Suchismita Das

Engineered anopheles immunity to Plasmodium infection.

A causative agent of human malaria, Plasmodium falciparum, is transmitted by Anopheles mosquitoes. The malaria parasite is under intensive attack from the mosquitos innate immune system during its sporogonic development. We have used genetic engineering to create immune-enhanced Anopheles stephensi mosquitoes through blood meal-inducible expression of a transgene encoding the IMD pathway-controlled NF-kB Rel2 transcription factor in the midgut and fat-body tissue. Transgenic mosquitoes showed greater resistance to Plasmodium and microbial infection as a result of timely concerted tissue-specific immune attacks involving multiple effectors. The relatively weak impact of this genetic modification on mosquito fitness under laboratory conditions encourages further investigation of this approach for malaria control.

Pathogenomics of Culex quinquefasciatus and Meta-Analysis of Infection Responses to Diverse Pathogens

Closing the Vector Circle The genome sequence of Culex quinquefasciatus offers a representative of the third major genus of mosquito disease vectors for comparative analysis. In a major international effort, Arensburger et al. (p. 86) uncovered divergences in the C. quinquefasciatus genome compared with the representatives of the other two genera Aedes aegypti and Anopheles gambiae. The main difference noted is the expansion of numbers of genes, particularly for immunity, oxidoreductive functions, and digestive enzymes, which may reflect specific aspects of the Culex life cycle. Bartholomay et al. (p. 88) explored infection-response genes in Culex in more depth and uncovered 500 immune response-related genes, similar to the numbers seen in Aedes, but fewer than seen in Anopheles or the fruit fly Drosophila melanogaster. The higher numbers of genes were attributed partly to expansions in those encoding serpins, C-type lectins, and fibrinogen-related proteins, consistent with greater immune surveillance and associated signaling needed to monitor the dangers of breeding in polluted, urbanized environments. Transcriptome analysis confirmed that inoculation with unfamiliar bacteria prompted strong immune responses in Culex. The worm and virus pathogens that the mosquitoes transmit naturally provoked little immune activation, however, suggesting that tolerance has evolved to any damage caused by replication of the pathogens in the insects. The genome of a third mosquito species reveals distinctions related to vector capacities and habitat preferences. The mosquito Culex quinquefasciatus poses a substantial threat to human and veterinary health as a primary vector of West Nile virus (WNV), the filarial worm Wuchereria bancrofti, and an avian malaria parasite. Comparative phylogenomics revealed an expanded canonical C. quinquefasciatus immune gene repertoire compared with those of Aedes aegypti and Anopheles gambiae. Transcriptomic analysis of C. quinquefasciatus genes responsive to WNV, W. bancrofti, and non-native bacteria facilitated an unprecedented meta-analysis of 25 vector-pathogen interactions involving arboviruses, filarial worms, bacteria, and malaria parasites, revealing common and distinct responses to these pathogen types in three mosquito genera. Our findings provide support for the hypothesis that mosquito-borne pathogens have evolved to evade innate immune responses in three vector mosquito species of major medical importance.

Anopheles Imd pathway factors and effectors in infection intensity-dependent anti-Plasmodium action.

The Anopheles gambiae immune response against Plasmodium falciparum, an etiological agent of human malaria, has been identified as a source of potential anti-Plasmodium genes and mechanisms to be exploited in efforts to control the malaria transmission cycle. One such mechanism is the Imd pathway, a conserved immune signaling pathway that has potent anti-P. falciparum activity. Silencing the expression of caspar, a negative regulator of the Imd pathway, or over-expressing rel2, an Imd pathway-controlled NFkappaB transcription factor, confers a resistant phenotype on A. gambiae mosquitoes that involves an array of immune effector genes. However, unexplored features of this powerful mechanism that may be essential for the implementation of a malaria control strategy still remain. Using RNA interference to singly or dually silence caspar and other components of the Imd pathway, we have identified genes participating in the anti-Plasmodium signaling module regulated by Caspar, each of which represents a potential target to achieve over-activation of the pathway. We also determined that the Imd pathway is most potent against the parasites ookinete stage, yet also has reasonable activity against early oocysts and lesser activity against late oocysts. We further demonstrated that caspar silencing alone is sufficient to induce a robust anti-P. falciparum response even in the relative absence of resident gut microbiota. Finally, we established the relevance of the Imd pathway components and regulated effectors TEP1, APL1, and LRIM1 in parasite infection intensity-dependent defense, thereby shedding light on the relevance of laboratory versus natural infection intensity models. Our results highlight the physiological considerations that are integral to a thoughtful implementation of Imd pathway manipulation in A. gambiae as part of an effort to limit the malaria transmission cycle, and they reveal a variety of previously unrecognized nuances in the Imd-directed immune response against P. falciparum.

