Mosquito Hemolymph Research Articles

Full maturation of in vitro Plasmodium falciparum oocysts using the AlgiMatrix 3D culture system

BackgroundPlasmodium falciparum oocysts undergo growth and maturation in a unique setting within the mosquito midgut, firmly situated between the epithelium and the basal lamina. This location exposes them to specific nutrient exchange and metabolic processes while in direct contact with the mosquito haemolymph. The limited availability of in vitro culture systems for growth of the various P. falciparum mosquito stages hampers study of their biology and impedes progress in combatting malaria.MethodsAn artificial in vitro environment was established to mimic this distinctive setting, transitioning from a 2D culture system to a 3D model capable of generating fully mature oocysts that give rise to in vitro sporozoites.ResultsA two-dimensional (2D) chamber slide was employed along with an extracellular matrix composed of type IV collagen, entactin, and gamma laminin. This matrix facilitated development of the optimal medium composition for cultivating mature P. falciparum oocysts in vitro. However, the limitations of this 2D culture system in replicating the in vivo oocyst environment prompted a refinement of the approach by optimizing a three-dimensional (3D) alginate matrix culture system. This new system offered improved attachment, structural support, and nutrient exchange for the developing oocysts, leading to their maturation and the generation of sporozoites.ConclusionsThis technique enables the in vitro growth of P. falciparum oocysts and sporozoites.

Killing of Plasmodium Sporozoites by Basic Amphipathic α-Helical Fusion Peptides.

Membranolytic molecules constitute the first line of innate immune defense against pathogenic microorganisms. Plasmodium sporozoites are potentially exposed to these cytotoxic molecules in the hemolymph and salivary glands of mosquitoes, as well as in the skin, blood, and liver of the mammalian host. Here, we show that sporozoites are resistant to bacteriolytic concentration of cecropin B, a cationic amphipathic antimicrobial insect peptide. Intriguingly, anti-tumoral cell-penetrating peptides derived from the anti-apoptotic protein AAC11 killed P. berghei and P. falciparum sporozoites. Using dynamic imaging, we demonstrated that the most cytotoxic peptide, called RT39, did not significantly inhibit the sporozoite motility until the occurrence of a fast permeabilization of the parasite membrane by the peptide. Concomitantly, the cytosolic fluorescent protein constitutively expressed by sporozoites leaked from the treated parasite body while To-Pro 3 and FITC-labeled RT39 internalized, respectively, binding to the nucleic acids and membranes of sporozoites. This led to an increase in the parasite granularity as assessed by flow cytometry. Most permeabilization events started at the parasite's posterior end, resulting in the appearance of a fluorescent dot in the anterior part of sporozoites. Understanding and exploiting the susceptibility of sporozoites and other plasmodial stages to membranolytic molecules might foster strategies to eliminate the parasite and block its transmission.

A zone-of-inhibition assay to screen for humoral antimicrobial activity in mosquito hemolymph.

In insects, antibacterial immunity largely depends on the activation of downstream signaling and effector responses, leading to the synthesis and secretion of soluble effector molecules, such as antimicrobial peptides (AMPs). AMPs are acute infection response peptides secreted into the hemolymph upon bacterial stimulation. The transcription of innate immunity genes encoding for AMPs is highly dependent on several signaling cascade pathways, such as the Toll pathway. In the African malaria mosquito, Anopheles gambiae, AMPs hold a special interest as their upregulation have been shown to limit the growth of malaria parasites, bacteria, and fungi. Most of the current knowledge on the regulation of insect AMPs in microbial infection have been obtained from Drosophila. However, largely due to the lack of convenient assays, the regulation of antimicrobial activity in mosquito hemolymph is still not completely understood. In this study, we report a zone of inhibition assay to identify the contribution of AMPs and components of the Toll pathway to the antimicrobial activity of A. gambiae hemolymph. As a proof of principle, we demonstrate that Micrococcus luteus challenge induces antimicrobial activity in the adult female mosquito hemolymph, which is largely dependent on defensin 1. Moreover, by using RNAi to silence Cactus, REL1, and MyD88, we showed that Cactus kd induces antimicrobial activity in the mosquito hemolymph, whereas the antimicrobial activity in REL1 kd and MyD88 kd is reduced after challenge. Finally, while injection itself is not sufficient to induce antimicrobial activity, our results show that it primes the response to bacterial challenge. Our study provides information that increases our knowledge of the regulation of antimicrobial activity in response to microbial infections in mosquitoes. Furthermore, this assay represents an ex vivo medium throughput assay that can be used to determine the upstream regulatory elements of antimicrobial activity in A. gambiae hemolymph.

