RNA interference is essential to modulating the pathogenesis of mosquito-borne viruses in the yellow fever mosquito Aedes aegypti.

While it has long been known that the transmission of mosquito-borne viruses depends on the establishment of persistent and nonlethal infections in the invertebrate host, specific roles for the insects' antiviral immune pathways in modulating the pathogenesis of viral infections is the subject of speculation and debate. Here, we show that a loss-of-function mutation in the Aedes aegypti Dicer-2 (Dcr-2) gene renders the insect acutely susceptible to a disease phenotype upon infection with pathogens in multiple virus families associated with important human diseases. Additional interrogation of the disease phenotype demonstrated that the virus-induced pathology is controlled through a canonical RNA interference (RNAi) pathway, which functions as a resistance mechanism. These results suggest comparatively modest contributions of proposed tolerance mechanisms to the fitness of A. aegypti infected with these pathogens. Similarly, the production of virus-derived piwi-interacting RNAs (vpiRNAs) was not sufficient to prevent the pathology associated with viral infections in Dcr-2 null mutants, also suggesting a less critical, or potentially secondary, role for vpiRNAs in antiviral immunity. These findings have important implications for understanding the ecological and evolutionary interactions occurring between A. aegypti and the pathogens they transmit to human and animal hosts.

Dual effector population modification gene-drive strains of the African malaria mosquitoes, Anopheles gambiae and Anopheles coluzzii.

Proposed genetic approaches for reducing human malaria include population modification, which introduces genes into vector mosquitoes to reduce or prevent parasite transmission. We demonstrate the potential of Cas9/guide RNA (gRNA)-based gene-drive systems linked to dual antiparasite effector genes to spread rapidly through mosquito populations. Two strains have an autonomous gene-drive system coupled to dual anti-Plasmodium falciparum effector genes comprising single-chain variable fragment monoclonal antibodies targeting parasite ookinetes and sporozoites in the African malaria mosquitoes Anopheles gambiae (AgTP13) and Anopheles coluzzii (AcTP13). The gene-drive systems achieved full introduction within 3 to 6 mo after release in small cage trials. Life-table analyses revealed no fitness loads affecting AcTP13 gene-drive dynamics but AgTP13 males were less competitive than wild types. The effector molecules reduced significantly both parasite prevalence and infection intensities. These data supported transmission modeling of conceptual field releases in an island setting that shows meaningful epidemiological impacts at different sporozoite threshold levels (2.5 to 10 k) for human infection by reducing malaria incidence in optimal simulations by 50 to 90% within as few as 1 to 2 mo after a series of releases, and by ≥90% within 3 mo. Modeling outcomes for low sporozoite thresholds are sensitive to gene-drive system fitness loads, gametocytemia infection intensities during parasite challenges, and the formation of potentially drive-resistant genome target sites, extending the predicted times to achieve reduced incidence. TP13-based strains could be effective for malaria control strategies following validation of sporozoite transmission threshold numbers and testing field-derived parasite strains. These or similar strains are viable candidates for future field trials in a malaria-endemic region.

CRISPR mediated transactivation in the human disease vector Aedes aegypti.

As a major insect vector of multiple arboviruses, Aedes aegypti poses a significant global health and economic burden. A number of genetic engineering tools have been exploited to understand its biology with the goal of reducing its impact. For example, current tools have focused on knocking-down RNA transcripts, inducing loss-of-function mutations, or expressing exogenous DNA. However, methods for transactivating endogenous genes have not been developed. To fill this void, here we developed a CRISPR activation (CRISPRa) system in Ae. aegypti to transactivate target gene expression. Gene expression is activated through pairing a catalytically-inactive ('dead') Cas9 (dCas9) with a highly-active tripartite activator, VP64-p65-Rta (VPR) and synthetic guide RNA (sgRNA) complementary to a user defined target-gene promoter region. As a proof of concept, we demonstrate that engineered Ae. aegypti mosquitoes harboring a binary CRISPRa system can be used to effectively overexpress two developmental genes, even-skipped (eve) and hedgehog (hh), resulting in observable morphological phenotypes. We also used this system to overexpress the positive transcriptional regulator of the Toll immune pathway known as AaRel1, which resulted in a significant suppression of dengue virus serotype 2 (DENV2) titers in the mosquito. This system provides a versatile tool for research pathways not previously possible in Ae. aegypti, such as programmed overexpression of endogenous genes, and may aid in gene characterization studies and the development of innovative vector control tools.

