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.

Bacterial Toxins Active against Mosquitoes: Mode of Action and Resistance.

Larvicides based on the bacteria Bacillus thuringiensis svar. israelensis (Bti) and Lysinibacillus sphaericus are effective and environmentally safe compounds for the control of dipteran insects of medical importance. They produce crystals that display specific and potent insecticidal activity against larvae. Bti crystals are composed of multiple protoxins: three from the three-domain Cry type family, which bind to different cell receptors in the midgut, and one cytolytic (Cyt1Aa) protoxin that can insert itself into the cell membrane and act as surrogate receptor of the Cry toxins. Together, those toxins display a complex mode of action that shows a low risk of resistance selection. L. sphaericus crystals contain one major binary toxin that display an outstanding persistence in field conditions, which is superior to Bti. However, the action of the Bin toxin based on its interaction with a single receptor is vulnerable for resistance selection in insects. In this review we present the most recent data on the mode of action and synergism of these toxins, resistance issues, and examples of their use worldwide. Data reported in recent years improved our understanding of the mechanism of action of these toxins, showed that their combined use can enhance their activity and counteract resistance, and reinforced their relevance for mosquito control programs in the future years.

Aedes aegypti continuously exposed to Bacillus thuringiensis svar. israelensis does not exhibit changes in life traits but displays increased susceptibility for Zika virus.

BACKGROUND: Aedes aegypti can transmit arboviruses worldwide, and Bacillus thuringiensis svar. israelensis (Bti)-based larvicides represent an effective tool for controlling this species. The safety of Bti and lack of resistance have been widely reported; however, little is known regarding the impact of the extensive use of these larvicides on the life traits of mosquitoes. Therefore, this study investigated biological parameters, including susceptibility to arbovirus, of an Ae. aegypti strain (RecBti) subjected to 29 generations of exposure to Bti compared with the RecL reference strain. METHODS: The biological parameters of individuals reared under controlled conditions were compared. Also, the viral susceptibility of females not exposed to Bti during their larval stage was analysed by oral infection and followed until 14 or 21 days post-infection (dpi). RESULTS: RecBti individuals did not display alterations in the traits that were assessed (fecundity, fertility, pupal weight, developmental time, emergence rate, sex ratio and haematophagic capacity) compared to RecL individuals. Females from both strains were susceptible to dengue serotype 2 (DENV-2) and Zika virus (ZIKV). However, RecBti females showed significantly higher rates of ZIKV infection compared with RecL females at 7 (90% versus 68%, Chi-square: χ2 = 7.27, df = 1, P = 0.006) and 14 dpi (100% versus 87%, Chi-square: χ2 = 7.69, df = 1, P = 0.005) and for dissemination at 7 dpi (83.3% versus 36%, Fisher's exact test: P < 0.0001, OR = 0.11, 95% CI 0.03-0.32). Quantification of DENV-2 and ZIKV viral particles produced statistically similar results for females from both strains. CONCLUSIONS: Prolonged exposure of Ae. aegypti larvae to Bti did not alter most of the evaluated biological parameters, except that RecBti females exhibited a higher vector susceptibility for ZIKV. This finding is related to a background of Bti exposure for several generations but not to a previous exposure of the tested females during the larval stage. This study highlights mosquito responses that could be associated with the chronic exposure to Bti in addition to the primary larvicidal effect elicited by this control agent.

Molecular and biological features of Culex quinquefasciatus homozygous larvae for two cqm1 alleles that confer resistance to Lysinibacillus sphaericus larvicides.

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.

In vivo nanoscale analysis of the dynamic synergistic interaction of Bacillus thuringiensis Cry11Aa and Cyt1Aa toxins in Aedes aegypti.

