The entomopathogen Bacillus sphaericus is an important tool for the vector control of Culex sp., and its effectiveness has been validated in field trials. The appearance of resistance to this bacterium, however, remains a threat to its use, and attempts have been made to understand the resistance mechanisms. Previous work showed that the resistance to B. sphaericus in a Culex quinquefasciatus colony is associated with the absence of the ≈ 60‐kDa binary toxin receptor in larvae midgut microvilli. Here, the gene encoding the C. quinquefasciatus toxin receptor, Cqm1, was cloned and sequenced from a susceptible colony. The deduced amino‐acid sequence confirmed its identity as an α‐glucosidase, and analysis of the corresponding gene sequence from resistant larvae implicated a 19‐nucleotide deletion as the basis for resistance. This deletion changes the ORF and originates a premature stop codon, which prevents the synthesis of the full‐length Cqm1. Expression of the truncated protein, however, was not detected when whole larvae extracts were probed with antibodies raised against an N‐terminal 45‐kDa recombinant fragment of Cqm1. It seems that the premature stop codon directs the mutated cqm1 to the nonsense‐mediated decay pathway of mRNA degradation. In‐gel assays confirmed that a single α‐glucosidase protein is missing from the resistant colony. Further in vitro affinity assays showed that the recombinant fragment binds to the toxin, and mapped the binding site to the N‐terminus of the receptor.

The glutathione S-transferases (GSTs) are enzymes involved in several distinct biological processes. In insects, the GSTs, especially delta and epsilon classes, play a key role in the metabolism of xenobiotics used to control insect populations. Here, we investigated its potential role in temephos resistance, examining the GSTE2 gene from susceptible (RecL) and resistant (RecR) strains of the mosquito Aedes aegypti, vector for several pathogenic arboviruses. Total GST enzymatic activity and the GSTE2 gene expression profile were evaluated, with the GSTE2 cDNA and genomic loci sequenced from both strains. Recombinant GSTE2 and mutants were produced in a heterologous expression system and assayed for enzyme kinetic parameters. These proteins also had their 3D structure predicted through molecular modeling. Our results showed that RecR has a profile of total GST enzymatic activity higher than RecL, with the expression of the GSTE2 gene in resistant larvae increasing six folds. Four exclusive RecR mutations were observed (L111S, I150V, E178A and A198E), which were absent in the laboratory susceptible strains. The enzymatic activity of the recombinant GSTE2 showed different kinetic parameters, with the GSTE2 RecR showing an enhanced ability to metabolize its substrate. The I150V mutation was shown to induce significant changes in catalytic parameters and a 3D modeling of GSTE2 mapped two of the RecR changes (L111S and I150V) near the enzyme's catalytic pocket, also implying an impact on its catalytic activity. Our results reinforce a potential role for GSTE2 in the metabolic resistance phenotype while contributing to the understanding of the molecular basis for the resistance mechanism.

Bin toxin fromBacillus sphaericusacts onCulex quinquefasciatuslarvae by binding to Cqm1 midgut-bound receptors, and disruption of thecqm1gene is the major cause of resistance. The goal of this work was to screen for a laboratory-selected resistancecqm1_RECallele in field populations in the city of Recife, Brazil, and to describe other resistance-associated polymorphisms in thecqm1gene. Thecqm1_RECallele 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 withcqm1_REC. Yet a third resistant allele (cqm1_REC-D25), displaying a 25-nt deletion, was identified in samples from a treated area exposed toB. 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.

The activity of the Bacillus sphaericus binary (Bin) toxin on Culex quinquefasciatus larvae depends on its specific binding to the Cqm1 receptor, a midgut membrane-bound α-glucosidase. A 19-nucleotide deletion in the cqm1 gene ( cqm1 _REC ) mediates high-level resistance to Bin toxin. Here, resistance in nontreated and B. sphaericus -treated field populations of C. quinquefasciatus was assessed through bioassays as well as a specific PCR assay designed to detect the cqm1 _REC allele in individual larvae. Resistance ratios at 90% lethal concentration, gathered through bioassays, were close to 1 and indicate that the selected populations had similar levels of susceptibility to B. sphaericus , comparable to that of a laboratory colony. A diagnostic PCR assay detected the cqm1 _REC allele in all populations investigated, and its frequency in two nontreated areas was 0.006 and 0.003, while the frequency in the B. sphaericus -treated population was significantly higher. Values of 0.053 and 0.055 were detected for two distinct sets of samples, and homozygote resistant larvae were found. Evaluation of Cqm1 expression in individual larvae through α-glucosidase assays corroborated the allelic frequency revealed by PCR. The data from this study indicate that the cqm1 _REC allele was present at a detectable frequency in nontreated populations, while the higher frequency in samples from the treated area is, perhaps, correlated with the exposure to B. sphaericus . This is the first report of the molecular detection of a biolarvicide resistance allele in mosquito populations, and it confirms that the PCR-based approach is suitable to track such alleles in target populations.

The eukaryotic initiation factor 4E (eIF4E) recognizes the mRNA cap structure and, together with eIF4G and eIF4A, form the eIF4F complex that regulates translation initiation in eukaryotes. In trypanosomatids, 2 eIF4E homologues (EIF4E3 and EIF4E4) have been shown to be part of eIF4F-like complexes with presumed roles in translation initiation. Both proteins possess unique N-terminal extensions, which can be targeted for phosphorylation. Here, we provide novel insights on the Leishmania infantum EIF4E4 function and regulation. We show that EIF4E4 is constitutively expressed throughout the parasite development but is preferentially phosphorylated in exponentially grown promastigote and amastigote life stages, hence correlating with high levels of translation. Phosphorylation targets multiple serine-proline or threonine-proline residues within the N-terminal extension of EIF4E4 but does not require binding to the EIF4E4's partner, EIF4G3, or to the cap structure. We also report that EIF4E4 interacts with PABP1 through 3 conserved boxes at the EIF4E4 N-terminus and that this interaction is a prerequisite for efficient EIF4E4 phosphorylation. EIF4E4 is essential for Leishmania growth and an EIF4E4 null mutant was only obtained in the presence of an ectopically provided wild type gene. Complementation for the loss of EIF4E4 with several EIF4E4 mutant proteins affecting either phosphorylation or binding to mRNA or to EIF4E4 protein partners revealed that, in contrast to other eukaryotes, only the EIF4E4-PABP1 interaction but neither the binding to EIF4G3 nor phosphorylation is essential for translation. These studies also demonstrated that the lack of both EIF4E4 phosphorylation and EIF4G3 binding leads to a non-functional protein. Altogether, these findings further highlight the unique features of the translation initiation process in trypanosomatid protozoa.