The Gram‐Negative Bacteria‐Binding Protein gene family: Its role in the innate immune system of Anopheles gambiae and in anti‐Plasmodium defence

Gram‐negative bacteria‐binding proteins (GNBPs) are pattern recognition receptors which contribute to the defensive response against Plasmodium infection in Anopheles. We have characterized the GNBP gene family in Anopheles gambiae at the molecular level, and show that they are functionally diverse components of the A. gambiae innate immune system. GNBPB4 is a major factor in the defence against a broad range of pathogens, while the other GNBPs have narrower defence specificities. GNBPB4 is associated with the regulation of immune signalling pathways and was found to interact with the Gram‐negative Escherichia coli and weakly co‐localized with Plasmodium berghei ookinetes in the mosquito midgut epithelium.

Molecular analysis of photic inhibition of blood-feeding in Anopheles gambiae

BackgroundAnopheles gambiae mosquitoes exhibit an endophilic, nocturnal blood feeding behavior. Despite the importance of light as a regulator of malaria transmission, our knowledge on the molecular interactions between environmental cues, the circadian oscillators and the host seeking and feeding systems of the Anopheles mosquitoes is limited.ResultsIn the present study, we show that the blood feeding behavior of mosquitoes is under circadian control and can be modulated by light pulses, both in a clock dependent and in an independent manner. Short light pulses (~2–5 min) in the dark phase can inhibit the blood-feeding propensity of mosquitoes momentarily in a clock independent manner, while longer durations of light stimulation (~1–2 h) can induce a phase advance in blood-feeding propensity in a clock dependent manner. The temporary feeding inhibition after short light pulses may reflect a masking effect of light, an unknown mechanism which is known to superimpose on the true circadian regulation. Nonetheless, the shorter light pulses resulted in the differential regulation of a variety of genes including those implicated in the circadian control, suggesting that light induced masking effects also involve clock components. Light pulses (both short and long) also regulated genes implicated in feeding as well as different physiological processes like metabolism, transport, immunity and protease digestions. RNAi-mediated gene silencing assays of the light pulse regulated circadian factors timeless, cryptochrome and three takeout homologues significantly up-regulated the mosquitos blood-feeding propensity. In contrast, gene silencing of light pulse regulated olfactory factors down-regulated the mosquitos propensity to feed on blood.ConclusionOur study show that the mosquitos feeding behavior is under circadian control. Long and short light pulses can induce inhibition of blood-feeding through circadian and unknown mechanisms, respectively, that involve the chemosensory system.

Engineered Aedes aegypti JAK/STAT Pathway-Mediated Immunity to Dengue Virus

We have developed genetically modified Ae. aegypti mosquitoes that activate the conserved antiviral JAK/STAT pathway in the fat body tissue, by overexpressing either the receptor Dome or the Janus kinase Hop by the blood feeding-induced vitellogenin (Vg) promoter. Transgene expression inhibits infection with several dengue virus (DENV) serotypes in the midgut as well as systemically and in the salivary glands. The impact of the transgenes Dome and Hop on mosquito longevity was minimal, but it resulted in a compromised fecundity when compared to wild-type mosquitoes. Overexpression of Dome and Hop resulted in profound transcriptome regulation in the fat body tissue as well as the midgut tissue, pinpointing several expression signatures that reflect mechanisms of DENV restriction. Our transcriptome studies and reverse genetic analyses suggested that enrichment of DENV restriction factor and depletion of DENV host factor transcripts likely accounts for the DENV inhibition, and they allowed us to identify novel factors that modulate infection. Interestingly, the fat body-specific activation of the JAK/STAT pathway did not result in any enhanced resistance to Zika virus (ZIKV) or chikungunya virus (CHIKV) infection, thereby indicating a possible specialization of the pathway’s antiviral role.

