Transgenic approaches in medical entomology: 2022 highlights.

Transgenesis has emerged as a powerful tool to control arthropod vectors and the diseases they transmit. Here, we highlight the latest developments on transgenic approaches in ticks, Anopheles and Aedes mosquitoes, based on recent findings and significant papers from 2022. We survey topics ranging from population replacement, population suppression, gene drive, sex ratio distortion, public engagement and capacity building, and gene editing in ticks. While presenting these advancements, we discuss the current challenges surrounding the application of arthropod transgenesis for the development of novel vector control strategies.

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.

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.

Mosquito transgenesis for malaria control.

Malaria is one of the deadliest diseases. Because of the ineffectiveness of current malaria-control methods, several novel mosquito vector-based control strategies have been proposed to supplement existing control strategies. Mosquito transgenesis and gene drive have emerged as promising tools for preventing the spread of malaria by either suppressing mosquito populations by self-destructing mosquitoes or replacing mosquito populations with disease-refractory populations. Here we review the development of mosquito transgenesis and its application for malaria control, highlighting the transgenic expression of antiparasitic effector genes, inactivation of host factor genes, and manipulation of miRNAs and lncRNAs. Overall, from a malaria-control perspective, mosquito transgenesis is not envisioned as a stand-alone approach; rather, its use is proposed as a complement to existing vector-control strategies.

Additional Feeding Reveals Differences in Immune Recognition and Growth of Plasmodium Parasites in the Mosquito Host.

Mosquitoes may feed multiple times during their life span in addition to those times needed to acquire and transmit malaria. To determine the impact of subsequent blood feeding on parasite development in Anopheles gambiae, we examined Plasmodium parasite infection with or without an additional noninfected blood meal. We found that an additional blood meal significantly reduced Plasmodium berghei immature oocyst numbers, yet had no effect on the human parasite Plasmodium falciparum These observations were reproduced when mosquitoes were fed an artificial protein meal, suggesting that parasite losses are independent of blood ingestion. We found that feeding with either a blood or protein meal compromises midgut basal lamina integrity as a result of the physical distention of the midgut, enabling the recognition and lysis of immature P. berghei oocysts by mosquito complement. Moreover, we demonstrate that additional feeding promotes P. falciparum oocyst growth, suggesting that human malaria parasites exploit host resources provided with blood feeding to accelerate their growth. This is in contrast to experiments with P. berghei, where the size of surviving oocysts is independent of an additional blood meal. Together, these data demonstrate distinct differences in Plasmodium species in evading immune detection and utilizing host resources at the oocyst stage, representing an additional, yet unexplored component of vectorial capacity that has important implications for the transmission of malaria.IMPORTANCE Mosquitoes must blood feed multiple times to acquire and transmit malaria. However, the impact of an additional mosquito blood meal following malaria parasite infection has not been closely examined. Here, we demonstrate that additional feeding affects mosquito vector competence; namely, additional feeding significantly limits Plasmodium berghei infection, yet has no effect on infection of the human parasite P. falciparum Our experiments support that these killing responses are mediated by the physical distension of the midgut and by temporary damage to the midgut basal lamina that exposes immature P. berghei oocysts to mosquito complement, while human malaria parasites are able to evade these killing mechanisms. In addition, we provide evidence that additional feeding promotes P. falciparum oocyst growth. This is in contrast to P. berghei, where oocyst size is independent of an additional blood meal. This suggests that human malaria parasites are able to exploit host resources provided by an additional feeding to accelerate their growth. In summary, our data highlight distinct differences in malaria parasite species in evading immune recognition and adapting to mosquito blood feeding. These observations have important, yet previously unexplored, implications for the impact of multiple blood meals on the transmission of malaria.

Emissivity of Building Materials for Infrared Measurements.

