RESUMEN

Improved understanding of mosquito-plant feeding interactions can reveal insights into the ecological dynamics of pathogen transmission. In wild malaria vectors Anopheles gambiae s.l. and An. funestus group surveyed in selected dryland ecosystems of Kenya, we found a low level of plant feeding (2.8%) using biochemical cold anthrone test but uncovered 14-fold (41%) higher rate via DNA barcoding targeting the chloroplast rbcL gene. Plasmodium falciparum positivity was associated with either reduced or increased total sugar levels and varied by mosquito species. Gut analysis revealed the mosquitoes to frequently feed on acacia plants (~ 89%) (mainly Vachellia tortilis) in the family Fabaceae. Chemical analysis revealed 1-octen-3-ol (29.9%) as the dominant mosquito attractant, and the sugars glucose, sucrose, fructose, talose and inositol enriched in the vegetative parts, of acacia plants. Nutritional analysis of An. longipalpis C with high plant feeding rates detected fewer sugars (glucose, talose, fructose) compared to acacia plants. These results demonstrate (i) the sensitivity of DNA barcoding to detect plant feeding in malaria vectors, (ii) Plasmodium infection status affects energetic reserves of wild anopheline vectors and (iii) nutrient content and olfactory cues likely represent potent correlates of acacia preferred as a host plant by diverse malaria vectors. The results have relevance in the development of odor-bait control strategies including attractive targeted sugar-baits.

Asunto(s)

Anopheles , Código de Barras del ADN Taxonómico , Ecosistema , Mosquitos Vectores , Plasmodium falciparum , Animales , Mosquitos Vectores/parasitología , Mosquitos Vectores/genética , Anopheles/parasitología , Anopheles/genética , Anopheles/metabolismo , Kenia , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Malaria/transmisión , Malaria/parasitología , Acacia/metabolismo , Acacia/parasitología , Acacia/genética , Conducta Alimentaria/fisiología , Ribulosa-Bifosfato Carboxilasa/metabolismo , Ribulosa-Bifosfato Carboxilasa/genética

RESUMEN

BACKGROUND: Fibrinogen-related protein 1 (frep1) is a member of the pattern-recognizing receptor family (PRR) which generates an innate immune response after recognizing the pattern associated molecular pattern (PAMP) that occurs on the surface of microorganisms. The main objective of this study is to characterize frep1 and its in-silico analysis in Anopheles stephensi. METHODS AND RESULT: The DNA was extracted from female Anopheles stephensi. PCR was performed for complete analysis of frep1 using specific primers. The gene sequence of frep1 was identified by Sanger sequencing. The bioinformatics structure analysis approach revealed the presence of 3 exons and 4 introns in the frep1. The sequence of frep1 was submitted to NCBI GeneBank with accession number ON817187.1. Quantitative real-time PCR was performed to analyze frep1 expression. At the developmental stage, frep1 is highly expressed in the L1 stage, egg, and adult female mosquito. In addition, frep1 is highly expressed in the tissue fat body, midgut, and salivary gland. After blood-fed, an upregulation of frep1 at 48 h in the midgut, and downregulation in fat body were observed at different time intervals. CONCLUSION: The genomic data of frep1 is encoded by 12,443 bp. The frep1 has a significant role in the early metamorphosis. Its expression in fat body and midgut suggests it could be important for fat metabolism and post-blood digestion. The conserved domain could be targeted for vector control. Further study is required to elucidate its function against malaria parasites to confirm its agonist role in malaria transmission.

Asunto(s)

Anopheles , Proteínas de Insectos , Malaria , Mosquitos Vectores , Anopheles/genética , Anopheles/metabolismo , Animales , Mosquitos Vectores/genética , Femenino , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Malaria/parasitología , Simulación por Computador , Fibrinógeno/metabolismo , Fibrinógeno/genética , Filogenia , Inmunidad Innata/genética , Secuencia de Aminoácidos