General control nonderepressible 1 interacts with cationic amino acid transporter 1 and affects Aedes aegypti fecundity

BackgroundThe amino acid transporter protein cationic amino acid transporter 1 (CAT1) is part of the nutrient sensor in the fat body of mosquitoes. A member of the SLC7 family of cationic amino acid transporters, it is paramount for the detection of elevated amino acid levels in the mosquito hemolymph after a blood meal and the subsequent changes in gene expression in the fat body.MethodsWe performed a re-annotation of Aedes aegypti cationic amino acid transporters (CATs) and selected the C-terminal tail of CAT1 to perform a yeast two-hybrid screen to identify putative interactors of this protein. One interesting interacting protein we identified was general control nonderepressible 1 (GCN1). We determined the expression pattern of GCN1 in several adult organs and structures using qRT-PCR and western blots. Finally, we knocked down GCN1 using double-stranded RNA and identified changes in downstream signaling intermediates and the effects of knockdown on vitellogenesis and fecundity.ResultsIn a screen for Ae. aegypti CAT1-interacting proteins we identified GCN1 as a putative interactor. GCN1 is highly expressed in the ovaries and fat body of the mosquito. We provide evidence that eukaryotic translation initiation factor 2 subunit alpha (eIF2α) phosphorylation changed during vitellogenesis and that RNA interference knockdown of GCN1 in whole mosquitoes reduced egg clutch sizes of treated mosquitoes relative to controls.ConclusionsAedes aegypti CAT1 and GCN1 are likely interacting partners and GCN1 is likely necessary for proper egg development. Our data suggest that GCN1 is part of a nutrient sensor mechanism in various mosquito tissues involved in vitellogenesis.Graphical

An ex vivo system for investigation of Plasmodium berghei invasion of the salivary gland of Anopheles stephensi mosquitoes

ABSTRACT Plasmodium sporozoites associated with the midgut and in the hemolymph of mosquitoes differ from sporozoites in the secretory cavities and ducts of the insects’ salivary glands in their transcriptome, proteome, motility, and infectivity. Using an ex vivo Anopheles stephensi salivary gland culture system incorporating simple microfluidics and transgenic Plasmodium berghei with the fluorescent protein gene mCherry under the transcriptional control of the Pbuis4 promoter whose expression served as a proxy for parasite maturation, we observed rapid parasite maturation in the absence of salivary gland invasion. While in vivo Pbuis4::mCherry expression was only detectable in sporozoites within the salivary glands (mature parasites) as expected, the simple exposure of P. berghei sporozoites to dissected salivary glands led to rapid parasite maturation as indicated by mCherry expression. These results suggest that previous efforts to develop ex vivo and in vitro systems for investigating sporozoite interactions with mosquito salivary glands have likely been unsuccessful in part because the maturation of sporozoites leads to a loss in the ability to invade salivary glands.

Hemocyte-specific FREP13 abrogates the exogenous bacterial population in the hemolymph and promotes midgut endosymbionts in Anopheles stephensi.

The immune blood cells "hemocytes" of mosquitoes impart a highly selective immune response against various microorganisms/pathogens. Among several immune effectors, fibrinogen-related proteins (FREPs) have been recognized as key modulators of cellular immune responses; however, their physiological relevance has not been investigated in detail. Our ongoing comparative RNA-sequencing analysis identified a total of 13 FREPs originating from naïve sugar-fed, blood-fed, bacterial challenged and Plasmodium vivax-infected hemocytes in Anopheles stephensi. Transcriptional profiling of the selected seven FREP transcripts showed distinct responses against different pathophysiological conditions, where an exclusive induction of FREP12 after 10days of P.vivax infection was observed. This represents a possible role of FREP12 in immunity against free circulating sporozoites and needs to be explored in the future. When challenged with live bacterial injection in the thorax, we observed a higher affinity of FREP13 and FREP65 toward Gram-negative and Gram-positive bacteria in the mosquito hemocytes, respectively. Furthermore, we observed increased bacterial survival and proliferation, which is likely compromised by the downregulation of TEP1, in FREP13 messenger RNA-depleted mosquito hemolymph. In contrast, after blood-feeding, we also noticed a significant delay of 24h in the enrichment of gut endosymbionts in the FREP13-silenced mosquitoes. Taken together, we conclude that hemocyte-specific FREP13 carries the unique ability of tissue-specific regulation, having an antagonistic antibacterial role in the hemolymph, and an agonistic role against gut endosymbionts.