The insecticidal Cry11Aa and Cyt1Aa proteins are produced by Bacillus thuringiensis as crystal inclusions. They work synergistically inducing high toxicity against mosquito larvae. It was proposed that these crystal inclusions are rapidly solubilized and activated in the gut lumen, followed by pore formation in midgut cells killing the larvae. In addition, Cyt1Aa functions as a Cry11Aa binding receptor, inducing Cry11Aa oligomerization and membrane insertion. Here, we used fluorescent labeled crystals, protoxins or activated toxins for in vivo localization at nano-scale resolution. We show that after larvae were fed solubilized proteins, these proteins were not accumulated inside the gut and larvae were not killed. In contrast, if larvae were fed soluble non-toxic mutant proteins, these proteins were found inside the gut bound to gut-microvilli. Only feeding with crystal inclusions resulted in high larval mortality, suggesting that they have a role for an optimal intoxication process. At the macroscopic level, Cry11Aa completely degraded the gastric caeca structure and, in the presence of Cyt1Aa, this effect was observed at lower toxin-concentrations and at shorter periods. The labeled Cry11Aa crystal protein, after midgut processing, binds to the gastric caeca and posterior midgut regions, and also to anterior and medium regions where it is internalized in ordered "net like" structures, leading finally to cell break down. During synergism both Cry11Aa and Cyt1Aa toxins showed a dynamic layered array at the surface of apical microvilli, where Cry11Aa is localized in the lower layer closer to the cell cytoplasm, and Cyt1Aa is layered over Cry11Aa. This array depends on the pore formation activity of Cry11Aa, since the non-toxic mutant Cry11Aa-E97A, which is unable to oligomerize, inverted this array. Internalization of Cry11Aa was also observed during synergism. These data indicate that the mechanism of action of Cry11Aa is more complex than previously anticipated, and may involve additional steps besides pore-formation activity.

Infestation of an endemic arbovirus area by sympatric populations of Aedes aegypti and Aedes albopictus in Brazil.

BACKGROUND Aedes aegypti and Aedes albopictus are the most important arbovirus vectors in the world. OBJECTIVES This study aimed to investigate and compare the infestation pattern of these species in a neighbourhood of Recife, Brazil, endemic for arboviruses in 2005 (T1) and 2013 (T2). METHODS Infestation, distribution and relative abundance of these sympatric species were recorded by egg collection using a network of 59 sentinel ovitraps (s-ovt) at fixed sampling stations for 12 months in T1 and T2. FINDINGS A permanent occupation pattern was detected which was characterised by the presence of egg-laying females of one or both species with a high ovitrap positivity index (94.3 to 100%) throughout both years analysed. In terms of abundance, the total of eggs collected was lower (p < 0.005) in T2 (146,153) than in T1 (281,103), although ovitraps still displayed a high index of positivity. The spatial distribution showed the presence of both species in 65.1% of the 148 s-ovt assessed, while a smaller number of traps exclusively contained Ae. aegypti (22%) or Ae. albopictus (13.2%) eggs. MAIN CONCLUSIONS Our comparative analysis demonstrated the robustness of the spatial occupation and permanence of Ae. aegypti and Ae. albopictus populations in this endemic urban area.

Functional Bacillus thuringiensis Cyt1Aa Is Necessary To Synergize Lysinibacillus sphaericus Binary Toxin (Bin) against Bin-Resistant and -Refractory Mosquito Species.

