shows that Asteglut1 participates in defense against P. berghei in An. stephensi. The regulation of Asteglut1 on vector competence might through modulating multiple biological processes, such as detoxification and immunity.
Project description:Anopheline mosquitoes are the major vectors of human malaria. Parasite-mosquito interactions are a critical aspect of disease transmission and a potential target for malaria control. Current investigations into parasite-mosquito interactions frequently assume that genetically resistant and susceptible mosquitoes exist in nature. Therefore, comparisons between the Plasmodium susceptibility profiles of different mosquito species may contribute to a better understanding of vectorial capacity. Anopheles stephensi is an important malaria vector in central and southern Asia and is widely used as a laboratory model of parasite transmission due to its high susceptibility to Plasmodium infection. In the present study, we identified a rodent malaria-refractory strain of A. stephensi mysorensis (Ehime) by comparative study of infection susceptibility. A very low number of oocysts develop in Ehime mosquitoes infected with P. berghei and P. yoelii, as determined by evaluation of developed oocysts on the basal lamina. A stage-specific study revealed that this reduced susceptibility was due to the impaired formation of ookinetes of both Plasmodium species in the midgut lumen and incomplete crossing of the midgut epithelium. There were no apparent abnormalities in the exflagellation of male parasites in the ingested blood or the maturation of oocysts after the rounding up of the ookinetes. Overall, these results suggest that invasive-stage parasites are eliminated in both the midgut lumen and epithelium in Ehime mosquitoes by strain-specific factors that remain unknown. The refractory strain newly identified in this report would be an excellent study system for investigations into novel parasite-mosquito interactions in the mosquito midgut.
Project description:The transmission of drug-resistant parasites by the mosquito may be influenced by the altered biological fitness of drug-resistant parasites and different immune reactions or metabolic change in the mosquito. At this point, little is known about the variations in mosquito immunity and metabolism when mosquitoes are infected with drug-resistant parasites. To understand the differential gene expression in Anopheles following infection with drug-resistant Plasmodium, we conducted a genome-wide transcriptomic profiling analysis of Anopheles stephensi following feeding on mice with drug-resistant or drug-sensitive P. yoelii, observed changes in gene expression profiles and identified transcripts affected by the drug-resistant parasite.To study the impact of drug-resistant Plasmodium infections on An. stephensi gene transcription, we analyzed the three major transition stages of Plasmodium infections: at 24 h and 13 and 19 days after blood-feeding. Six cDNA libraries (R-As24h, R-As13d, R-As19d,S-As24h, S-As13dand S-As19d) were constructed, and RNA sequencing was subsequently performed. In total, approximately 50.1 million raw reads, 47.9 million clean reads and 7.18G clean bases were obtained. Following differentially expressed gene (DEG) analysis, GO enrichment analysis of DEGs, and functional classification by KEGG, we showed that the variations in gene expression in An. stephensi infected by the drug-resistant P. yoelii NSM occurred mainly at 13 days after blood meal during sporozoite migration through the hemolymph. The differentially expressed genes included those functioning in some important immune reaction and iron metabolism pathways, such as pattern recognition receptors, regulators of the JNK pathway, components of the phagosome pathway, regulators of the melanization response, activators of complement reactions, insulin signaling cascade members, oxidative stress and detoxification proteins.Our study shows that drug-resistant P. yoelii NSM has an impact on the transcript abundance levels of An.stephensi mostly at 13 days after blood meal during sporozoite migration through the hemolymph and that most differentially expressed genes were downregulated. Our results highlight the need for a better understanding of selective pressures from these differentially expressed genes of the drug-resistant Plasmodium in the mosquito and the different transmission patterns of drug-resistant Plasmodium by Anopheles.
Project description:The endosymbiont Wolbachia wAlbB induces refractoriness to Plasmodium falciparum in Anopheles stephensi, the primary mosquito vector of human malaria in the Middle East and South Asia. However, it remains unknown whether such refractoriness can be extended to other malaria species. In particular, it was reported that under very specific conditions, wAlbB can enhance Plasmodium infection in some hosts. Here, we measured the impact of wAlbB on the rodent malaria parasite Plasmodium berghei in A. stephensi by comparing the load of oocysts and sporozoites in midguts and salivary glands, respectively, between wAlbB-infected and -uninfected mosquitoes. To investigate whether wAlbB modulated mosquito immune defense against parasites, we compared the expression of the immune genes, which were previously reported to involve in antimalarial response, in both midguts and the remaining carcass tissues of mosquitoes. The stable association of wAlbB with A. stephensi resulted in reduction of parasites by more than half at the oocyst stage, and up to 91.8% at the sporzoite stage. The anti-plasmodium immune genes, including TEP1, LRIM1, Toll pathway gene Rel1 and the effector Defensin 1, were induced by wAlbB in different mosquito body tissues. These findings suggest that immune priming is a potential cause of wAlbB-mediated antimalarial response in A. stephensi. More importantly, no evidence was found for any enhancement of Plasmodium infection in A. stephensi stably infected with wAlbB. We discuss these findings with possible implementations of Wolbachia for malaria control in disease endemic areas.