C-type lectin 4 regulates broad-spectrum melanization-based refractoriness to malaria parasites.

Anopheles gambiae melanization-based refractoriness to the human malaria parasite Plasmodium falciparum has rarely been observed in either laboratory or natural conditions, in contrast to the rodent model malaria parasite Plasmodium berghei that can become completely melanized by a TEP1 complement-like system-dependent mechanism. Multiple studies have shown that the rodent parasite evades this defense by recruiting the C-type lectins CTL4 and CTLMA2, while permissiveness to the human malaria parasite was not affected by partial depletion of these factors by RNAi silencing. Using CRISPR/Cas9-based CTL4 knockout, we show that A. gambiae can mount melanization-based refractoriness to the human malaria parasite, which is independent of the TEP1 complement-like system and the major anti-Plasmodium immune pathway Imd. Our study indicates a hierarchical specificity in the control of Plasmodium melanization and proves CTL4 as an essential host factor for P. falciparum transmission and one of the most potent mosquito-encoded malaria transmission-blocking targets.

The Aedes aegypti siRNA pathway mediates broad-spectrum defense against human pathogenic viruses and modulates antibacterial and antifungal defenses.

The mosquito's innate immune system defends against a variety of pathogens, and the conserved siRNA pathway plays a central role in the control of viral infections. Here, we show that transgenic overexpression of Dicer2 (Dcr2) or R2d2 resulted in an accumulation of 21-nucleotide viral sequences that was accompanied by a significant suppression of dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV) replication, thus indicating the broad-spectrum antiviral response mediated by the siRNA pathway that can be applied for the development of novel arbovirus control strategies. Interestingly, overexpression of Dcr2 or R2d2 regulated the mRNA abundance of a variety of antimicrobial immune genes, pointing to additional functions of DCR2 and R2D2 as well as cross-talk between the siRNA pathway and other immune pathways. Accordingly, transgenic overexpression of Dcr2 or R2d2 resulted in a lesser proliferation of the midgut microbiota and increased resistance to bacterial and fungal infections.

mosGILT controls innate immunity and germ cell development in Anopheles gambiae.

Gene-edited mosquitoes lacking a gamma-interferon-inducible lysosomal thiol reductase-like protein, namely (mosGILTnull) have lower Plasmodium infection, which is linked to impaired ovarian development and immune activation. The transcriptome of mosGILTnull Anopheles gambiae was therefore compared to wild type (WT) mosquitoes by RNA-sequencing to delineate mosGILT-dependent pathways. Compared to WT mosquitoes, mosGILTnull A. gambiae demonstrated altered expression of genes related to oogenesis, 20-hydroxyecdysone synthesis, as well as immune-related genes. Serendipitously, the zero population growth gene, zpg, an essential regulator of germ cell development was found to be one of the most downregulated genes in mosGILTnull mosquitoes. These results provide a crucial missing link between two previous studies on the role of zpg and mosGILT in ovarian development. This study further demonstrates that mosGILT has the potential to serve as a target for the biological control of mosquito vectors and to influence the Plasmodium life cycle within the vector.

Correlation between small-cell lung cancer serum protein/peptides determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and chemotherapy efficacy.

BACKGROUND: Currently, no effective measures are available to predict the curative efficacy of small-cell lung cancer (SCLC) chemotherapy. We expect to develop a method for effectively predicting the SCLC chemotherapy efficacy and prognosis in clinical practice in order to offer more pertinent therapeutic protocols for individual patients. METHODS: We adopted matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and ClinPro Tools system to detect serum samples from 154 SCLC patients with different curative efficacy of standard chemotherapy and analyze the different peptides/proteins of SCLC patients to discover predictive tumor markers related to chemotherapy efficacy. Ten peptide/protein peaks were significantly different in the two groups. RESULTS: A genetic algorithm model consisting of four peptides/proteins was developed from the training group to separate patients with different chemotherapy efficacies. Among them, three peptides/proteins (m/z 3323.35, 6649.03 and 6451.08) showed high expression in the disease progression group, whereas the peptide/protein at m/z 4283.18 was highly expressed in the disease response group. The classifier exhibited an accuracy of 91.4% (53/58) in the validation group. The survival analysis showed that the median progression-free survival (PFS) of 30 SCLC patients in disease response group was 9.0 months; in 28 cases in disease progression group, the median PFS was 3.0 months, a statistically significant difference (χ2 = 46.98, P < 0.001). The median overall survival (OS) of the two groups was 13.0 months and 7.0 months, a statistically significant difference (χ2 = 40.64, P < 0.001). CONCLUSIONS: These peptides/proteins may be used as potential biological markers for prediction of the curative efficacy and prognosis for SCLC patients treated with standard regimen chemotherapy.