RESUMEN

The versatility of cytochrome P450 reductase (CPR) in transferring electrons to P450s from other closely related species has been extensively exploited, e.g., by using An. gambiae CPR (AgCPR), as a homologous surrogate, to validate the role of An. funestus P450s in insecticide resistance. However, genomic variation between the AgCPR and An. funestus CPR (AfCPR) suggests that the full metabolism spectrum of An. funestus P450s might be missed when using AgCPR. To test this hypothesis, we expressed AgCPR and AfCPR side-by-side with CYP6P9a and CYP6P9b and functionally validated their role in the detoxification of insecticides from five different classes. Major variations were observed within the FAD- and NADP-binding domains of AgCPR and AfCPR, e.g., the coordinates of the second FAD stacking residue AfCPR-Y456 differ from that of AgCPR-His456. While no significant differences were observed in the cytochrome c reductase activities, when co-expressed with their endogenous AfCPR, the P450s significantly metabolized higher amounts of permethrin and deltamethrin, with CYP6P9b-AfCPR membrane metabolizing α-cypermethrin as well. Only the CYP6P9a-AfCPR membrane significantly metabolized DDT (producing dicofol), bendiocarb, clothianidin, and chlorfenapyr (bioactivation into tralopyril). This demonstrates the broad substrate specificity of An. funestus CYP6P9a/-b, capturing their role in conferring cross-resistance towards unrelated insecticide classes, which can complicate resistance management.

Asunto(s)

Anopheles , Resistencia a los Insecticidas , Insecticidas , NADPH-Ferrihemoproteína Reductasa , Piretrinas , Anopheles/genética , Anopheles/efectos de los fármacos , Anopheles/enzimología , Anopheles/metabolismo , Animales , Resistencia a los Insecticidas/genética , NADPH-Ferrihemoproteína Reductasa/metabolismo , NADPH-Ferrihemoproteína Reductasa/genética , Insecticidas/farmacología , Insecticidas/metabolismo , Piretrinas/farmacología , Piretrinas/metabolismo , Oxidación-Reducción , Proteínas de Insectos/metabolismo , Proteínas de Insectos/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Especificidad por Sustrato , Nitrilos/metabolismo , Nitrilos/farmacología , Permetrina/farmacología

RESUMEN

With the spread of resistance to long-established insecticides targeting Anopheles malaria vectors, understanding the actions of compounds newly identified for vector control is essential. With new commercial vector-control products containing neonicotinoids under development, we investigate the actions of 6 neonicotinoids (imidacloprid, thiacloprid, clothianidin, dinotefuran, nitenpyram and acetamiprid) on 13 Anopheles gambiae nicotinic acetylcholine receptor (nAChR) subtypes produced by expression of combinations of the Agα1, Agα2, Agα3, Agα8 and Agß1 subunits in Xenopus laevis oocytes, the Drosophila melanogaster orthologues of which we have previously shown to be important in neonicotinoid actions. The presence of the Agα2 subunit reduces neonicotinoid affinity for the mosquito nAChRs, whereas the Agα3 subunit increases it. Crystal structures of the acetylcholine binding protein (AChBP), an established surrogate for the ligand-binding domain, with dinotefuran bound, shows a unique target site interaction through hydrogen bond formation and CH-N interaction at the tetrahydrofuran ring. This is of interest as dinotefuran is also under trial as the toxic element in baited traps. Multiple regression analyses show a correlation between the efficacy of neonicotinoids for the Agα1/Agα2/Agα8/Agß1 nAChR, their hydrophobicity and their rate of knockdown of adult female An. gambiae, providing new insights into neonicotinoid features important for malaria vector control.

Asunto(s)

Anopheles , Guanidinas , Insecticidas , Mosquitos Vectores , Neonicotinoides , Nitrocompuestos , Receptores Nicotínicos , Animales , Anopheles/metabolismo , Anopheles/genética , Anopheles/efectos de los fármacos , Neonicotinoides/farmacología , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/química , Insecticidas/farmacología , Insecticidas/química , Nitrocompuestos/farmacología , Nitrocompuestos/química , Guanidinas/farmacología , Mosquitos Vectores/efectos de los fármacos , Mosquitos Vectores/genética , Xenopus laevis , Ligandos , Piridinas/farmacología , Malaria/transmisión , Malaria/parasitología , Tiazoles/farmacología , Tiazoles/química , Tiazoles/metabolismo , Tiazinas/farmacología , Tiazinas/química , Oocitos/metabolismo , Oocitos/efectos de los fármacos , Femenino , Proteínas de Insectos/metabolismo , Proteínas de Insectos/genética , Proteínas de Insectos/química , Imidazoles/farmacología , Imidazoles/química