Mosquito Innate Immunity

Mosquitoes live under the endless threat of infections from different kinds of pathogens such as bacteria, parasites, and viruses. The mosquito defends itself by employing both physical and physiological barriers that resist the entry of the pathogen and the subsequent establishment of the pathogen within the mosquito. However, if the pathogen does gain entry into the insect, the insect mounts a vigorous innate cellular and humoral immune response against the pathogen, thereby limiting the pathogen’s propagation to nonpathogenic levels. This happens through three major mechanisms: phagocytosis, melanization, and lysis. During these processes, various signaling pathways that engage intense mosquito–pathogen interactions are activated. A critical overview of the mosquito immune system and latest information about the interaction between mosquitoes and pathogens are provided in this review. The conserved, innate immune pathways and specific anti-pathogenic strategies in mosquito midgut, hemolymph, salivary gland, and neural tissues for the control of pathogen propagation are discussed in detail.

Detection of antibacterial protein in Bacillus sphaericus-treated Culex pipiens (Diptera: Culicidae)

Antimicrobial peptides (AMPs) constitute a major arm of defense in mosquitoes against microbes. The purpose of this study was to determine which of the peptides are produced in the mosquito hemolymph after bacterial treatment. Culex pipiens (Diptera: Culicidae) larvae, collected from drainage canal in Suez Governorate, Egypt, were treated with LC80 of Bacillus sphaericus strain 2362 (0.035 ppm). The hemolymph of bacteria-treated Cx. pipiens mosquitoes and non-treated mosquitoes as control were extracted and undergo electrophoresis, using a Bio-Rad Mini-protean II cell. In Cx. pipiens mosquitoes emerging from bacteria-treated larvae, a band of protein was detected at 165 kDa. This protein was more likely to be Thioester-containing protein 1 (TEP1). Understanding mosquito resistance mechanism to bacterial control is crucial to build up programs to overcome such resistance.

A mosquito hemolymph odorant-binding protein family member specifically binds juvenile hormone

Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes, Culex, and Anopheles mosquito species. Using isothermal titration calorimetry, we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary "long" D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.

Late-phase immune responses limiting oocyst survival are independent of TEP1 function yet display strain specific differences in Anopheles gambiae

BackgroundThere is emerging evidence that mosquito anti-Plasmodium immunity is multimodal with distinct mechanisms for killing malaria parasites at either the ookinete or oocyst stages. Early-phase responses targeting the ookinete require complement-like components circulating in the mosquito hemolymph that result in TEP1-mediated lysis or melanization. Additional responses mediated by the LL3 and STAT pathways limit oocyst survival through unknown mechanisms that require mosquito hemocyte function. While previous experiments argue that these mechanisms of parasite killing are independent, the transient nature of gene-silencing has rendered these experiments inconclusive. To address this issue, we outline experiments using a TALEN-derived TEP1 mutant line to examine the role of TEP1 in the Anopheles gambiae late-phase immune response.ResultsDespite higher early oocyst numbers in the TEP1 mutant line, no differences in oocyst survival were observed when compared to control mosquitoes, suggesting that TEP1 function is independent of the late-phase immune response. To further validate this phenotype in the TEP1 mutant, oocyst survival was evaluated in the TEP1 mutant background by silencing either LL3 or STAT-A. Surprisingly, only STAT-A silenced mosquitoes were able to reconstitute the late-phase immune phenotype increasing oocyst survival in the TEP1 mutant line. Additional experiments highlight significant differences in LL3 expression in the M/S hybrid genetic background of the TEP1 mutant line compared to that of the Keele strain (M form) of An. gambiae, and demonstrate that LL3 is not required for granulocyte differentiation in the M/S hybrid G3 genetic background in response to malaria parasite infection.ConclusionsThrough the combination of genetic experiments utilizing genetic mutants and reverse genetic approaches, new information has emerged regarding the mechanisms of mosquito late-phase immunity. When combined with previously published experiments, the body of evidence argues that Plasmodium oocyst survival is TEP1 independent, thus establishing that the mechanisms of early- and late-phase immunity are distinct. Moreover, we identify that the known components that mediate oocyst survival are susceptible to strain-specific differences depending on their genetic background and provide further evidence that the signals that promote hemocyte differentiation are required to limit oocyst survival. Together, this study provides new insights into the mechanisms of oocyst killing and the importance of genetics in shaping mosquito vector competence.