Glyphosate inhibits melanization and increases susceptibility to infection in insects.

Melanin, a black-brown pigment found throughout all kingdoms of life, has diverse biological functions including UV protection, thermoregulation, oxidant scavenging, arthropod immunity, and microbial virulence. Given melanin's broad roles in the biosphere, particularly in insect immune defenses, it is important to understand how exposure to ubiquitous environmental contaminants affects melanization. Glyphosate-the most widely used herbicide globally-inhibits melanin production, which could have wide-ranging implications in the health of many organisms, including insects. Here, we demonstrate that glyphosate has deleterious effects on insect health in 2 evolutionary distant species, Galleria mellonella (Lepidoptera: Pyralidae) and Anopheles gambiae (Diptera: Culicidae), suggesting a broad effect in insects. Glyphosate reduced survival of G. mellonella caterpillars following infection with the fungus Cryptococcus neoformans and decreased the size of melanized nodules formed in hemolymph, which normally help eliminate infection. Glyphosate also increased the burden of the malaria-causing parasite Plasmodium falciparum in A. gambiae mosquitoes, altered uninfected mosquito survival, and perturbed the microbial composition of adult mosquito midguts. Our results show that glyphosate's mechanism of melanin inhibition involves antioxidant synergy and disruption of the reaction oxidation-reduction balance. Overall, these findings suggest that glyphosate's environmental accumulation could render insects more susceptible to microbial pathogens due to melanin inhibition, immune impairment, and perturbations in microbiota composition, potentially contributing to declines in insect populations.

Broad spectrum immunomodulatory effects of Anopheles gambiae microRNAs and their use for transgenic suppression of Plasmodium.

Malaria, caused by the protozoan parasite Plasmodium and transmitted by Anopheles mosquitoes, represents a major threat to human health. Plasmodium's infection cycle in the Anopheles vector is critical for transmission of the parasite between humans. The midgut-stage bottleneck of infection is largely imposed by the mosquito's innate immune system. microRNAs (miRNAs, small noncoding RNAs that bind to target RNAs to regulate gene expression) are also involved in regulating immunity and the anti-Plasmodium defense in mosquitoes. Here, we characterized the mosquito's miRNA responses to Plasmodium infection using an improved crosslinking and immunoprecipitation (CLIP) method, termed covalent ligation of endogenous Argonaute-bound RNAs (CLEAR)-CLIP. Three candidate miRNAs' influence on P. falciparum infection and midgut microbiota was studied through transgenically expressed miRNA sponges (miR-SPs) in midgut and fat body tissues. MiR-SPs mediated conditional depletion of aga-miR-14 or aga-miR-305, but not aga-miR-8, increased mosquito resistance to both P. falciparum and P. berghei infection, and enhanced the mosquitoes' antibacterial defenses. Transcriptome analysis revealed that depletion of aga-miR-14 or aga-miR-305 resulted in an increased expression of multiple immunity-related and anti-Plasmodium genes in mosquito midguts. The overall fitness cost of conditionally expressed miR-SPs was low, with only one of eight fitness parameters being adversely affected. Taken together, our results demonstrate that targeting mosquito miRNA by conditional expression of miR-SPs may have potential for the development of malaria control through genetically engineered mosquitoes.

CRISPR/Cas9 -mediated gene knockout of Anopheles gambiae FREP1 suppresses malaria parasite infection.

Plasmodium relies on numerous agonists during its journey through the mosquito vector, and these agonists represent potent targets for transmission-blocking by either inhibiting or interfering with them pre- or post-transcriptionally. The recently developed CRISPR/Cas9-based genome editing tools for Anopheles mosquitoes provide new and promising opportunities for the study of agonist function and for developing malaria control strategies through gene deletion to achieve complete agonist inactivation. Here we have established a modified CRISPR/Cas9 gene editing procedure for the malaria vector Anopheles gambiae, and studied the effect of inactivating the fibrinogen-related protein 1 (FREP1) gene on the mosquito's susceptibility to Plasmodium and on mosquito fitness. FREP1 knockout mutants developed into adult mosquitoes that showed profound suppression of infection with both human and rodent malaria parasites at the oocyst and sporozoite stages. FREP1 inactivation, however, resulted in fitness costs including a significantly lower blood-feeding propensity, fecundity and egg hatching rate, a retarded pupation time, and reduced longevity after a blood meal.