Infrared thermography (IRT) is a technique increasingly used in building inspection. If in many applications it is sufficient to analyze the thermal patterns, others exist in which the exact determination of the surface temperature is a fundamental aspect. In these circumstances, the emissivity of the surfaces assumes special relevance, being probably the most important property in the definition of the boundary conditions. However, information on the uncertainty involved in its measurement, as well as the conditions that influence it, is scarce. This article presents an innovative contribution both to the characterization of the emissivity of various construction materials, and to the discussion of emissivity measurement procedures and the attendant uncertainty. In this sense, three experimental campaigns were carried out: T.I, preliminary tests to assess the initial conditions required for an accurate IRT measurement of the emissivity (reference tape and position of the camera); T.II, assessment of the emissivity of nine different building materials, in dry conditions, using the emissometer and the IRT and black tape methods; and T.III, assessment of the emissivity of three materials during the drying process. The results confirmed that emissivity is a crucial parameter for the accurate measurement of surface temperature. Emissivity measurements carried out with IRT (black tape method) and with the emissometer returned meaningful differences when compared with the values available in the literature. This disagreement led to surface temperature differences of up to 7 °C (emissometer versus reference values). This research also highlighted that the moisture content of the materials influences the emissivity values, with fluctuations that can be greater than 10%, and that the effect of moisture is visible even for low values of moisture content.

Versatile transgenic multistage effector-gene combinations for Plasmodium falciparum suppression in Anopheles.

The malaria parasite's complex journey through the Anopheles mosquito vector provides multiple opportunities for targeting Plasmodium with recombinant effectors at different developmental stages and different host tissues. We have designed and expressed transgenes that efficiently suppress Plasmodium infection by targeting the parasite with multiple independent endogenous and exogenous effectors at multiple infection stages to potentiate suppression and minimize the probability for development of resistance to develop. We have also addressed the fitness impact of transgene expression on the mosquito. We show that highly potent suppression can be achieved by targeting both pre-oocyst stages by transgenically overexpressing either the endogenous immune deficiency immune pathway transcription factor Rel2 or a polycistronic mRNA encoding multiple antiparasitic effectors and simultaneously targeting the sporozoite stages with an anti-sporozoite single-chain antibody fused to the antiparasitic protein Scorpine. Expression of the selected endogenous effector systems appears to pose a lower fitness cost than does the use of foreign genes.

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.

Diverse Host and Restriction Factors Regulate Mosquito-Pathogen Interactions.

Mosquitoes transmit diseases that seriously impact global human health. Despite extensive knowledge of the life cycles of mosquito-borne parasites and viruses within their hosts, control strategies have proven insufficient to halt their spread. An understanding of the relationships established between such pathogens and the host tissues they inhabit is therefore paramount for the development of new strategies that specifically target these interactions, to prevent the pathogens' maturation and transmission. Here we present an updated account of the antagonists and host factors that affect the development of Plasmodium, the parasite causing malaria, and mosquito-borne viruses, such as dengue virus and Zika virus, within their mosquito vectors, and we discuss the similarities and differences between Plasmodium and viral systems, looking toward the elucidation of new targets for disease control.

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.

New rapid one-step PCR diagnostic assay for Plasmodium falciparum infective mosquitoes.

An essential component of malaria vector control programmes is the detection of Plasmodium falciparum within its mosquito vectors, particularly in the salivary glands where the infective sporozoites reside. Several protocols have been developed for this purpose; however they require dissection of mosquito specimens prior to analysis. Here, a novel one-step RT-qPCR TaqMan diagnostic assay was developed for mosquitoes with infective Plasmodium falciparum sporozoites in the salivary glands. It is based on detection of the sporozoite-specific Pfslarp and Pfplp1 gene transcripts. These transcripts were chosen based on bioinformatics analysis, and experimentally verified to be overexpressed in the salivary gland sporozoite stage of the parasite compared to other mosquito parasite stages. The proof of principle and the performance of the assay were demonstrated using RNAlater preserved mosquito samples. Tests of analytical sensitivity showed the novel TaqMan assay to be 100% accurate, although its performance in the field needs to be further demonstrated. This method has no requirement for dissection and post-PCR processing and thus is simple and rapid to perform in individual mosquitoes or mosquito pools. It can be used in single or multiplex formats also targeting additional markers expressed in different tissues, such as detoxification enzymes associated with insecticide resistance.