RESUMEN

BACKGROUND: Malaria, a global health concern, is caused by parasites of the Plasmodium genus, which undergo gametogenesis in the midgut of mosquitoes after ingestion of an infected blood meal. The resulting male and female gametes fuse to form a zygote, which differentiates into a motile ookinete. After traversing the midgut epithelium, the ookinete differentiates into an oocyst on the basal side of the epithelium. METHODS: Membrane proteins with increased gene expression levels from the gamete to oocyst stages in P. berghei were investigated utilizing PlasmoDB, the functional genomic database for Plasmodium spp. Based on this analysis, we selected the 184-kDa membrane protein, Pb184, for further study. The expression of Pb184 was further confirmed through immunofluorescence staining, following which we examined whether Pb184 is involved in fertilization using antibodies targeting the C-terminal region of Pb184 and biotin-labeled C-terminal region peptides of Pb184. RESULTS: Pb184 is expressed on the surface of male and female gametes. The antibody inhibited zygote and ookinete formation in vitro. When mosquitoes were fed on parasite-infected blood containing the antibody, oocyst formation decreased on the second day after feeding. Synthesized biotin-labeled peptides matching the C-terminal region of Pb184 bound to the female gamete and the residual body of male gametes, and inhibited differentiation into ookinetes in the in vitro culture system. CONCLUSIONS: These results may be useful for the further studying the fertilization mechanism of Plasmodium protozoa. There is also the potential for their application as future tools to prevent malaria transmission.

Asunto(s)

Fertilización , Plasmodium berghei , Proteínas Protozoarias , Plasmodium berghei/genética , Plasmodium berghei/metabolismo , Animales , Femenino , Masculino , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Ratones , Células Germinativas/metabolismo , Malaria/parasitología , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Cigoto/metabolismo , Anopheles/parasitología , Anopheles/metabolismo , Oocistos/metabolismo , Gametogénesis/genética

RESUMEN

Mosquito saliva plays a crucial physiological role in both sugar and blood feeding by helping sugar digestion and exerting antihemostatic functions. During meal acquisition, mosquitoes are exposed to the internalization of external microbes. Since mosquitoes reingest significant amounts of saliva during feeding, we hypothesized that salivary antimicrobial components may participate in the protection of mouthparts, the crop, and the gut by inhibiting bacterial growth. To identify novel potential antimicrobials from mosquito saliva, we selected 11 candidates from Anopheles coluzzii salivary transcriptomic datasets and obtained them either using a cell-free transcription/translation expression system or, when feasible, via chemical synthesis. Hyp6.2 and hyp13, which were predicted to be produced as propeptides and cleaved in shorter mature forms, showed the most interesting results in bacterial growth inhibition assays. Hyp6.2 (putative mature form, 35 amino acid residues) significantly inhibited the growth of Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli and Serratia marcescens) bacteria. Hyp13 (short form, 19 amino acid residues) dose-dependently inhibited E. coli and S. marcescens growth, inducing membrane disruption in both Gram-positive and Gram-negative bacteria as indicated with scanning electron microscopy. In conclusion, we identified two A. coluzzii salivary peptides inhibiting Gram-positive and Gram-negative bacteria growth and possibly contributing to the protection of mosquito mouthparts and digestive tracts from microbial infection during and/or after feeding.

Asunto(s)

Anopheles , Péptidos Antimicrobianos , Mosquitos Vectores , Saliva , Anopheles/metabolismo , Animales , Saliva/metabolismo , Péptidos Antimicrobianos/farmacología , Péptidos Antimicrobianos/metabolismo , Péptidos Antimicrobianos/química , Malaria , Escherichia coli/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Proteínas de Insectos/metabolismo , Proteínas de Insectos/genética

RESUMEN

The incorporation of phytoactive compounds in the management of malarial vectors holds promise for the development of innovative and efficient alternatives. Nevertheless, the molecular and physiological responses that these bioactive substances induce remain underexplored. This present study investigated the toxicity of different concentrations of aqueous and methanol extracts of Ocimum tenuiflorum against larvae of Anopheles gambiae (sensu stricto) and unraveled the possible underlying molecular pathways responsible for the observed physiological effects. FTIR and GCMS analyses of phytoactive compounds in aqueous and methanol crude extracts of O. tenuiflorum showed the presence of OH stretching vibration, C = C stretching modes of aromatics and methylene rocking vibration; ring deformation mode with high levels of trans-ß-ocimene, 3,7-dimethyl-1,3,6-octatriene in aqueous extract and 4-methoxy-benzaldehyde, 1,3,5-trimethyl-cyclohexane and o-cymene in methanol extract. The percentage mortality upon exposure to methanol and aqueous extracts of O. tenuiflorum were 21.1% and 26.1% at 24 h, 27.8% and 36.1% at 48 h and 36.1% and 45% at 72 h respectively. Using reverse transcription quantitative polymerase chain reaction (RT-qPCR), down-regulation of ABC transporter, overexpression of CYP6M2, Hsp70, and α-esterase, coupled with significantly increased levels of SOD, CAT, and GSH, were observed in An. gambiae (s.s.) exposed to aqueous and methanol extracts of O. tenuiflorum as compared to the control. Findings from this study have significant implications for our understanding of how An. gambiae (s.s.) larvae detoxify phytoactive compounds.