In vivo gene expression profiling of the entomopathogenic fungus Beauveria bassiana elucidates its infection stratagems in Anopheles mosquito.

The use of entomopathogenic fungi to control mosquitoes is a promising tool for reducing vector-borne disease transmission. To better understand infection stratagems of insect pathogenic fungi, we analyzed the global gene expression profiling of Beauveria bassiana at 36, 60, 84 and 108 h after topical infection of Anopheles stephensi adult mosquitoes using RNA sequencing (RNA-Seq). A total of 5,354 differentially expressed genes (DEGs) are identified over the course of fungal infection. When the fungus grows on the mosquito cuticle, up-regulated DEGs include adhesion-related genes involved in cuticle attachment, Pth11-like GPCRs hypothesized to be involved in host recognition, and extracellular enzymes involved in the degradation and penetration of the mosquito cuticle. Once in the mosquito hemocoel, the fungus evades mosquito immune system probably through up-regulating expression of β-1,3-glucan degrading enzymes and chitin synthesis enzymes for remodeling of cell walls. Moreover, six previous unknown SSCP (small secreted cysteine-rich proteins) are significantly up-regulated, which may serve as "effectors" to suppress host defense responses. B. bassiana also induces large amounts of antioxidant genes to mitigate host-generated exogenous oxidative stress. At late stage of infection, B. bassiana activates a broad spectrum of genes including nutrient degrading enzymes, some transporters and metabolism pathway components, to exploit mosquito tissues and hemolymph as a nutrient source for hyphal growth. These findings establish an important framework of knowledge for further comprehensive elucidation of fungal pathogenesis and molecular mechanism of Beauveria-mosquito interactions.

Transgenic Expression of the Anti-parasitic Factor TEP1 in the Malaria Mosquito Anopheles gambiae

Mosquitoes genetically engineered to be resistant to Plasmodium parasites represent a promising novel approach in the fight against malaria. The insect immune system itself is a source of anti-parasitic genes potentially exploitable for transgenic designs. The Anopheles gambiae thioester containing protein 1 (TEP1) is a potent anti-parasitic protein. TEP1 is secreted and circulates in the mosquito hemolymph, where its activated cleaved form binds and eliminates malaria parasites. Here we investigated whether TEP1 can be used to create malaria resistant mosquitoes. Using a GFP reporter transgene, we determined that the fat body is the main site of TEP1 expression. We generated transgenic mosquitoes that express TEP1r, a potent refractory allele of TEP1, in the fat body and examined the activity of the transgenic protein in wild-type or TEP1 mutant genetic backgrounds. Transgenic TEP1r rescued loss-of-function mutations, but did not increase parasite resistance in the presence of a wild-type susceptible allele. Consistent with previous reports, TEP1 protein expressed from the transgene in the fat body was taken up by hemocytes upon a challenge with injected bacteria. Furthermore, although maturation of transgenic TEP1 into the cleaved form was impaired in one of the TEP1 mutant lines, it was still sufficient to reduce parasite numbers and induce parasite melanization. We also report here the first use of Transcription Activator Like Effectors (TALEs) in Anopheles gambiae to stimulate expression of endogenous TEP1. We found that artificial elevation of TEP1 expression remains moderate in vivo and that enhancement of endogenous TEP1 expression did not result in increased resistance to Plasmodium. Taken together, our results reveal the difficulty of artificially influencing TEP1-mediated Plasmodium resistance, and contribute to further our understanding of the molecular mechanisms underlying mosquito resistance to Plasmodium parasites.

Molecular expression of aquaporin mRNAs in the northern house mosquito, Culex pipiens