Anopheles gambiae Lacking AgTRIO Inefficiently Transmits Plasmodium berghei to Mice.

Antibodies to AgTRIO, a mosquito salivary protein, partially reduce the initial Plasmodium burden in mice. We therefore silenced AgTRIO in mosquitoes and determined the relative contribution of AgTRIO to the ability of Anopheles gambiae to transmit Plasmodium berghei to mice. RNA interference-mediated silencing of AgTRIO inA. gambiae resulted in a 60% reduction in AgTRIO expression. The decrease in AgTRIO expression did not alter the burden of Plasmodium sporozoites in mosquito salivary glands. When experimentally injected into mice, sporozoites from AgTRIO-silenced mosquitoes colonized the liver less effectively than sporozoites from control mosquitoes. Silencing of AgTRIO did not decrease the infectivity of sporozoites in vitro or influence the expression of genes associated with Plasmodium cell adhesion or traversal activity. AgTRIO decreased the expression of proinflammation cytokines by splenocytes in vitro Moreover, in vivo, AgTRIO decreased the expression of TNF-α when coinjected with sporozoites into the skin and there was more TNF-α expression at the bite site of AgTRIO knockdown mosquitoes than at the bite site of control mosquitoes. AgTRIO therefore influences the local environment in the vertebrate host, which facilitates Plasmodium sporozoite infection in mice.

Cytoplasmic actin is an extracellular insect immune factor which is secreted upon immune challenge and mediates phagocytosis and direct killing of bacteria, and is a Plasmodium Antagonist.

Actin is a highly versatile, abundant, and conserved protein, with functions in a variety of intracellular processes. Here, we describe a novel role for insect cytoplasmic actin as an extracellular pathogen recognition factor that mediates antibacterial defense. Insect actins are secreted from cells upon immune challenge through an exosome-independent pathway. Anopheles gambiae actin interacts with the extracellular MD2-like immune factor AgMDL1, and binds to the surfaces of bacteria, mediating their phagocytosis and direct killing. Globular and filamentous actins display distinct functions as extracellular immune factors, and mosquito actin is a Plasmodium infection antagonist.

Anopheles Midgut FREP1 Mediates Plasmodium Invasion.

Malaria transmission depends on sexual stage Plasmodium parasites successfully invading Anopheline mosquito midguts following a blood meal. However, the molecular mechanisms of Plasmodium invasion of mosquito midguts have not been fully elucidated. Previously, we showed that genetic polymorphisms in the fibrinogen-related protein 1 (FREP1) gene are significantly associated with Plasmodium falciparum infection in Anopheles gambiae, and FREP1 is important for Plasmodium berghei infection of mosquitoes. Here we identify that the FREP1 protein is secreted from the mosquito midgut epithelium and integrated as tetramers into the peritrophic matrix, a chitinous matrix formed inside the midgut lumen after a blood meal feeding. Moreover, we show that the FREP1 can directly bind Plasmodia sexual stage gametocytes and ookinetes. Notably, ablating FREP1 expression or targeting FREP1 with antibodies significantly decreases P. falciparum infection in mosquito midguts. Our data support that the mosquito-expressed FREP1 mediates mosquito midgut invasion by multiple species of Plasmodium parasites via anchoring ookinetes to the peritrophic matrix and enabling parasites to penetrate the peritrophic matrix and the epithelium. Thus, targeting FREP1 can limit malaria transmission.

Chromobacterium Csp_P reduces malaria and dengue infection in vector mosquitoes and has entomopathogenic and in vitro anti-pathogen activities.