Asunto(s)

Transportadoras de Casetes de Unión a ATP , Anopheles , Antioxidantes , Proteínas HSP70 de Choque Térmico , Ocimum , Extractos Vegetales , Animales , Anopheles/efectos de los fármacos , Anopheles/genética , Anopheles/metabolismo , Extractos Vegetales/farmacología , Antioxidantes/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Larva/efectos de los fármacos , Larva/metabolismo , Proteínas de Insectos/metabolismo , Proteínas de Insectos/genética , Estrés Fisiológico/efectos de los fármacos

RESUMEN

BACKGROUND: Hemocytes are immune cells that patrol the mosquito hemocoel and mediate critical cellular defense responses against pathogens. However, despite their importance, a comprehensive transcriptome of these cells was lacking because they constitute a very small fraction of the total cells in the insect, limiting the study of hemocyte differentiation and immune function. RESULTS: In this study, an in-depth hemocyte transcriptome was built by extensive bulk RNA sequencing and assembly of hemocyte RNAs from adult A. gambiae female mosquitoes, based on approximately 2.4 billion short Illumina and about 9.4 million long PacBio high-quality reads that mapped to the A. gambiae PEST genome (P4.14 version). A total of 34,939 transcripts were annotated including 4,020 transcripts from novel genes and 20,008 novel isoforms that result from extensive differential splicing of transcripts from previously annotated genes. Most hemocyte transcripts identified (89.8%) are protein-coding while 10.2% are non-coding RNAs. The number of transcripts identified in the novel hemocyte transcriptome is twice the number in the current annotation of the A. gambiae genome (P4.14 version). Furthermore, we were able to refine the analysis of a previously published single-cell transcriptome (scRNAseq) data set by using the novel hemocyte transcriptome as a reference to re-define the hemocyte clusters and determine the path of hemocyte differentiation. Unsupervised pseudo-temporal ordering using the Tools for Single Cell Analysis software uncovered a novel putative prohemocyte precursor cell type that gives rise to prohemocytes. Pseudo-temporal ordering with the Monocle 3 software, which analyses changes in gene expression during dynamic biological processes, determined that oenocytoids derive from prohemocytes, a cell population that also gives rise to the granulocyte lineage. CONCLUSION: A high number of mRNA splice variants are expressed in hemocytes, and they may account for the plasticity required to mount efficient responses to many different pathogens. This study highlights the importance of a comprehensive set of reference transcripts to perform robust single-cell transcriptomic data analysis of cells present in low abundance. The detailed annotation of the hemocyte transcriptome will uncover new facets of hemocyte development and function in adult dipterans and is a valuable community resource for future studies on mosquito cellular immunity.

Asunto(s)

Anopheles , Animales , Femenino , Anopheles/genética , Anopheles/metabolismo , Hemocitos , Perfilación de la Expresión Génica , Transcriptoma , Proteínas/metabolismo

RESUMEN

Serine protease cascades regulate important insect immune responses, including melanization and Toll pathway activation. In the context of melanization, central components of these cascades are clip domain serine proteases (CLIPs) including the catalytic, clip domain serine proteases (cSPs) and their non-catalytic homologs (cSPHs). Here, we define partially the structural hierarchy of An. gambiae cSPs of the CLIPB family, central players in melanization, and characterize their relative contributions to bacterial melanization and to mosquito susceptibility to bacterial infections. Using in vivo genetic analysis we show that the protease cascade branches downstream of the cSPs CLIPB4 and CLIPB17 into two branches one converging on CLIPB10 and the second on CLIPB8. We also show that the contribution of key cSPHs to melanization in vivo in response to diverse microbial challenges is more significant than any of the individual cSPs, possibly due to partial functional redundancy among the latter. Interestingly, we show that the key cSPH CLIPA8 which is essential for the efficient activation cleavage of CLIPBs in vivo is efficiently cleaved itself by several CLIPBs in vitro, suggesting that cSPs and cSPHs regulate signal amplification and propagation in melanization cascades by providing positive reinforcement upstream and downstream of each other.