Aquaporins (AQPs) are a family of membrane-bound proteins, originally described as water-channels, that broadly exist in prokaryotic and eukaryotic cells. However, some AQPs can transport small molecules (e.g. urea, glycerol) along with or preferentially to water. Previous work in the mosquitoes Aedes aegypti and Anopheles gambiae have characterized the molecular expression of one or more AQP genes and shown that they are involved in water homeostasis after blood feeding, tolerance to dehydration and heat stresses, and development of Plasmodium falciparum in the mosquito. However, the molecular expression of AQPs in the northern house mosquito Culex pipiens, the primary vector of West Nile virus, is presently unknown. Here we characterize the relative mRNA expression of six AQP genes in C. pipiens. We used quantitative RT-PCR (qPCR) to compare AQP mRNA levels 1) among different life stages and sexes, 2) between blood fed and non-blood fed adult females, 3) between adult females injected with phosphate-buffered saline (PBS) and non-injected females, and 4) between adult females exposed to a low vs. high relative humidity. We found that: 1) four AQP mRNAs (Drip, Bib, Eglp1, Aqp12L) were differentially expressed between larval and adult life stages, but not between sexes; 2) after a blood meal, the expression of two AQP mRNAs (Drip and Aqp12L) were down-regulated and one AQP mRNA (Eglp2) was up-regulated; 3) the expression of two AQP mRNAs (Drip and Prip) were down-regulated and one AQP mRNA (Eglp1) was up regulated after injecting the hemolymph of mosquitoes with PBS; and 4) the expression of Prip and Eglp2 mRNAs were both down-regulated in response to a low relative humidity. Our study is the first to characterize the molecular expression patterns of AQPs in C. pipiens, thus providing a foundation for future research elucidating the physiological and functional roles of AQPs in this important disease vector.

A de novo transcriptome of the Malpighian tubules in non-blood-fed and blood-fed Asian tiger mosquitoes Aedes albopictus: insights into diuresis, detoxification, and blood meal processing.

Background. In adult female mosquitoes, the renal (Malpighian) tubules play an important role in the post-prandial diuresis, which removes excess ions and water from the hemolymph of mosquitoes following a blood meal. After the post-prandial diuresis, the roles that Malpighian tubules play in the processing of blood meals are not well described.Methods. We used a combination of next-generation sequencing (paired-end RNA sequencing) and physiological/biochemical assays in adult female Asian tiger mosquitoes (Aedes albopictus) to generate molecular and functional insights into the Malpighian tubules and how they may contribute to blood meal processing (3–24 h after blood ingestion).Results/Discussion. Using RNA sequencing, we sequenced and assembled the first de novo transcriptome of Malpighian tubules from non-blood-fed (NBF) and blood-fed (BF) mosquitoes. We identified a total of 8,232 non-redundant transcripts. The Malpighian tubules of NBF mosquitoes were characterized by the expression of transcripts associated with active transepithelial fluid secretion/diuresis (e.g., ion transporters, water channels, V-type H+-ATPase subunits), xenobiotic detoxification (e.g., cytochrome P450 monoxygenases, glutathione S-transferases, ATP-binding cassette transporters), and purine metabolism (e.g., xanthine dehydrogenase). We also detected the expression of transcripts encoding sodium calcium exchangers, G protein coupled-receptors, and septate junctional proteins not previously described in mosquito Malpighian tubules. Within 24 h after a blood meal, transcripts associated with active transepithelial fluid secretion/diuresis exhibited a general downregulation, whereas those associated with xenobiotic detoxification and purine catabolism exhibited a general upregulation, suggesting a reinvestment of the Malpighian tubules’ molecular resources from diuresis to detoxification. Physiological and biochemical assays were conducted in mosquitoes and isolated Malpighian tubules, respectively, to confirm that the transcriptomic changes were associated with functional consequences. In particular, in vivo diuresis assays demonstrated that adult female mosquitoes have a reduced diuretic capacity within 24 h after a blood meal. Moreover, biochemical assays in isolated Malpighian tubules showed an increase in glutathione S-transferase activity and the accumulation of uric acid (an end product of purine catabolism) within 24 h after a blood meal. Our data provide new insights into the molecular physiology of Malpighian tubules in culicine mosquitoes and reveal potentially important molecular targets for the development of chemical and/or gene-silencing insecticides that would disrupt renal function in mosquitoes.

Endogenously-expressed NH2-terminus of circumsporozoite protein interferes with sporozoite invasion of mosquito salivary glands.

