RESUMO
BACKGROUND: Culex quinquefasciatus resistance to the binary toxin from Lysinibacillus sphaericus larvicides can occur because of mutations in the cqm1 gene that prevents the expression of the toxin receptor, Cqm1 α-glucosidase. In a resistant laboratory-selected colony maintained for more than 250 generations, cqm1REC and cqm1REC-2 resistance alleles were identified. The major allele initially found, cqm1REC , became minor and was replaced by cqm1REC-2 . This study aimed to investigate the features associated with homozygous larvae for each allele to understand the reasons for the allele replacement and to generate knowledge on resistance to microbial larvicides. RESULTS: Homozygous larvae for each allele were compared. Both larvae displayed the same level of resistance to the binary toxin (3500-fold); therefore, a change in phenotype was not the reason for the replacement observed. The lack of Cqm1 expression did not reduce the total specific α-glucosidase activity for homozygous cqm1REC and cqm1REC-2 larvae, which were statistically similar to the susceptible strain, using artificial or natural substrates. The expression of eight Cqm1 paralog α-glucosidases was demonstrated in resistant and susceptible larvae. Bioassays in which cqm1REC or cqm1REC-2 homozygous larvae were reared under stressful conditions showed that most adults produced were cqm1REC-2 homozygous (69%). Comparatively, in the offspring of a heterozygous sub-colony reared under optimal conditions for 20 generations, the cqm1REC allele assumed a higher frequency (0.72). CONCLUSION: Homozygous larvae for each allele exhibited a similar resistant phenotype. However, they presented specific advantages that might favor their selection and can be used in designing resistance management practices. © 2021 Society of Chemical Industry.
Assuntos
Toxinas Bacterianas , Culex , Proteínas de Insetos/genética , alfa-Glucosidases/genética , Alelos , Animais , Bacillaceae , Culex/enzimologia , Culex/genética , Larva/genéticaRESUMO
Culex quinquefasciatus is a successful invasive species broadly distributed in subtropical regions, including Brazil. It is an extremely annoying mosquito due to its nocturnal biting behavior, in high-density populations and it is a potential bridge between sylvatic arbovirus from birds to man in urban territories. Herein, we present a review concerning the methods of chemical control employed against Cx. quinquefasciatus in Brazil since the 1950's and insecticide resistance data registered in the literature. As there is no specific national programme for Cx. quinquefasciatus control in Brazil, the selection of insecticide resistance is likely due in part to the well-designed chemical campaigns against Aedes aegypti and the elevated employment of insecticides by households and private companies. There are very few publications about insecticide resistance in Cx. quinquefasciatus from Brazil when compared to Ae. aegypti. Nevertheless, resistance to organophosphates, carbamate, DDT, pyrethroids and biolarvicides has been registered in Cx. quinquefasciatus populations from distinct localities of the country. Concerning physiological mechanisms selected for resistance, distinct patterns of esterases, as well as mutations in the acetylcholinesterase (ace-1) and voltage-gated sodium channel (NaV) genes, have been identified in natural populations. Given environmental changes and socioeconomical issues in the cities, in recent years we have been experiencing an increase in the number of disease cases caused by arboviruses, which may involve Cx. quinquefasciatus participation as a key vector. It is urgent to better understand the efficiency and susceptibility status to insecticides, as well as the genetic background of known resistant mechanisms already present in Cx. quinquefasciatus populations for an effective and rapid chemical control when eventually required.
Assuntos
Culex/efeitos dos fármacos , Resistência a Inseticidas , Inseticidas/farmacologia , Animais , Brasil , Culex/enzimologia , Culex/genética , Humanos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Mosquitos Vetores/efeitos dos fármacos , Mosquitos Vetores/enzimologia , Mosquitos Vetores/genéticaRESUMO
A binary mosquitocidal toxin composed of a three-domain Cry-like toxin (Cry48Aa) and a binary-like toxin (Cry49Aa) was identified in Lysinibacillus sphaericus. Cry48Aa/Cry49Aa has action on Culex quinquefasciatus larvae, in particular, to those that are resistant to the Bin Binary toxin, which is the major insecticidal factor from L. sphaericus-based biolarvicides, indicating that Cry48Aa/Cry49Aa interacts with distinct target sites in the midgut and can overcome Bin toxin resistance. This study aimed to identify Cry48Aa/Cry49Aa ligands in C. quinquefasciatus midgut through binding assays and mass spectrometry. Several proteins, mostly from 50 to 120 kDa, bound to the Cry48Aa/Cry49Aa toxin were revealed by toxin overlay and pull-down assays. These proteins were identified against the C. quinquefasciatus genome and after analysis a set of 49 proteins were selected which includes midgut bound proteins such as aminopeptidases, amylases, alkaline phosphatases in addition to molecules from other classes that can be potentially involved in this toxin's mode of action. Among these, some proteins are orthologs of Cry receptors previously identified in mosquito larvae, as candidate receptors for Cry48Aa/Cry49Aa toxin. Further investigation is needed to evaluate the specificity of their interactions and their possible role as receptors.