Asunto(s)

Anopheles , Infecciones Bacterianas , Animales , Anopheles/genética , Anopheles/metabolismo , Anopheles/microbiología , Serina Proteasas , Serina Endopeptidasas/genética , Serina Endopeptidasas/química , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo

RESUMEN

Phenoloxidase (PO) catalyzed melanization and other insect immune responses are mediated by serine proteases (SPs) and their noncatalytic homologs (SPHs). Many of these SP-like proteins have a regulatory clip domain and are called CLIPs. In most insects studied so far, PO precursors are activated by a PAP (i.e., PPO activating protease) and its cofactor of clip-domain SPHs. Although melanotic encapsulation is a well-known refractory mechanism of mosquitoes against malaria parasites, it is unclear if a cofactor is required for PPO activation. In Anopheles gambiae, CLIPA4 is 1:1 orthologous to Manduca sexta SPH2; CLIPs A5-7, A12-14, A26, A31, A32, E6, and E7 are 11:4 orthologous to M. sexta SPH1a, 1b, 4, and 101, SPH2 partners in the cofactors. Here we produced proCLIPs A4, A6, A7Δ, A12, and activated them with CLIPB9 or M. sexta PAP3. A. gambiae PPO2 and PPO7 were expressed in Escherichia coli for use as PAP substrates. CLIPB9 was mutated to CLIPB9Xa by including a Factor Xa cleavage site. CLIPA7Δ was a deletion mutant with a low complexity region removed. After PAP3 or CLIPB9Xa processing, CLIPA4 formed a high Mr complex with CLIPA6, A7Δ or A12, which assisted PPO2 and PPO7 activation. High levels of specific PO activity (55-85 U/µg for PO2 and 1131-1630 U/µg for PO7) were detected in vitro, indicating that cofactor-assisted PPO activation also occurs in this species. The cleavage sites and mechanisms for complex formation and cofactor function are like those reported in M. sexta and Drosophila melanogaster. In conclusion, these data suggest that the three (and perhaps more) SPHI-II pairs may form cofactors for CLIPB9-mediated activation of PPOs for melanotic encapsulation in A. gambiae.

Asunto(s)

Anopheles , Manduca , Animales , Serina Proteasas/metabolismo , Anopheles/metabolismo , Drosophila melanogaster/metabolismo , Serina Endopeptidasas , Catecol Oxidasa/genética , Catecol Oxidasa/metabolismo , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Monofenol Monooxigenasa , Manduca/metabolismo , Proteínas de Insectos/metabolismo , Hemolinfa

RESUMEN

An active immune response is energetically demanding and requires reallocation of nutrients to support resistance to and tolerance of infection. Insulin signaling is a critical global regulator of metabolism and whole-body homeostasis in response to nutrient availability and energetic needs, including those required for mobilization of energy in support of the immune system. In this review, we share findings that demonstrate interactions between innate immune activity and insulin signaling primarily in the insect model Drosophila melanogaster as well as other insects like Bombyx mori and Anopheles mosquitos. These studies indicate that insulin signaling and innate immune activation have reciprocal effects on each other, but that those effects vary depending on the type of pathogen, route of infection, and nutritional status of the host. Future research will be required to further understand the detailed mechanisms by which innate immunity and insulin signaling activity impact each other.

Asunto(s)

Anopheles , Insulina , Animales , Insulina/metabolismo , Drosophila melanogaster , Inmunidad Innata , Insectos , Anopheles/metabolismo

RESUMEN

The Anopheles mosquito is one of thousands of species in which sex differences play a central part in their biology, as only females need a blood meal to produce eggs. Sex differentiation is regulated by sex chromosomes, but their presence creates a dosage imbalance between males (XY) and females (XX). Dosage compensation (DC) can re-equilibrate the expression of sex chromosomal genes. However, because DC mechanisms have only been fully characterized in a few model organisms, key questions about its evolutionary diversity and functional necessity remain unresolved1. Here we report the discovery of a previously uncharacterized gene (sex chromosome activation (SOA)) as a master regulator of DC in the malaria mosquito Anopheles gambiae. Sex-specific alternative splicing prevents functional SOA protein expression in females. The male isoform encodes a DNA-binding protein that binds the promoters of active X chromosomal genes. Expressing male SOA is sufficient to induce DC in female cells. Male mosquitoes lacking SOA or female mosquitoes ectopically expressing the male isoform exhibit X chromosome misregulation, which is compatible with viability but causes developmental delay. Thus, our molecular analyses of a DC master regulator in a non-model organism elucidates the evolutionary steps that lead to the establishment of a chromosome-specific fine-tuning mechanism.