Immune Regulation of Plasmodium Is Anopheles Species Specific and Infection Intensity Dependent.

Malaria parasite ookinetes must traverse the vector mosquito midgut epithelium to transform into sporozoite-producing oocysts. The Anopheles innate immune system is a key regulator of this process, thereby determining vector competence and disease transmission. The role of Anopheles innate immunity factors as agonists or antagonists of malaria parasite infection has been previously determined using specific single Anopheles-Plasmodium species combinations. Here we show that the two C-type lectins CTL4 and CTLMA2 exert differential agonistic and antagonistic regulation of parasite killing in African and South American Anopheles species. The C-type lectins regulate both parasite melanization and lysis through independent mechanisms, and their implication in parasite melanization is dependent on infection intensity rather than mosquito-parasite species combination. We show that the leucine-rich repeat protein LRIM1 acts as an antagonist on the development of Plasmodium ookinetes and as a regulator of oocyst size and sporozoite production in the South American mosquito Anopheles albimanus Our findings explain the rare observation of human Plasmodium falciparum melanization and define a key factor mediating the poor vector competence of Anopheles albimanus for Plasmodium berghei and Plasmodium falciparumIMPORTANCE Malaria, one of the world's deadliest diseases, is caused by Plasmodium parasites that are vectored to humans by the bite of Anopheles mosquitoes. The mosquito's innate immune system is actively engaged in suppressing Plasmodium infection. Studies on mosquito immunity revealed multiple factors that act as either facilitators or inhibitors of Plasmodium infection, but these findings were mostly based on single Anopheles-Plasmodium species combinations, not taking into account the diversity of mosquito and parasite species. We show that the functions of CTL4 and CTLMA2 have diverged in different vector species and can be both agonistic and antagonistic for Plasmodium infection. Their protection against parasite melanization in Anopheles gambiae is dependent on infection intensity, rather than the mosquito-parasite combination. Importantly, we describe for the first time how LRIM1 plays an essential role in Plasmodium infection of Anopheles albimanus, suggesting it is a key regulator of the poor vector competence of this species.

The Anopheles FBN9 immune factor mediates Plasmodium species-specific defense through transgenic fat body expression.

Mosquitoes have a multifaceted innate immune system that is actively engaged in warding off various pathogens, including the protozoan malaria parasite Plasmodium. Various immune signaling pathways and effectors have been shown to mediate a certain degree of defense specificity against different Plasmodium species. A key pattern recognition receptor of the Anopheles gambiae immune system is the fibrinogen domain-containing immunolectin FBN9, which has been shown to be transcriptonally induced by Plasmodium infection, and to mediate defense against both rodent and human malaria parasites and bacteria. Here we have further studied the defense specificity of FBN9 using a transgenic approach, in which FBN9 is overexpressed in the fat body tissue after a blood meal through a vitellogenin promoter. Interestingly, the Vg-FBN9 transgenic mosquitoes showed increased resistance only to the rodent parasite P. berghei, and not to the human parasite P. falciparum, pointing to differences in the mosquito's defense mechanisms against the two parasite species. The Vg-FBN9 transgenic mosquitoes were also more resistant to infection with both Gram-positive and Gram-negative bacteria and showed increased longevity when infected with P. berghei. Our study points to the importance of both experimentally depleting and enriching candidate anti-Plasmodium effectors in functional studies in order to ascertain their suitability for the development of transgenic mosquito-based malaria control strategies.

Mosquito gut antiparasitic and antiviral immunity.

Mosquitoes are responsible for the transmission of diseases with a serious impact on global human health, such as malaria and dengue. All mosquito-transmitted pathogens complete part of their life cycle in the insect gut, where they are exposed to mosquito-encoded barriers and active factors that can limit their development. Here we present the current understanding of mosquito gut immunity against malaria parasites, filarial worms, and viruses such as dengue, Chikungunya, and West Nile. The most recently proposed immune mediators involved in intestinal defenses are discussed, as well as the synergies identified between the recognition of gut microbiota and the mounting of the immune response.