Protocol for Dengue Infections in Mosquitoes (A. aegypti) and Infection Phenotype Determination

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of the virus in the mosquito tissues. This protocol is routinely used for studying mosquito-virus interactions, especially for identification of novel host factors that are able to determine vector competence. The entire experiment must be conducted in a BSL2 laboratory. Similar to Plasmodium falciparum infections, proper attire including gloves and lab coat must be worn at all times. After the experiment, all the materials that came in contact with the virus need to be treated with 75% ethanol and bleached before proceeding with normal washing. All other materials need to be autoclaved before discarding them.

Bacteria- and IMD Pathway-Independent Immune Defenses against Plasmodium falciparum in Anopheles gambiae

The mosquito Anopheles gambiae uses its innate immune system to control bacterial and Plasmodium infection of its midgut tissue. The activation of potent IMD pathway-mediated anti-Plasmodium falciparum defenses is dependent on the presence of the midgut microbiota, which activate this defense system upon parasite infection through a peptidoglycan recognition protein, PGRPLC. We employed transcriptomic and reverse genetic analyses to compare the P. falciparum infection-responsive transcriptomes of septic and aseptic mosquitoes and to determine whether bacteria-independent anti-Plasmodium defenses exist. Antibiotic treated aseptic mosquitoes mounted molecular immune responses representing a variety of immune functions upon P. falciparum infection. Among other immune factors, our analysis uncovered a serine protease inhibitor (SRPN7) and Clip-domain serine protease (CLIPC2) that were transcriptionally induced in the midgut upon P. falciparum infection, independent of bacteria. We also showed that SRPN7 negatively and CLIPC2 positively regulate the anti-Plasmodium defense, independently of the midgut-associated bacteria. Co-silencing assays suggested that these two genes may function together in a signaling cascade. Neither gene was regulated, nor modulated, by infection with the rodent malaria parasite Plasmodium berghei, suggesting that SRPN7 and CLIPC2 are components of a defense system with preferential activity towards P. falciparum. Further analysis using RNA interference determined that these genes do not regulate the anti-Plasmodium defense mediated by the IMD pathway, and both factors act as agonists of the endogenous midgut microbiota, further demonstrating the lack of functional relatedness between these genes and the bacteria-dependent activation of the IMD pathway. This is the first study confirming the existence of a bacteria-independent, anti-P. falciparum defense. Further exploration of this anti-Plasmodium defense will help clarify determinants of immune specificity in the mosquito, and expose potential gene and/or protein targets for malaria intervention strategies based on targeting the parasite in the mosquito vector.

Continuous exposure to Plasmodium results in decreased susceptibility and transcriptomic divergence of the Anopheles gambiae immune system

BackgroundPlasmodium infection has been shown to compromise the fitness of the mosquito vector, reducing its fecundity and longevity. However, from an evolutionary perspective, the impact of Plasmodium infection as a selective pressure on the mosquito is largely unknown.ResultsIn the present study we have addressed the effect of a continuous Plasmodium berghei infection on the resistance to infection and global gene expression in Anopheles gambiae.Exposure of A. gambiae to P. berghei-infected blood and infection for 16 generations resulted in a decreased susceptibility to infection, altered constitutive expression levels for approximately 2.4% of the mosquitos total transcriptome and a lower basal level of immune genes expression, including several anti-Plasmodium factors. The infection-responsiveness for several defense genes was elevated in the P. berghei exposed mosquito colonies.ConclusionOur study establishes the existence of a selective pressure exerted by the parasite P. berghei on the malaria vector A. gambiae that results in a decreased permissiveness to infection and changes in the mosquito transcriptome regulation that suggest a decreased constitutive immune gene activity but a more potent immune response upon Plasmodium challenge.