Assuntos
Proteínas de Bactérias/metabolismo , Culex/enzimologia , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Inseticidas/metabolismo , Animais , Toxinas de Bacillus thuringiensis , Culex/genética , Trato Gastrointestinal/enzimologia , Larva/enzimologia , LigantesRESUMO
Culex (Melanoconion) pedroiSirivanakarn & Belkin 1980 and Culex (Melanoconion) ribeirensisForattini & Sallum 1985 are two morphologically very similar species of the Pedroi subgroup of mosquitoes in the Spissipes section of the subgenus Melanoconion of the genus Culex L. 1758. We carried out an analysis of the mitochondrial cytochrome c oxidase I (COI) DNA variation between these two species. The recent observation of sympatric coexistence in a forested area of Rio de Janeiro State (Brazil) triggered the need to validate these two species previously identified based on morphology. We concluded that the COI is a useful tool for identification of Cx. pedroi and Cx. ribeirensis.
Assuntos
Culex/classificação , Complexo IV da Cadeia de Transporte de Elétrons/genética , Proteínas de Insetos/genética , Mitocôndrias/genética , Animais , Brasil , Culex/enzimologia , Culex/genética , DNA Mitocondrial/genética , Variação Genética , Mitocôndrias/enzimologiaRESUMO
Cqm1 and Aam1 are α-glucosidases (EC 3.2.1.20) expressed in Culex quinquefasciatus and Aedes aegypti larvae midgut, respectively. These orthologs share high sequence similarity but while Cqm1 acts as a receptor for the Binary (Bin) insecticidal toxin from Lysinibacillus sphaericus, Aam1 does not bind the toxin, rendering Ae. aegypti refractory to this bacterium. Aam1 is heavily glycosylated, contrasting to Cqm1, but little is known regarding how glycosylation impacts on its function. This study aimed to compare the N-glycosylation patterns and the catalytic activities of Aam1 and Cqm1. Mutant proteins were generated where predicted Aam1 N-glycosylation sites (N-PGS) were either inserted into Cqm1 or abrogated in Aam1. The mutants validated four N-PGS which were found to localize externally on the Aam1 structure. These Aam1 and Cqm1 mutants maintained their Bin binding properties, confirming that glycosylation has no role in this interaction. The α-glucosidase activity of both proteins was next investigated, with Aam1 having a remarkably higher catalytic efficiency, influenced by changes in glycosylation. Molecular dynamics showed that glycosylated and nonglycosylated Aam1 models displayed distinct patterns that could influence their catalytic activity. Differential N-glycosylation may then be associated with higher catalytic efficiency in Aam1, enhancing the functional diversity of related orthologs.
Assuntos
Aedes/enzimologia , Culex/enzimologia , alfa-Glucosidases/metabolismo , Animais , Glicosilação , Bacilos Gram-Positivos , Simulação de Dinâmica MolecularRESUMO
The Cqm1 α-glucosidase, expressed within the midgut of Culex quinquefasciatus mosquito larvae, is the receptor for the Binary toxin (Bin) from the entomopathogen Lysinibacillus sphaericus. Mutations of the Cqm1 α-glucosidase gene cause high resistance levels to this bacterium in both field and laboratory populations, and a previously described allele, cqm1REC, was found to be associated with a laboratory-resistant colony (R2362). This study described the identification of a novel resistance allele, cqm1REC-2, that was co-selected with cqm1REC within the R2362 colony. The two alleles display distinct mutations but both generate premature stop codons that prevent the expression of midgut-bound Cqm1 proteins. Using a PCR-based assay to monitor the frequency of each allele during long-term maintenance of the resistant colony, cqm1REC was found to predominate early on but later was replaced by cqm1REC-2 as the most abundant resistance allele. Homozygous larvae for each allele were then generated that displayed similar high-resistance phenotypes with equivalent low levels of transcript and lack of protein expression for both cqm1REC and cqm1REC-2. In progeny from a cross of homozygous individuals for each allele at a 1 : 1 ratio, analyzed for ten subsequent generations, cqm1REC showed a higher frequency than cqm1REC-2. The replacement of cqm1REC by cqm1REC -2 observed in the R2362 colony, kept for 210 generations, indicates changes in fitness related to traits that are unknown but linked to these two alleles, and constitutes a unique example of evolution of resistance within a controlled laboratory environment.