Asunto(s)

Empalme Alternativo , Anopheles , Compensación de Dosificación (Genética) , Proteínas de Insectos , Caracteres Sexuales , Diferenciación Sexual , Cromosoma X , Animales , Femenino , Masculino , Anopheles/genética , Anopheles/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Diferenciación Sexual/genética , Cromosoma X/genética , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo

RESUMEN

Identifying the mechanisms by which bacterial pathogens kill host cells is fundamental to understanding how to control and prevent human and animal disease. In the case of Bacillus thuringiensis (Bt), such knowledge is critical to using the bacterium to kill insect vectors that transmit human and animal disease. For the Cry4B toxin produced by Bt, its capacity to kill Anopheles gambiae, the primary mosquito vector of malaria, is the consequence of a variety of signaling activities. We show here that Cry4B, acting as first messenger, binds specifically to the bitopic cadherin BT-R3 G-protein-coupled receptor (GPCR) localized in the midgut of A. gambiae, activating the downstream second messenger cyclic adenosine monophosphate (cAMP). The direct result of the Cry4B-BT-R3 binding is the release of αs from the heterotrimeric αßγ-G-protein complex and its activation of adenylyl cyclase (AC). The upshot is an increased level of cAMP, which activates protein kinase A (PKA). The functional impact of cAMP-PKA signaling is the stimulation of Na+/K+-ATPase (NKA) which serves as an Na+/K+ pump to maintain proper gradients of extracellular Na+ and intracellular K+. Increased level of cAMP amplifies NKA and upsets normal ion concentration gradients. NKA, as a scaffolding protein, accelerates the first messenger signal to the nucleus, generating additional BT-R3 molecules and promoting their exocytotic trafficking to the cell membrane. Accumulation of BT-R3 on the cell surface facilitates recruitment of additional toxin molecules which, in turn, amplify the original signal in a cascade-like manner. This report provides the first evidence of a bacterial toxin using NKA via AC/PKA signaling to execute cell death.

Asunto(s)

Anopheles , Bacillus thuringiensis , Animales , Humanos , Bacillus thuringiensis/metabolismo , Anopheles/metabolismo , Adenosina Trifosfatasas/metabolismo , Mosquitos Vectores , Adenilil Ciclasas/metabolismo , Muerte Celular , ATPasa Intercambiadora de Sodio-Potasio/metabolismo

RESUMEN

Understanding development and genetic regulation in the Anopheles gambiae germline is essential to engineer effective genetic control strategies targeting this malaria mosquito vector. These include targeting the germline to induce sterility or using regulatory sequences to drive transgene expression for applications such as gene drive. However, only very few germline-specific regulatory elements have been characterised with the majority showing leaky expression. This has been shown to considerably reduce the efficiency of current genetic control strategies, which rely on regulatory elements with more tightly restricted spatial and/or temporal expression. Meiotic silencing of the sex chromosomes limits the flexibility of transgene expression to develop effective sex-linked genetic control strategies. Here, we build on our previous study, dissecting gametogenesis into four distinct cell populations, using single-cell RNA sequencing to define eight distinct cell clusters and associated germline cell-types using available marker genes. We reveal overexpression of X-linked genes in a distinct cluster of pre-meiotic cells and document the onset of meiotic silencing of the X chromosome in a subcluster of cells in the latter stages of spermatogenesis. This study provides a comprehensive dataset, characterising the expression of distinct cell types through spermatogenesis and widening the toolkit for genetic control of malaria mosquitoes.

Asunto(s)

Anopheles , Malaria , Animales , Masculino , Anopheles/metabolismo , Espermatogénesis/genética , Cromosoma X/genética , Cromosomas Sexuales

RESUMEN

Contact insecticides are primarily used for the control of Anopheles malaria vectors. These chemicals penetrate mosquito legs and other appendages; the first barriers to reaching their neuronal targets. An ATP-Binding Cassette transporter from the H family (ABCH2) is highly expressed in Anopheles coluzzii legs, and further induced upon insecticide exposure. RNAi-mediated silencing of the ABCH2 caused a significant increase in deltamethrin mortality compared to control mosquitoes, coincident with a corresponding increase in 14C-deltamethrin penetration. RT-qPCR analysis and immunolocalization revealed ABCH2 to be mainly localized in the legs and head appendages, and more specifically, the apical part of the epidermis, underneath the cuticle. To unravel the molecular mechanism underlying the role of ABCH2 in modulating pyrethroid toxicity, two hypotheses were investigated: An indirect role, based on the orthology with other insect ABCH transporters involved with lipid transport and deposition of CHC lipids in Anopheles legs which may increase cuticle thickness, slowing down the penetration rate of deltamethrin; or the direct pumping of deltamethrin out of the organism. Evaluation of the leg cuticular hydrocarbon (CHC) content showed no affect by ABCH2 silencing, indicating this protein is not associated with the transport of leg CHCs. Homology-based modeling suggested that the ABCH2 half-transporter adopts a physiological homodimeric state, in line with its ability to hydrolyze ATP in vitro when expressed on its own in insect cells. Docking analysis revealed a deltamethrin pocket in the homodimeric transporter. Furthermore, deltamethrin-induced ATP hydrolysis in ABCH2-expressing cell membranes, further supports that deltamethrin is indeed an ABCH2 substrate. Overall, our findings pinpoint ABCH2 participating in deltamethrin toxicity regulation.

