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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.

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.

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.

Moisture is one of the major causes of problems in buildings, and it can compromise their performance. Infrared thermography (IRT) is a non-destructive testing technology that can be used to assess the humidification phenomenon and, thus, prevent some of the problems caused by moisture. The images obtained by IRT reflect the thermal patterns of the surface under study and can be evaluated using a quantitative approach, which allows not only the traditional visualization of the thermal patterns but also quantification of surface temperatures and/or their differences. The relevance of this work is related to the discussion of the strengths and weaknesses of several methods to quantitatively assess the humidification phenomenon using IRT. For that purpose, the partial humidification by the bottom surface of a lightweight concrete specimen was considered as a case study. To evaluate the thermal gradients, the evolution of the thermal imaging throughout the measurement period and the definition of the areas particularly affected by moisture, a methodology that included a pre-processing phase for data reduction, followed by a data processing phase, were implemented. In the data processing, different statistical and numerical methods were tested. The results of the statistical descriptive analysis highlighted the time variation of the surface temperature, both when considering the entire specimen and when considering only specific areas. The variability of the temperatures at certain moments of the experiment could be observed in the box-plot representation. The image subtraction proved to be an interesting technique to quantify the temperature differences if the first image was used as reference. A thermal index, TI, was proposed to assess the cooling rate. The index highlighted the initial instant when the effect of moisture on the surface temperature was detectable.

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.

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 falciparum . IMPORTANCE 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. 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.

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. 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.

Graphical Abstract

The Plasmodium infection cycle in mosquitoes relies on numerous host factors in the vector midgut, which can be targeted with therapeutics. The mosquito prefoldin complex is needed to fold proteins and macromolecular complexes properly. Here we show that the conserved Anopheles mosquito prefoldin (PFDN)–chaperonin system is a potent transmission-blocking target for multiple Plasmodium species. Silencing any prefoldin subunit or its CCT/TRiC partner via RNA interference reduces Plasmodium falciparum oocyst loads in the mosquito midgut, as does co-feeding mosquitoes with PFDN6-specific antibody and gametocytes. Inhibition of the PFDN–CCT/TRiC chaperonin complex results in the loss of epithelial and extracellular matrix integrity, which triggers microorganism-mediated anti- Plasmodium immune priming and compromises the parasite’s laminin-based immune evasion. Mouse malaria transmission-blocking vaccine and antibody co-feeding assays support its potential as a multispecies transmission-blocking target for P. falciparum and Plasmodium vivax . Further study is needed to determine the potential of this system as a transmission-blocking vaccine target. Disrupting the prefoldin–chaperonin complex in various species of Anopheles mosquitoes results in a compromised intestinal barrier leading to an immune response that inhibits the malaria parasite.

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.