Assuntos
Bacillaceae/patogenicidade , Culex/genética , Culex/microbiologia , Animais , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Cruzamentos Genéticos , Culex/enzimologia , Evolução Molecular , Feminino , Frequência do Gene , Genes de Insetos , Infecções por Bactérias Gram-Positivas/genética , Infecções por Bactérias Gram-Positivas/microbiologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Larva/enzimologia , Larva/genética , Larva/microbiologia , Masculino , Mutação , Seleção Genética , alfa-Glucosidases/genética , alfa-Glucosidases/metabolismoRESUMO
BACKGROUND: Culex quinquefasciatus is a hematophagous insect from the Culicidae family that feeds on the blood of humans, dogs, birds and livestock. This species transmits a wide variety of pathogens between humans and animals. The midgut environment is the first location of pathogen-vector interactions for blood-feeding mosquitoes and the expression of specific peptidases in the early stages of feeding could influence the outcome of the infection. Trypsin-like serine peptidases belong to a multi-gene family that can be expressed in different isoforms under distinct physiological conditions. However, the confident assignment of the trypsin genes that are expressed under each condition is still a challenge due to the large number of trypsin-coding genes in the Culicidae family and most likely because they are low abundance proteins. METHODS: We used zymography for the biochemical characterization of the peptidase profile of the midgut from C. quinquefasciatus females fed on sugar. Protein samples were also submitted to SDS-PAGE followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for peptidase identification. The peptidases sequences were analyzed with bioinformatics tools to assess their distinct features. RESULTS: Zymography revealed that trypsin-like serine peptidases were responsible for the proteolytic activity in the midgut of females fed on sugar diet. After denaturation in SDS-PAGE, eight trypsin-like serine peptidases were identified by LC-MS/MS. These peptidases have structural features typical of invertebrate digestive trypsin peptidases but exhibited singularities at the protein sequence level such as: the presence of different amino acids at the autocatalytic motif and substrate binding regions as well as different number of disulfide bounds. Data mining revealed a group of trypsin-like serine peptidases that are specific to C. quinquefasciatus when compared to the culicids genomes sequenced so far. CONCLUSION: We demonstrated that proteomics approaches combined with bioinformatics tools and zymographic analysis can lead to the functional annotation of trypsin-like serine peptidases coding genes and aid in the understanding of the complexity of peptidase expression in mosquitoes.
Assuntos
Culex/enzimologia , Trato Gastrointestinal/enzimologia , Serina Endopeptidases/metabolismo , Sacarose/metabolismo , Sequência de Aminoácidos , Ração Animal , Animais , Feminino , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Dados de Sequência Molecular , Serina Endopeptidases/genéticaRESUMO
Despite the established role of Culex quinquefasciatus as a vector of various neurotropic viruses, such as the Rift Valley and West Nile viruses, as well as lymphatic filariasis, little is known regarding the organism's reproductive physiology. As in other oviparous animals, vitellogenin, the most important source of nutrients for the embryo development, is digested by intracellular proteases. Using mass spectrometry, we have identified two cathepsin B homologues partially purified by self-proteolysis of Cx. quinquefasciatus total egg extract. The transcriptional profile of these two cathepsin B homologues was determined by quantitative RT-PCR, and the enzymatic activity associated with the peptidase was determined in ovaries after female engorgement. According to the VectorBase (vectorbase.org) annotation, both cathepsin B homologues shared approximately 66% identity in their amino acid sequences. The two cathepsin B genes are expressed simultaneously in the fat body of the vitellogenic females, and enzymatic activity was detected within the ovaries, suggesting an extra-ovarian origin. Similar to the transcriptional profile of vitellogenin, cathepsin B transcripts were shown to accumulate post-blood meal and reached their highest expression at 36 h PBM. However, while vitellogenin expression decreased drastically at 48 h PBM, the expression of the cathepsins increased until 84 h PBM, at which time the females of our colony were ready for oviposition. The similarity between their transcriptional profiles strongly suggests a role for the cathepsin B homologues in vitellin degradation.