Asunto(s)

Anopheles , Insecticidas , Malaria , Animales , Anopheles/metabolismo , Resistencia a los Insecticidas , Mosquitos Vectores/genética , Insecticidas/farmacología , Nitrilos/toxicidad , Nitrilos/metabolismo , Adenosina Trifosfato/metabolismo , Control de Mosquitos

RESUMEN

Malaria needs new strategies for its control. Plasmodium spp., the causative agent of malaria, is transmitted by mosquitoes. These parasites develop into oocysts and sporozoites in the body of the mosquitoes. A deeper understanding of oocysts that produce the infectious form of the parasite, sporozoites, can facilitate the development of novel countermeasures. However, the isolation of Plasmodium oocysts is challenging as these are formed between midgut epithelial cells and basal lamina after gametocytes enter the mosquito's body through blood feeding. Further research on oocysts has been impeded by issues related to oocyst isolation. Therefore, in this study, we injected Plasmodium into mosquitoes-an artificial and unique method-and aimed to clarify how oocysts were formed in mosquitoes after Plasmodium injection and whether free oocysts were formed from the mosquito tissue. Plasmodium berghei (ANKA strain) ookinetes cultured in vitro were injected into the thoracic body cavity (hemocoel) of female and male Anopheles stephensi mosquitoes. Oocysts were formed in the body of female and male mosquitoes at 14 days post injection. In addition, oocysts formed as a result of injection developed into sporozoites, which were infectious to mice. These findings suggest that P. berghei can complete its developmental stage in mosquitoes by injection. Some of the oocysts formed were free from mosquito tissue, and it was possible to collect oocysts with minimal contamination of mosquito tissue. These free oocysts can be used for investigating oocyst proteins and metabolism.

Asunto(s)

Anopheles , Malaria , Masculino , Femenino , Animales , Ratones , Oocistos , Anopheles/metabolismo , Anopheles/parasitología , Malaria/veterinaria , Plasmodium berghei

RESUMEN

Mosquitoes rely mainly on the sense of smell to decipher their environment and locate suitable food sources, hosts for blood feeding and oviposition sites. The molecular bases of olfaction involve multigenic families of olfactory proteins that have evolved to interact with a narrow set of odorants that are critical for survival. Understanding the complex interplay between diversified repertoires of olfactory proteins and ecologically-relevant odorant signals, which elicit important behaviors, is fundamental for the design of novel control strategies targeting the sense of smell of disease vector mosquitoes. Previously, large multigene families of odorant receptor and ionotropic receptor proteins, as well as a subset of odorant-binding proteins have been shown to mediate the selectivity and sensitivity of the mosquito olfactory system. In this study, we identify a mosquito-specific antennal protein (MSAP) gene as a novel molecular actor of odorant reception. MSAP is highly conserved across mosquito species and is transcribed at an extremely high level in female antennae. In order to understand its role in the mosquito olfactory system, we generated knockout mutant lines in Anopheles gambiae, and performed comparative analysis of behavioral and physiological responses to human-associated odorants. We found that MSAP promotes female mosquito attraction to human odor and enhances the sensitivity of the antennae to a variety of odorants. These findings suggest that MSAP is an important component of the mosquito olfactory system, which until now has gone completely unnoticed.