A mosquito mediator of parasite-induced immune priming.

Immune memory is a central feature of the mammalian adaptive immune system. The more primitive innate immune system of insects has also been shown to comprise memory, or immune priming. A recent study has shed new light on how Plasmodium primes the mosquito immune system for greater resistance to a subsequent infection with the same pathogen.

Hemozoin activates the innate immune system and reduces Plasmodium berghei infection in Anopheles gambiae.

BACKGROUND: Malaria is a worldwide infectious disease caused by Plasmodium parasites and transmitted by female Anopheles mosquitoes. The malaria vector mosquito Anopheles can trigger effective mechanisms to control completion of the Plasmodium lifecycle; the mosquito immune response to the parasite involves several pathways which are not yet well characterized. Plasmodium metabolite hemozoin has emerged as a potent immunostimulator of mammalian tissues. In this study, we aim to investigate the role of this parasite's by-product as stimulator of Anopheles gambiae immunity to Plasmodium berghei. METHODS: Female mosquitoes were inoculated with hemozoin and the Plasmodium infection rate and intensity were measured. Differences between treatments were detected by Zero-inflated models. Microarray transcription analysis was performed to assess gene expression response to hemozoin. Genome-wide analysis results were confirmed by stimulation of Anopheles gambiae tissues and cells with hemozoin and silencing of REL2-F and its negative regulator Caspar. RESULTS: Gene expression profiles revealed that hemozoin activates several immunity genes, including pattern recognition receptors (PRRs) and antimicrobial peptides (AMPs). Importantly, we found that the Immune deficiency (Imd) pathway Nuclear Factor-kappaB (NF-κB) transcription factor REL2, in its full-length form REL2-F, was induced upon hemozoin treatment. CONCLUSIONS: We have for the first time shown the impact of hemozoin treatment in Plasmodium infection, reducing both rate and intensity of the infection. We propose that hemozoin boosts the innate immunity in Anopheles, activating key effector genes involved in mosquito resistance to Plasmodium, and this activation is REL2-mediated.

Human CHCHD4 mitochondrial proteins regulate cellular oxygen consumption rate and metabolism and provide a critical role in hypoxia signaling and tumor progression.

Increased expression of the regulatory subunit of HIFs (HIF-1α or HIF-2α) is associated with metabolic adaptation, angiogenesis, and tumor progression. Understanding how HIFs are regulated is of intense interest. Intriguingly, the molecular mechanisms that link mitochondrial function with the HIF-regulated response to hypoxia remain to be unraveled. Here we describe what we believe to be novel functions of the human gene CHCHD4 in this context. We found that CHCHD4 encodes 2 alternatively spliced, differentially expressed isoforms (CHCHD4.1 and CHCHD4.2). CHCHD4.1 is identical to MIA40, the homolog of yeast Mia40, a key component of the mitochondrial disulfide relay system that regulates electron transfer to cytochrome c. Further analysis revealed that CHCHD4 proteins contain an evolutionarily conserved coiled-coil-helix-coiled-coil-helix (CHCH) domain important for mitochondrial localization. Modulation of CHCHD4 protein expression in tumor cells regulated cellular oxygen consumption rate and metabolism. Targeting CHCHD4 expression blocked HIF-1α induction and function in hypoxia and resulted in inhibition of tumor growth and angiogenesis in vivo. Overexpression of CHCHD4 proteins in tumor cells enhanced HIF-1α protein stabilization in hypoxic conditions, an effect insensitive to antioxidant treatment. In human cancers, increased CHCHD4 expression was found to correlate with the hypoxia gene expression signature, increasing tumor grade, and reduced patient survival. Thus, our study identifies a mitochondrial mechanism that is critical for regulating the hypoxic response in tumors.