BackgroundThe circumsporozoite protein is the most abundant polypeptide expressed by sporozoites, the malaria parasite stage capable of infecting humans. Sporozoite invasion of mosquito salivary glands prior to transmission is likely mediated by a receptor/ligand-like interaction of the parasites with the target tissues, and the amino (NH2)-terminal portion of CSP is involved in this interaction but not the TSR region on the carboxyl (C)-terminus. Peptides based on the NH2-terminal domain could compete with the parasites for the salivary gland receptors and thus inhibit penetration.MethodsPeptides based on the NH2-terminus and TSR domains of the CSP from avian or human malaria parasites, Plasmodium gallinaceum and Plasmodium falciparum, respectively, were expressed endogenously in mosquito haemolymph using a transient (Sindbis virus-mediated) or stable (piggyBac-mediated transgenesis) system.ResultsTransient endogenous expression of partial NH2-terminus peptide from P. falciparum CSP in P. gallinaceum-infected Aedes aegypti resulted in a reduced number of sporozoites in the salivary glands. When a transgenic approach was used to express a partial CSP NH2-terminal domain from P. gallinaceum the number of sporozoites in the salivary glands did not show a difference when compared to controls. However, a significant difference could be observed when mosquitoes with a lower infection were analysed. The same result could not be observed with mosquitoes endogenously expressing peptides based on the TSR domain from either P. gallinaceum or P. falciparum.ConclusionThese results support the conclusion that CSP partial NH2-terminal domain can be endogenously expressed to promote a competition for the receptor used by sporozoites to invade salivary glands, and they could be used to block this interaction and reduce parasite transmission. The same effect cannot be obtained with peptides based on the TSR domain.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1207-8) contains supplementary material, which is available to authorized users.

Coordinated changes in JH biosynthesis and JH hemolymph titers in Aedes aegypti mosquitoes

Juvenile hormone III (JH) is synthesized by the corpora allata (CA) and plays a key role in mosquito development and reproduction. A decrease in JH titer during the last instar larvae allows pupation and metamorphosis to proceed. As the anti-metamorphic role of JH comes to an end, the CA of the late pupa once again synthesizes JH, which plays an essential role in orchestrating reproductive maturation. In spite of the importance of Aedes aegypti as a vector, a detailed study of the changes of JH hemolymph titers during the gonotrophic cycle has never been performed. In the present studies, using a high performance liquid chromatography coupled to a fluorescent detector (HPLC–FD) method, we measured changes in JH levels in the hemolymph of female mosquitoes during the pupal and adult stages. Our results revealed tightly concomitant changes in JH biosynthesis and JH hemolymph titers during the gonotrophic cycle of female mosquito. Feeding high sugar diets resulted in an increase of JH titers, and mating also modified JH titers in hemolymph. In addition these studies confirmed that JH titer in mosquitoes is fundamentally determined by the rate of biosynthesis in the CA.

Impact of trehalose transporter knockdown onAnopheles gambiaestress adaptation and susceptibility toPlasmodium falciparuminfection

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.

The CLIP-Domain Serine Protease Homolog SPCLIP1 Regulates Complement Recruitment to Microbial Surfaces in the Malaria Mosquito Anopheles gambiae

The complement C3-like protein TEP1 of the mosquito Anopheles gambiae is required for defense against malaria parasites and bacteria. Two forms of TEP1 are present in the mosquito hemolymph, the full-length TEP1-F and the proteolytically processed TEP1cut that is part of a complex including the leucine-rich repeat proteins LRIM1 and APL1C. Here we show that the non-catalytic serine protease SPCLIP1 is a key regulator of the complement-like pathway. SPCLIP1 is required for accumulation of TEP1 on microbial surfaces, a reaction that leads to lysis of malaria parasites or triggers activation of a cascade culminating with melanization of malaria parasites and bacteria. We also demonstrate that the two forms of TEP1 have distinct roles in the complement-like pathway and provide the first evidence for a complement convertase-like cascade in insects analogous to that in vertebrates. Our findings establish that core principles of complement activation are conserved throughout the evolution of animals.

DsRed2 transient expression in Culex quinquefasciatus mosquitoes

Culex quinquefasciatus mosquitoes have been successfully genetically modified only once, despite the efforts of several laboratories to transform and establish a stable strain. We have developed a transient gene expression method, in Culex, that delivers plasmid DNA directly to the mosquito haemolymph and additional tissues. We were able to express DsRed2 fluorescent protein in adult Cx. quinquefasciatus mosquitoes by injecting plasmids directly into their thorax. The expression of DsRed2 in adult Cx. quinquefasciatus mosquitoes is an important stepping stone to genetic transformation and the potential use of new control strategies and genetic interactions.

DsRed2 transient expression in Culex quinquefasciatus mosquitoes

Culex quinquefasciatus mosquitoes have been successfully genetically modified only once, despite the efforts of several laboratories to transform and establish a stable strain. We have developed a transient gene expression method, in Culex, that delivers plasmid DNA directly to the mosquito haemolymph and additional tissues. We were able to express DsRed2 fluorescent protein in adult Cx. quinquefasciatus mosquitoes by injecting plasmids directly into their thorax. The expression of DsRed2 in adult Cx. quinquefasciatus mosquitoes is an important stepping stone to genetic transformation and the potential use of new control strategies and genetic interactions.