The binary (Bin) toxin from Lysinibacillus sphaericus is effective to mosquito larvae, but its utilization is threatened by the development of insect resistance. Bin toxin is composed of the BinB subunit required for binding to midgut receptors and the BinA subunit that causes toxicity after cell internalization, mediated by BinB. Culex quinquefasciatus resistance to this toxin is caused by mutations that prevent expression of Bin toxin receptors in the midgut. Previously, it was shown that the Cyt1Aa toxin from Bacillus thuringiensis subsp. israelensis restores Bin toxicity to Bin-resistant C. quinquefasciatus and to Aedes aegypti larvae, which are naturally devoid of functional Bin receptors. Our goal was to elucidate the mechanism involved in Cyt1Aa synergism with Bin in such larvae. In vivo assays showed that the mixture of Bin toxin, or its BinA subunit, with Cyt1Aa was effective to kill resistant larvae. However, no specific binding interaction between Cyt1Aa and the Bin toxin, or its subunits, was observed. The synergy between Cyt1Aa and Bin toxins is dependent on functional Cyt1Aa, as demonstrated by using the nontoxic Cyt1AaV122E mutant toxin affected in oligomerization and membrane insertion, which was unable to synergize Bin toxicity in resistant larvae. The synergism correlated with the internalization of Bin or BinA into anterior and medium midgut epithelial cells, which occurred only in larvae treated with wild-type Cyt1Aa toxin. This toxin is able to overcome failures in the binding step involving BinB receptor by allowing the internalization of Bin toxin, or its BinA subunit, into the midgut cells.IMPORTANCE One promising management strategy for mosquito control is the utilization of a mixture of L. sphaericus and B. thuringiensis subsp. israelensis insecticidal toxins. From this set, Bin and Cyt1Aa toxins synergize and display toxicity to resistant C. quinquefasciatus and to A. aegypti larvae, whose midgut cells lack Bin toxin receptors. Our data set provides evidence that functional Cyt1Aa is essential for internalization of Bin or its BinA subunit into such cells, but binding interaction between Bin and Cyt1Aa is not observed. Thus, this mechanism contrasts with that for the synergy between Cyt1Aa and the B. thuringiensis subsp. israelensis Cry toxins, where active Cyt1Aa is not necessary but a specific binding between Cry and Cyt1Aa is required. Our study established the initial molecular basis of the synergy between Bin and Cyt1Aa, and these findings enlarge our knowledge of their mode of action, which could help to develop improved strategies to cope with insect resistance.

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.

A differential transcriptional profile by Culex quinquefasciatus larvae resistant to Lysinibacillus sphaericus IAB59 highlights genes and pathways associated with the resistance phenotype.

BACKGROUND: The study of the mechanisms by which larvae of the Culex quinquefasciatus mosquito survive exposure to the entomopathogen Lysinibacillus sphaericus has benefited substantially from the generation of laboratory-selected colonies resistant to this bacterium. One such colony, RIAB59, was selected after regular long-term exposure of larvae to the L. sphaericus IAB59 strain. This strain is characterized by its ability to produce the well known Binary (Bin) toxin, and the recently characterized Cry48Aa/Cry49Aa toxin, able to kill Bin-resistant larvae. Resistance to Bin is associated with the depletion of its receptor, Cqm1 α-glucosidase, from the larvae midgut. This study aimed to identify novel molecules and pathways associated with survival of the RIAB59 larvae and the resistance phenotype. METHODS: A transcriptomic approach and bioinformatic tools were used to compare the profiles derived from the midguts of larvae resistant and susceptible to L. sphaericus IAB59. RESULTS: The RNA-seq profiles identified 1355 differentially expressed genes (DEGs), with 673 down- and 682 upregulated transcripts. One of the most downregulated DEGs was cqm1, which validates the approach. Other strongly downregulated mRNAs encode the enzyme pantetheinase, apolipoprotein D, lipases, heat-shock proteins and a number of lesser known and hypothetical polypeptides. Among the upregulated DEGs, the top most encodes a peroxisomal enzyme involved in lipid metabolism, while others encode enzymes associated with juvenile hormone synthesis, ion channels, DNA binding proteins and defense polypeptides. Further analyses confirmed a strong downregulation of several enzymes involved in lipid catabolism while the assignment of DEGs into metabolic pathways highlighted the upregulation of those related to DNA synthesis and maintenance, confirmed by their clustering into related protein networks. Several other pathways were also identified with mixed profiles of down- and upregulated transcripts. Quantitative RT-PCR confirmed the changes in levels seen for selected mRNAs. CONCLUSIONS: Our transcriptome-wide dataset revealed that the RIAB59 colony, found to be substantially more resistant to Bin than to the Cry48Aa/Cry49Aa toxin, developed a differential expression profile as well as metabolic features co-selected during the long-term adaptation to IAB59 and that are most likely linked to Bin resistance.