Assuntos
Catepsina B/metabolismo , Culex/enzimologia , Proteínas do Ovo/metabolismo , Sequência de Aminoácidos , Animais , Catepsina B/classificação , Catepsina B/genética , Bases de Dados Genéticas , Eletroforese em Gel de Poliacrilamida , Feminino , Hidrólise , Dados de Sequência Molecular , Ovário/metabolismo , Óvulo/metabolismo , Peptídeos/análise , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNA , Espectrometria de Massas em Tandem , Transcriptoma , Vitelogeninas/metabolismoRESUMO
BACKGROUND: Culex quinquefasciatus is the vector of many agents of human diseases, including Wuchereria bancrofti, the parasite that causes bancroftian filariasis, an endemic disease in Pernambuco State, Brazil. Although temephos is not currently used to control C. quinquefasciatus, the species might be under a selection process from incidental exposure to this compound. This study aimed to evaluate the susceptibility status of C. quinquefasciatus to temephos, using bioassays, and to investigate its putative resistance mechanisms through biochemical assays and screening of the G119S mutation in the acetylcholinesterase gene, which is associated with organophosphate resistance, carried out by PCR and sequencing. RESULTS: The results showed that only mosquitoes from Santa Cruz do Capibaribe (SC) had an alteration in their susceptibility status (RR = 7.2-fold), while the other populations were all susceptible to the insecticide. Biochemical assays showed increased activity for all esterases in SC, as well as evidence of acetylcholinesterase insensitivity. The G119S mutation was detected in this population with a frequency of 0.11, but it was not found in the remaining populations. CONCLUSION: These data show that mechanisms of temephos resistance have been selected in natural C. quinquefasciatus populations from Pernambuco, which could undermine future control actions.
Assuntos
Culex/efeitos dos fármacos , Inseticidas/toxicidade , Temefós/toxicidade , Acetilcolinesterase/genética , Acetilcolinesterase/metabolismo , Animais , Bioensaio , Brasil , Culex/enzimologia , Culex/genética , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Controle de Mosquitos , MutaçãoRESUMO
BACKGROUND: The mosquito Culex quinquefasciatu s, a widespread insect in tropical and sub-tropical regions of the world, is a vector of multiple arboviruses and parasites, and is considered an important risk to human and veterinary health. Proteolytic enzymes play crucial roles in the insect physiology including the modulation of embryonic development and food digestion. Therefore, these enzymes represent important targets for the development of new control strategies. This study presents zymographic characterization and comparative analysis of the proteolytic activity found in eggs, larval instars and pupae of Culex quinquefasciatus. METHODS: The proteolytic profiles of eggs, larvae and pupa of Cx. quinquefasciatus were characterized by SDS-PAGE co-polymerized with 0.1% gelatin, according to the pH, temperature and peptidase inhibitor sensitivity. In addition, the proteolytic activities were characterized in solution using 100 µM of the fluorogenic substrate Z-Phe-Arg-AMC. RESULTS: Comparison of the proteolytic profiles by substrate-SDS-PAGE from all preimaginal stages of the insect revealed qualitative and quantitative differences in the peptidase expression among eggs, larvae and pupae. Use of specific inhibitors revealed that the proteolytic activity from preimaginal stages is mostly due to trypsin-like serine peptidases that display optimal activity at alkaline pH. In-solution, proteolytic assays of the four larval instars using the fluorogenic substrate Z-Phe-Arg-AMC in the presence or absence of a trypsin-like serine peptidase inhibitor confirmed the results obtained by substrate-SDS-PAGE analysis. The trypsin-like serine peptidases of the four larval instars were functional over a wide range of temperatures, showing activities at 25°C and 65°C, with an optimal activity between 37°C and 50°C. CONCLUSION: The combined use of zymography and in-solution assays, as performed in this study, allowed for a more detailed analysis of the repertoire of proteolytic enzymes in preimaginal stages of the insect. Finally, differences in the trypsin-like serine peptidase profile of preimaginal stages were observed, suggesting that such enzymes exert specific functions during the different stages of the life cycle of the insect.