Plasmodium and dengue virus, the causative agents of the two most devastating vector-borne diseases, malaria and dengue, are transmitted by the two most important mosquito vectors, Anopheles gambiae and Aedes aegypti, respectively. Insect-bacteria associations have been shown to influence vector competence for human pathogens through multi-faceted actions that include the elicitation of the insect immune system, pathogen sequestration by microbes, and bacteria-produced anti-pathogenic factors. These influences make the mosquito microbiota highly interesting from a disease control perspective. Here we present a bacterium of the genus Chromobacterium (Csp_P), which was isolated from the midgut of field-caught Aedes aegypti. Csp_P can effectively colonize the mosquito midgut when introduced through an artificial nectar meal, and it also inhibits the growth of other members of the midgut microbiota. Csp_P colonization of the midgut tissue activates mosquito immune responses, and Csp_P exposure dramatically reduces the survival of both the larval and adult stages. Ingestion of Csp_P by the mosquito significantly reduces its susceptibility to Plasmodium falciparum and dengue virus infection, thereby compromising the mosquito's vector competence. This bacterium also exerts in vitro anti-Plasmodium and anti-dengue activities, which appear to be mediated through Csp_P -produced stable bioactive factors with transmission-blocking and therapeutic potential. The anti-pathogen and entomopathogenic properties of Csp_P render it a potential candidate for the development of malaria and dengue control strategies.

Impact of trehalose transporter knockdown on Anopheles gambiae stress adaptation and susceptibility to Plasmodium falciparum infection.

Anopheles gambiae is a major vector mosquito for Plasmodium falciparum, the deadly pathogen causing most human malaria in sub-Saharan Africa. Synthesized in the fat body, trehalose is the predominant sugar in mosquito hemolymph. It not only provides energy but also protects the mosquito against desiccation and heat stresses. Trehalose enters the mosquito hemolymph by the trehalose transporter AgTreT1. In adult female A. gambiae, AgTreT1 is predominantly expressed in the fat body. We found that AgTreT1 expression is induced by environmental stresses such as low humidity or elevated temperature. AgTreT1 RNA silencing reduces the hemolymph trehalose concentration by 40%, and the mosquitoes succumb sooner after exposure to desiccation or heat. After an infectious blood meal, AgTreT1 RNA silencing reduces the number of P. falciparum oocysts in the mosquito midgut by over 70% compared with mock-injected mosquitoes. These data reveal important roles for AgTreT1 in stress adaptation and malaria pathogen development in a major vector mosquito. Thus, AgTreT1 may be a potential target for malaria vector control.

Exploring Anopheles gut bacteria for Plasmodium blocking activity.

Malaria parasite transmission requires the successful development of Plasmodium gametocytes into flagellated microgametes upon mosquito blood ingestion, and the subsequent fertilization of microgametes and macrogametes for the development of motile zygotes, called ookinetes, which invade and transverse the Anopheles vector mosquito midgut at around 18-36 h after blood ingestion. Within the mosquito midgut, the malaria parasite has to withstand the mosquito's innate immune response and the detrimental effect of its commensal bacterial flora. We have assessed the midgut colonization capacity of five gut bacterial isolates from field-derived, and two from laboratory colony, mosquitoes and their effect on Plasmodium development in vivo and in vitro, along with their impact on mosquito survival. Some bacterial isolates activated the mosquito's immune system, affected the mosquito's lifespan, and were capable of blocking Plasmodium development. We have also shown that the ability of these bacteria to inhibit the parasites is likely to involve different mechanisms and factors. A Serratia marcescens isolate was particularly efficient in colonizing the mosquitoes' gut, compromising mosquito survival and inhibiting both Plasmodium sexual- and asexual-stage through secreted factors, thereby rendering it a potential candidate for the development of a malaria transmission intervention strategy.

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 parasite's 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.

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 mosquito's 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.

Development of an anti-Pfs230 monoclonal antibody as a Plasmodium falciparum gametocyte blocker.

Vector control is a crucial strategy for malaria elimination by preventing infection and reducing disease transmission. Most gains have been achieved through insecticide-treated nets (ITNs) and indoor residual spraying (IRS), but the emergence of insecticide resistance among Anopheles mosquitoes calls for new tools to be applied. Here, we present the development of a highly effective murine monoclonal antibody, targeting the N-terminal region of the Plasmodium falciparum gametocyte antigen Pfs230, that can decrease the infection prevalence by > 50% when fed to Anopheles mosquitoes with gametocytes in an artificial membrane feeding system. We used a standard mouse immunization protocol followed by protein interaction and parasite-blocking validation at three distinct stages of the monoclonal antibody development pipeline: post-immunization, post-hybridoma generation, and final validation of the monoclonal antibody. We evaluated twenty antibodies identifying one (mAb 13G9) with high Pfs230-affinity and parasite-blocking activity. This 13G9 monoclonal antibody could potentially be developed into a transmission-blocking single-chain antibody for expression in transgenic mosquitoes.