Long-term exposure of Aedes aegypti to Bacillus thuringiensis svar. israelensis did not involve altered susceptibility to this microbial larvicide or to other control agents.

BACKGROUND: Bacillus thuringiensis svar. israelensis (Bti) is an effective and safe biolarvicide to control Aedes aegypti. Its mode of action based on four protoxins disfavors resistance; however, control in endemic areas that display high mosquito infestation throughout the year requires continuous larvicide applications, which imposes a strong selection pressure. Therefore, this study aimed to investigate the effects of an intensive Bti exposure on an Ae. aegypti strain (RecBti), regarding its susceptibility to Bti and two of its protoxins tested individually, to other control agents temephos and diflubenzuron, and its profile of detoxifying enzymes. METHODS: The RecBti strain was established using a large egg sample (10,000) from Recife city (Brazil) and more than 290,000 larvae were subjected to Bti throughout 30 generations. Larvae susceptibility to larvicides and the activity of detoxifying enzymes were determined by bioassays and catalytic assays, respectively. The Rockefeller strain was the reference used for these evaluations. RESULTS: Bti exposure yielded an average of 74% mortality at each generation. Larvae assessed in seven time points throughout the 30 generations were susceptible to Bti crystal (resistance ratio RR ≤ 2.8) and to its individual toxins Cry11Aa and Cry4Ba (RR ≤ 4.1). Early signs of altered susceptibility to Cry11Aa were detected in the last evaluations, suggesting that this toxin was a marker of the selection pressure imposed. RecBti larvae were also susceptible (RR ≤ 1.6) to the other control agents, temephos and diflubenzuron. The activity of the detoxifying enzymes α- and ß-esterases, glutathione-S-transferases and mixed-function oxidases was classified as unaltered in larvae from two generations (F19 and F25), except for a ß-esterases increase in F25. CONCLUSIONS: Prolonged exposure of Ae. aegypti larvae to Bti did not evolve into resistance to the crystal, and no cross-resistance with temephos and diflubenzuron were recorded, which supports their sustainable use with Bti for integrated control practices. The unaltered activity of most detoxifying enzymes suggests that they might not play a major role in the metabolism of Bti toxins, therefore resistance by this mechanism is unlikely to occur. This study also highlights the need to establish suitable criteria to classify the status of larval susceptibility/resistance.

Insecticide susceptibility of Aedes albopictus and Ae. aegypti from Brazil and the Swiss-Italian border region.

BACKGROUND: Aedes aegypti and Ae. albopictus are two highly invasive mosquito species, both vectors of several viruses, including dengue, chikungunya and Zika. While Ae. aegypti is the primary vector in the tropics and sub-tropics, Ae. albopictus is increasingly under the public health watch as it has been implicated in arbovirus-transmission in more temperate regions, including continental Europe. Vector control using insecticides is the pillar of most control programmes; hence development of insecticide resistance is of great concern. As part of a Brazilian-Swiss Joint Research Programme we set out to assess whether there are any signs of existing or incipient insecticide resistance primarily against the larvicide Bacillus thuringiensis svar. israelensis (Bti), but also against currently applied and potentially alternative insecticides in our areas, Recife (Brazil) and the Swiss-Italian border region. METHODS: Following World Health Organization guidelines, dose-response curves for a range of insecticides were established for both colonized and field caught Ae. aegypti and Ae. albopictus. The larvicides included Bti, two of its toxins, Cry11Aa and Cry4Ba, Lysinibacillus sphaericus, Vectomax CG®, a formulated combination of Bti and L. sphaericus, and diflubenzuron. In addition to the larvicides, the Swiss-Italian Ae. albopictus populations were also tested against five adulticides (bendiocarb, dichlorodiphenyltrichloroethane, malathion, permethrin and λ-cyhalothrin). RESULTS: Showing a similar dose-response, all mosquito populations were fully susceptible to the larvicides tested and, in particular, to Bti which is currently used both in Brazil and Switzerland. In addition, there were no signs of incipient resistance against Bti as larvae were equally susceptible to the individual toxins, Cry11Aa and Cry4Ba. The field-caught Swiss-Italian populations were susceptible to the adulticides tested but DDT mortality rates showed signs of reduced susceptibility. CONCLUSIONS: The insecticides currently used for mosquito control in Switzerland and Brazil are still effective against the target populations. The present study provides an important reference as relatively few insecticide susceptibility surveys have been carried out with Ae. albopictus.

