Isolation of the monooxygenase complex from Rhipicephalus (Boophilus) microplus:Clues to understanding acaricide resistance

The monooxygenase complex is composed of three key proteins, a cytochrome P450 (CYP), the cytochrome P450 oxidoreductase (CPR) and cytochrome b5 and plays a key role in the metabolism and detoxification of xenobiotic substances, including pesticides. In addition, overexpression of these components has been linked to pesticide resistance in several important vectors of disease. Despite this, the monooxygenase complex has not been isolated from the Southern cattle tick Rhipicephalus (Boophilus) microplus, a major disease vector in livestock. Using bioinformatics 115 transcriptomic sequences were analyzed to identify putative pesticide metabolizing CYPs. RACE-PCR was used to amplify the full length sequence of one CYP; CYP3006G8 which displays a high degree of homology to members of the CYP6 and 9 subfamilies, known to metabolize pyrethroids. mRNA expression levels of CYP3006G8 were investigated in 11 strains of R. microplus with differing resistance profiles by qPCR, the results of which indicated a correlation with pyrethroid metabolic resistance. In addition to this gene, the sequences for CPR and cytochrome b5 were also identified and subsequently isolated from R. microplus using PCR. CYP3006G8 is only the third CYP gene isolated from R. microplus and the first to putatively metabolize pesticides. The initial results of expression analysis suggest that CYP3006G8 metabolizes pyrethroids but further biochemical characterization is required to confirm this. Differences in the kinetic parameters of human and mosquito CPR in terms of NADPH binding have been demonstrated and could potentially be used to design species specific pesticides. Similar differences in the tick CPR would confirm that this is a characteristic of heamatophagous arthropods

Transcriptome-Based Identification of a Functional Fasciola hepatica Carboxylesterase B

Bioinformatics analysis of the complete transcriptome of Fasciola hepatica, identified a total of ten putative carboxylesterase transcripts, including a 3146 bp mRNA transcript coding a 2205 bp open reading frame that translates into a protein of 735 amino acids, resulting in a predicted protein mass of 83.5 kDa and a putative carboxylesterase B enzyme. The gene coding for this enzyme was found in two reported F. hepatica complete genomes stretching 23,230 bp, containing two exons of 1282 and 1864 bp, respectively, as well as a 20,084 bp intron between the exons. The enzymatic activity was experimentally assayed on F. hepatica protein extracts by SDS-PAGE zymograms using synthetic chromogenic substrates, confirming both the theoretical molecular weight and carboxylesterase enzymatic activity. Further bioinformatics predicted that this enzyme is an integral component of the cellular membrane that should be active as a 167 kDa homodimer complex and polyacrylamide gel electrophoresis (PAGE) zymograms experiments confirmed the analysis. Additional bioinformatics analysis showed that DNA sequences that code for this particular enzyme are highly conserved in other parasitic trematodes, although they are labeled hypothetical proteins.Fil: Pedroza Gómez, Yaretzi J.. Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias; MéxicoFil: Cossio Bayugar, Raquel. Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias; MéxicoFil: Aguilar Díaz, Hugo. Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias; MéxicoFil: Scarcella, Silvana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Reynaud, Enrique. Universidad Nacional Autónoma de México; MéxicoFil: del Rayo Sanchez Carbente, María. Universidad Nacional Autónoma de México; MéxicoFil: Narváez Padilla, Verónica. Universidad Nacional Autónoma de México; MéxicoFil: Miranda Miranda, Estefan. Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias; Méxic

Relationship between acaricide resistance and acetylcholinesterase gene polymorphisms in the cattle tick

In this study, we aimed to develop a comprehensive methodology for identifying amino acid polymorphisms in acetylcholinesterase transcript 2 (AChE2) in acaricide-resistant Rhipicephalus microplus ticks. This included assessing AChE2 expression levels through qPCR and conducting 3D modeling to evaluate the interaction between acaricides and AChE2 using docking techniques. The study produced significant results, demonstrating that acaricide-resistant R. microplus ticks exhibit significantly higher levels of AChE expression than susceptible reference ticks. In terms of amino acid sequence, we identified 9 radical amino acid substitutions in AChE2 from acaricide-resistant ticks, when compared to the gene sequence of the susceptible reference strain. To further understand the implications of these substitutions, we utilized 3D acaricide-AChE2 docking modeling to examine the interaction between the acaricide and the AChE2 catalytic site. Our models suggest that these amino acid polymorphisms alter the configuration of the binding pocket, thereby contributing to differences in acaricide interactions and ultimately providing insights into the acaricide-resistance phenomenon in R. microplus

