A Culex quinquefasciatus strain resistant to the binary toxin from Lysinibacillus sphaericus displays altered enzyme activities and energy reserves.

BACKGROUND: The resistance of a Culex quinquefasciatus strain to the binary (Bin) larvicidal toxin from Lysinibacillus sphaericus is due to the lack of expression of the toxin's receptors, the membrane-bound Cqm1 α-glucosidases. A previous transcriptomic profile of the resistant larvae showed differentially expressed genes coding Cqm1, lipases, proteases and other genes involved in lipid and carbohydrate metabolism. This study aimed to investigate the metabolic features of Bin-resistant individuals by comparing the activity of some enzymes, energy reserves, fertility and fecundity to a susceptible strain. METHODS: The activity of specific enzymes was recorded in midgut samples from resistant and susceptible larvae. The amount of lipids and reducing sugars was determined for larvae and adults from both strains. Additionally, the fecundity and fertility parameters of these strains under control and stress conditions were examined. RESULTS: Enzyme assays showed that the esterase activities in the midgut of resistant larvae were significantly lower than susceptible ones using acetyl-, butyryl- and heptanoyl-methylumbelliferyl esthers as substrates. The α-glucosidase activity was also reduced in resistant larvae using sucrose and a synthetic substrate. No difference in protease activities as trypsins, chymotrypsins and aminopeptidases was detected between resistant and susceptible larvae. In larval and adult stages, the resistant strain showed an altered profile of energy reserves characterized by significantly reduced levels of lipids and a greater amount of reducing sugars. The fertility and fecundity of females were similar for both strains, indicating that those changes in energy reserves did not affect these reproductive parameters. CONCLUSIONS: Our dataset showed that Bin-resistant insects display differential metabolic features co-selected with the phenotype of resistance that can potentially have effects on mosquito fitness, in particular, due to the reduced lipid accumulation.

Culex quinquefasciatus Resistant to the Binary Toxin from Lysinibacillus sphaericus Displays a Consistent Downregulation of Pantetheinase Transcripts.

Culex quinquefasciatus resistance to the binary (Bin) toxin, the major larvicidal component from Lysinibacillus sphaericus, is associated with mutations in the cqm1 gene, encoding the Bin-toxin receptor. Downregulation of the cqm1 transcript was found in the transcriptome of larvae resistant to the L. sphaericus IAB59 strain, which produces both the Bin toxin and a second binary toxin, Cry48Aa/Cry49Aa. Here, we investigated the transcription profiles of two other mosquito colonies having Bin resistance only. These confirmed the cqm1 downregulation and identified transcripts encoding the enzyme pantetheinase as the most downregulated mRNAs in both resistant colonies. Further quantification of these transcripts reinforced their strong downregulation in Bin-resistant larvae. Multiple genes were found encoding this enzyme in Cx. quinquefasciatus and a recombinant pantetheinase was then expressed in Escherichia coli and Sf9 cells, with its presence assessed in the midgut brush border membrane of susceptible larvae. The pantetheinase was expressed as a ~70 kDa protein, potentially membrane-bound, which does not seem to be significantly targeted by glycosylation. This is the first pantetheinase characterization in mosquitoes, and its remarkable downregulation might reflect features impacted by co-selection with the Bin-resistant phenotype or potential roles in the Bin-toxin mode of action that deserve to be investigated.

Aedes aegypti Strain Subjected to Long-Term Exposure to Bacillus thuringiensis svar. israelensis Larvicides Displays an Altered Transcriptional Response to Zika Virus Infection.

Bacillus thuringiensis svar. israelensis (Bti) larvicides are effective in controlling Aedes aegypti; however, the effects of long-term exposure need to be properly evaluated. We established an Ae. aegypti strain that has been treated with Bti for 30 generations (RecBti) and is still susceptible to Bti, but females exhibited increased susceptibility to Zika virus (ZIKV). This study compared the RecBti strain to a reference strain regarding: first, the relative transcription of selected immune genes in ZIKV-challenged females (F30) with increased susceptibility detected in a previous study; then, the whole transcriptomic profile using unchallenged females (F35). Among the genes compared by RT-qPCR in the ZIKV-infected and uninfected females from RecBti (F30) and the reference strain, hop, domeless, relish 1, defensin A, cecropin D, and gambicin showed a trend of repression in RecBti infected females. The transcriptome of RecBti (F35) unchallenged females, compared with a reference strain by RNA-seq, showed a similar profile and only 59 differentially expressed genes were found among 9202 genes analyzed. Our dataset showed that the long-term Bti exposure of the RecBti strain was associated with an alteration of the expression of genes potentially involved in the response to ZIKV infection in challenged females, which is an important feature found under this condition.

Discovery of 1,2,4-oxadiazole derivatives as a novel class of noncompetitive inhibitors of 3-hydroxykynurenine transaminase (HKT) from Aedes aegypti.

The mosquito Aedes aegypti is the vector of arboviruses such as Zika, Chikungunya, dengue and yellow fever. These infectious diseases have a major impact on public health. The unavailability of effective vaccines or drugs to prevent or treat most of these diseases makes vector control the main form of prevention. One strategy to promote mosquito population control is the use of synthetic insecticides to inhibit key enzymes in the metabolic pathway of these insects, particularly during larval stages. One of the main targets of the kynurenine detoxification pathway in mosquitoes is the enzyme 3-hydroxykynurenine transaminase (HKT), which catalyzes the conversion of 3-hydroxykynurenine (3-HK) into xanthurenic acid (XA). In this work, we report eleven newly synthesized oxadiazole derivatives and demonstrate that these compounds are potent noncompetitive inhibitors of HKT from Ae. aegypti. The present data provide direct evidence that HKT can be explored as a molecular target for the discovery of novel larvicides against Ae. aegypti. More importantly, it ensures that structural information derived from the HKT 3D-structure can be used to guide the development of more potent inhibitors.

Co-selection and replacement of resistance alleles to Lysinibacillus sphaericus in a Culex quinquefasciatus colony.

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.

Conjugal transfer of a toxin-coding megaplasmid from Bacillus thuringiensis subsp. israelensis to mosquitocidal strains of Bacillus sphaericus.

Both Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis produce mosquitocidal toxins during sporulation and are extensively used in the field for control of mosquito populations. All the known toxins of the latter organism are known to be encoded on a large plasmid, pBtoxis. In an attempt to combine the best properties of the two bacteria, an erythromycin resistance-marked pBtoxis plasmid was transferred to B. sphaericus by a mating technique. The resulting transconjugant bacteria were significantly more toxic to Aedes aegypti mosquitoes and were able to overcome resistance to B. sphaericus in a resistant colony of Culex quinquefasciatus, apparently due to the production of Cry11A but not Cry4A or Cry4B. The stability of the plasmid in the B. sphaericus host was moderate during vegetative growth, but segregational instability was observed, which led to substantial rates of plasmid loss during sporulation.

Integrated control measures against Culex quinquefasciatus, the vector of filariasis in Recife

Integrated control measures against Culex quinquefasciastus have been implemented in a pilot urban area in Recife, Brazil. About 3,000 breeding sites found within the operational area were responsible for very high mosquito densities recorded during the pretrial period. Physical control measures have been applied to cess pits before starting a series of 37 treatments of the other sites with Bacillus sphaericus strain 2362, over 27 months. In spite of the difficulties due to environmental conditions, very significant reductions in preimaginal population of C. quinquefasciatus were achieved and, as a consequence, low adult mosquito densities were maintained for a relatively long period of time. Entomological and environmental data gathered in this pilot project can contribute to design an integrated mosquito control program in Recife city.