Identification of Cry48Aa/Cry49Aa toxin ligands in the midgut of Culex quinquefasciatus larvae.

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.

N-glycosylation influences the catalytic activity of mosquito α-glucosidases associated with susceptibility or refractoriness to Lysinibacillus sphaericus.

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.

A new allele conferring resistance to Lysinibacillus sphaericus is detected in low frequency in Culex quinquefasciatus field populations.

BACKGROUND: The Cqm1 α-glucosidase of Culex quinquefasciatus larvae acts as the midgut receptor for the binary toxin of the biolarvicide Lysinibacillus sphaericus. Mutations within the cqm1 gene can code for aberrant polypeptides that can no longer be properly expressed or bind to the toxin, leading to insect resistance. The cqm1 REC and cqm1 REC-2 alleles were identified in a laboratory selected colony and both displayed mutations that lead to equivalent phenotypes of refractoriness to L. sphaericus. cqm1 REC was first identified as the major resistance allele in this colony but it was subsequently replaced by cqm1 REC-2 , suggesting the better adaptive features of the second allele. The major aim of this study was to evaluate the occurrence of cqm1 REC-2 and track its origin in field populations where cqm1 REC was previously identified. METHODS: The screening of the cqm1 REC-2 allele was based on more than 2000 C. quinquefasciatus larvae from five localities in the city of Recife, Brazil and used a multiplex PCR assay that is also able to identify cqm1 REC . Full-length sequencing of the cqm1 REC-2 and selected cqm1 samples was performed to identify further polymorphisms between these alleles. RESULTS: The cqm1 REC-2 allele was found in field samples, specifically in two heterozygous individuals from a single locality with an overall frequency and distribution much lower than that observed for cqm1 REC . The full-length sequences from these two cqm1 REC-2 copies were almost identical to the cqm1 REC-2 derived from the resistant colony but displayed more than 30 SNPs when compared with cqm1 and cqm1 REC . The cqm1 REC and cqm1 REC-2 resistant alleles were found to be associated with two distinct sets of wild-type cqm1 variants found in field populations. CONCLUSIONS: The cqm1 REC-2 allele occurs in populations in Recife and was probably already present in the samples used to establish the laboratory resistant colony. The data generated indicates that cqm1 REC-2 can be selected in field populations, although its low frequency and distribution in Recife suggest that cqm1 REC-2 presents a lower risk of selection compared to cqm1 REC .

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.

Non conserved residues between Cqm1 and Aam1 mosquito α-glucosidases are critical for the capacity of Cqm1 to bind the Binary toxin from Lysinibacillus sphaericus.

The Binary (Bin) toxin from the entomopathogenic bacterium Lysinibacillus sphaericus acts on larvae of the culicid Culex quinquefasciatus through its binding to Cqm1, a midgut-bound α-glucosidase. Specific binding by the BinB subunit to the Cqm1 receptor is essential for toxicity however the toxin is unable to bind to the Cqm1 ortholog from the refractory species Aedes aegypti (Aam1). Here, to investigate the molecular basis for the interaction between Cqm1 and BinB, recombinant Cqm1 and Aam1 were first expressed as soluble forms in Sf9 cells. The two proteins were found to display the same glycosilation patterns and BinB binding properties as the native α-glucosidases. Chimeric constructs were then generated through the exchange of reciprocal fragments between the corresponding cqm1 and aam1 cDNAs. Subsequent expression and binding experiments defined a Cqm1 segment encompassing residues S129 and A312 as critical for the interaction with BinB. Through site directed mutagenesis experiments, replacing specific sets of residues from Cqm1 with those of Aam1, the 159GG160 doublet was required for this interaction. Molecular modeling mapped these residues to an exposed loop within the Cqm1's structure, compatible with a target site for BinB and providing a possible explanation for its lack of binding to Aam1.