Efficacy of Entomopathogenic Staphylococcus Bacteria as a Biocontrol Agent against Rhipicephalus microplus Ticks: Assessing Reproductive Inhibition and Mortality Rates

Rhipicephalus microplus is a persistent ectoparasite of cattle that causes bovine anaplasmosis and babesiosis, causing economic losses worldwide. Chemical treatment is the primary method for tick control, but the emergence of pesticide-resistant ticks is a major challenge. Alternative biocontrol strategies utilizing entomopathogenic microorganisms are being explored. This study aimed to validate the species identification and assess the efficacy of four strains of Staphylococcus bacteria (S. shinii S1 and S-2, S. succinus, and S. xylosus) previously reported as being entomopathogenic to R. microplus ticks. According to the bioassays, S. shinii S-1 exhibited the greatest degree of reproductive inhibition (47%), followed by S. succinus (44.3%) at a concentration of 1 × 108 cfu/mL. S. xylosus displayed decreased reproductive inhibition (6.3%). In an additional bioassay, S. shinii S-1 exhibited a significant larval mortality of 67.63%, followed by S. succinus with 66.75%, S. shinni S-2 with 64.61%, and S. xylosus with 28.18% mortality. The common signs of infection observed on these ticks included swelling, yellowish exudate on the hypostome, and reduced limb mobility and color change, except for S. succinus, which did not cause color changes. These bacteria were naturally found on bovine skin. However, further studies are needed to confirm their potential as promising alternatives or complementary agents to existing acaricidal compounds

In Situ Localization of <i>Staphylococcus shinii</i> and <i>Staphylococcus succinus</i> in Infected <i>Rhipicephalus microplus</i> Ticks: Implications for Biocontrol Strategies

Rhipicephalus microplus is a blood-sucking parasite that causes heavy infestations on cattle and is a vector for severe tick-borne diseases, such as anaplasmosis and babesiosis, and poses a significant threat to the cattle industry. Cattle ticks show increasing acaricide resistance, which creates an additional problem concerning the inefficient chemical control of tick populations in cattle-grazing areas, necessitating the exploration of alternative tick biocontrol methods. Our study aimed to demonstrate the acaropathogenic efficacy of two bacterial species during experimental infections on R. microplus. Our experimental data confirmed that S. shinii and S. succinus exhibited significant acaropathogenic properties against R. microplus, as demonstrated by the tracking of fluorescent-labeled bacteria within the engorged-tick body. Our experiments revealed that both bacterial species could infect the hemolymph, salivary glands, and vestibular vagina of the tick, inducing histological changes in the affected organs that may impair feeding as well as reproductive capabilities. Gené’s organ infection was detected only in S. succinus. Our findings offer valuable insights for developing biocontrol strategies to manage Rhipicephalus microplus populations effectively

Natural occurrence of lethal aspergillosis in the cattle tick<i>Rhipicephalus (Boophilus) microplus</i>(Acari:Ixodidae)

SUMMARYThe purpose of this study was to describe an unreported entomopathogenic fungus that naturally infects the cattle tickRhipicephalus (Boophilus) microplus(Acari: Ixodidae). Engorged female ticks, showed symptoms of fungal infection after controlled tick infestation of cattle. Infected ticks developed a distinctive dark colour, a pale mould grew over the cuticle and the ticks eventually died covered with fungal conidiophores. The responsible fungus was isolated and cultured on mycological medium and submitted to microscopic morphology, biochemical phenotyping and 18S rRNA ribotyping analyses, which identified it as aflatoxin-producingAspergillus flavus. Spores from the cultured fungus were experimentally sprayed over healthy engorged female ticks, obtaining an 80% prevalence of experimental infection of healthy ticks and their egg masses, the larval progeny after incubation under laboratory conditions was also infected. These results demonstrate thatA. flavusis the causative agent of the natural fungal disease of the cattle tickR. microplusdescribed here.</jats:p