Asunto(s)

Anopheles , Malaria , Receptores Odorantes , Humanos , Femenino , Animales , Odorantes , Anopheles/metabolismo , Mosquitos Vectores/genética , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Olfato/fisiología

RESUMEN

Insect Odorant Binding Proteins (OBPs) constitute important components of their olfactory apparatus, as they are essential for odor recognition. OBPs undergo conformational changes upon pH change, altering their interactions with odorants. Moreover, they can form heterodimers with novel binding characteristics. Anopheles gambiae OBP1 and OBP4 were found capable of forming heterodimers possibly involved in the specific perception of the attractant indole. In order to understand how these OBPs interact in the presence of indole and to investigate the likelihood of a pH-dependent heterodimerization mechanism, the crystal structures of OBP4 at pH 4.6 and 8.5 were determined. Structural comparison to each other and with the OBP4-indole complex (3Q8I, pH 6.85) revealed a flexible N-terminus and conformational changes in the α4-loop-α5 region at acidic pH. Fluorescence competition assays showed a weak binding of indole to OBP4 that becomes further impaired at acidic pH. Additional Molecular Dynamic and Differential Scanning Calorimetry studies displayed that the influence of pH on OBP4 stability is significant compared to the modest effect of indole. Furthermore, OBP1-OBP4 heterodimeric models were generated at pH 4.5, 6.5, and 8.5, and compared concerning their interface energy and cross-correlated motions in the absence and presence of indole. The results indicate that the increase in pH may induce the stabilization of OBP4 by increasing its helicity, thereby enabling indole binding at neutral pH that further stabilizes the protein and possibly promotes the creation of a binding site for OBP1. A decrease in interface stability and loss of correlated motions upon transition to acidic pH may provoke the heterodimeric dissociation allowing indole release. Finally, we propose a potential OBP1-OBP4 heterodimer formation/disruption mechanism induced by pH change and indole binding.

Asunto(s)

Anopheles , Receptores Odorantes , Animales , Odorantes , Anopheles/química , Anopheles/metabolismo , Receptores Odorantes/química , Sitios de Unión , Indoles/química , Concentración de Iones de Hidrógeno , Proteínas de Insectos/metabolismo

RESUMEN

Among several proteins participating in the olfactory perception process of insects, Odorant Binding Proteins (OBPs) are today considered valid targets for the discovery of compounds that interfere with their host-detection behavior. The 3D structures of Anopheles gambiae mosquito AgamOBP1 in complex with the known synthetic repellents DEET and Icaridin have provided valuable information on the structural characteristics that govern their selective binding. However, no structure of a plant-derived repellent bound to an OBP has been available until now. Herein, we present the novel three-dimensional crystal structures of AgamOBP5 in complex with two natural phenolic monoterpenoid repellents, Carvacrol and Thymol, and the MPD molecule. Structural analysis revealed that both monoterpenoids occupy a binding site (Site-1) by adopting two alternative conformations. An additional Carvacrol was also bound to a secondary site (Site-2) near the central cavity entrance. A protein-ligand hydrogen-bond network supplemented by van der Waals interactions spans the entire binding cavity, bridging α4, α6, and α3 helices and stabilizing the overall structure. Fluorescence competition and Differential Scanning Calorimetry experiments verified the presence of two binding sites and the stabilization effect on AgamOBP5. While Carvacrol and Thymol bind to Site-1 with equal affinity in the submicromolar range, they exhibit a significantly lower and distinct binding capacity for Site-2 with Kd's of ~7 µΜ and ~18 µΜ, respectively. Finally, a comparison of AgamOBP5 complexes with the AgamOBP4-Indole structure revealed that variations of ligand-interacting aminoacids such as A109T, I72M, A112L, and A105T cause two structurally similar and homologous proteins to display different binding specificities.

Asunto(s)

Anopheles , Repelentes de Insectos , Receptores Odorantes , Animales , Repelentes de Insectos/química , Repelentes de Insectos/metabolismo , Timol/metabolismo , Ligandos , Anopheles/química , Anopheles/metabolismo , Monoterpenos/metabolismo , Receptores Odorantes/química

RESUMEN

During their development in mosquitoes, malaria parasites undergo massive losses that are in part due to a potent antiparasitic response mounted by the vector. The most efficient and best-characterized response relies on a complement-like system particularly effective against parasites as they cross the mosquito midgut epithelium. While our vision of the molecular and cellular events that lead to parasite elimination is still partial, our understanding of the steps triggering complement activation at the surface of invading parasites has considerably progressed, not only through the identification of novel contributing genes, but also with the recent in-depth characterization of the different mosquito blood cell types, and the ability to track them in live mosquitoes. Here, we propose a simple model based on the time of invasion to explain how parasites may escape complement-like responses during midgut infection.

Asunto(s)

Anopheles , Antiinfecciosos , Parásitos , Animales , Anopheles/metabolismo , Anopheles/parasitología , Antiparasitarios/metabolismo , Mosquitos Vectores/parasitología , Proteínas del Sistema Complemento , Sistema Digestivo/parasitología , Antiinfecciosos/metabolismo