The susceptibility of Aedes aegypti populations displaying temephos resistance to Bacillus thuringiensis israelensis: a basis for management.

BACKGROUND: Aedes aegypti is the vector of dengue virus, and its control is essential to prevent disease transmission. Among the agents available to control this species, biolarvicides based on Bacillus thuringiensis serovar israelensis (Bti) are an effective alternative to replace the organophosphate temephos for controlling populations that display resistance to this insecticide. The major goal of this study was to determine the baseline susceptibility of Brazilian Ae. aegypti populations to Bti, taking into account their background in terms of larvicide exposure, status of temephos resistance and the level of activity of detoxifying enzymes involved in metabolic resistance to insecticides. METHODS: Population samples were established under insectarium conditions. Larval susceptibility to temephos and Bti was evaluated through bioassays and lethal concentrations of these compounds were determined. Biochemical assays were performed to determine the specific activity of five detoxifying enzymes in these samples. RESULTS: Fourteen populations were characterized and, except for one case, all displayed resistance to temephos. Most populations were classified as highly resistant. The populations also showed increased activity of one or more detoxifying enzymes (glutathione-S-transferases, esterases and mixed function oxidases), regardless of their temephos resistance status. All populations analyzed were susceptible to Bti, and the lethal concentrations were similar to those detected in two laboratory susceptible colonies. The response to Bti showed little variation. A maximum resistance ratio of 2.1 was observed in two untreated populations, while in two Bti-treated populations, the maximum resistance ratio was 1.9. No positive correlation was found between temephos resistance, increased activity of detoxifying enzymes, and susceptibility to Bti. CONCLUSIONS: Data from this study show that all populations were susceptible to Bti, including twelve untreated and two treated populations that had been exposed to this agent for more than ten years. The temephos resistance and increased activity of detoxifying enzymes observed in thirteen populations was not correlated with changes in susceptibility to Bti. Our data show a lack of cross-resistance between these two compounds; thus, Bti can be used in an integrated control program to fight Ae. aegypti and counteract the temephos resistance that was found among all populations analyzed.

Novel mutations associated with resistance to Bacillus sphaericus in a polymorphic region of the Culex quinquefasciatus cqm1 gene.

Bin toxin from Bacillus sphaericus acts on Culex quinquefasciatus larvae by binding to Cqm1 midgut-bound receptors, and disruption of the cqm1 gene is the major cause of resistance. The goal of this work was to screen for a laboratory-selected resistance cqm1(REC) allele in field populations in the city of Recife, Brazil, and to describe other resistance-associated polymorphisms in the cqm1 gene. The cqm1(REC) allele was detected in the four nontreated populations surveyed at frequencies from 0.001 to 0.017, and sequence analysis from these samples revealed a novel resistant allele (cqm1(REC-D16)) displaying a 16-nucletotide (nt) deletion which is distinct from the 19-nt deletion associated with cqm1(REC). Yet a third resistant allele (cqm1(REC-D25)), displaying a 25-nt deletion, was identified in samples from a treated area exposed to B. sphaericus. A comparison of the three deletion events revealed that all are located within the same 208-nt region amplified during the screening procedure. They also introduce equivalent frameshifts in the sequence and generate the same premature stop codon, leading to putative transcripts encoding truncated proteins which are unable to locate to the midgut epithelium. The populations analyzed in this study contained a variety of alleles with mutations disrupting the function of the corresponding Bin toxin receptor. Their locations reveal a hot spot that can be exploited to assess the resistance risk through DNA screening.