RESUMO

To conduct differential gene expression analysis, ovaries from the cattle tick Rhipicephalus microplus were dissected at three distinct developmental stages (preingurgitated, immature ingurgitated, and mature ingurgitated). Additionally, undissected intact mature males and complete ingurgitated female ticks without ovaries (carcasses) were also collected to serve as reference samples for analysis. To perform total RNA purification, tissue from ten individuals representing each of the five previously described conditions was pooled. mRNA was isolated from the purified total RNA using the oligo (dT) method. Following fragmentation, double stranded cDNA was synthesized and ligated to sequencing adapters. Suitable-sized fragments were subsequently used for PCR amplification. Libraries were analyzed and quantified using an Agilent 2100 Bioanalyzer and an ABI StepOnePlus Real-Time PCR System. A total of 45.64 Gb bases were sequenced using the Illumina HiSeq sequencing platform. After assembling the samples and correcting for abundance, we obtained 82,877 unigenes. The total length, average length, N50, and GC content of the unigenes were 89,754,828 bp,1,082 bp,2,068 bp and 49.04 % respectively. For functional annotation, the unigenes were aligned with 7 functional databases. The number of unigenes identified in the functional databases were as follows: 32,518 (NR:39.24 %), 10,259 (NT:12.38 %), 23,624 (Swissprot:28.50 %), 22,203 (KOG:26.79 %), 25,072 (KEGG:30.25 %), 17,435(GO:21.04 %), and 23,220 (InterPro:28.02 %). Unigene candidate coding regions (CDS) among the unigenes were predicted using TransDecoder software and 42,143 CDS were detected. We also detected 10,522 simple sequence repeats (SSRs) distributed on 8,126 unigenes, and predicted 4,672 transcription factors (TF) coding unigenes. Our data can be used to identify genes that are important for male and female tick and arachnid reproduction and tick general physiology.

RESUMO

BACKGROUND: Hormone receptors exert their function through binding with their ligands, which results in cellular signaling activation mediated by genomic or non-genomic mechanisms. The intrinsic molecular communication of tick Rhipicephalus microplus and its host Bos taurus comprises an endocrine regulation involving hormones. In the present study, we performed a molecular and in silico analysis of a Membrane Associated Progesterone Receptor in R. microplus (RmMAPRC). METHODS: The RmMAPRC protein sequence was analyzed with bioinformatics tools, and its structure was characterized by three-dimensional (3D) modeling and molecular docking. A semi-quantitative reverse transcription and polymerase chain reaction (sqRT-PCR) assessed the RmMAPRC gene presence and relative expression in tick organs and embryonic cells. RESULTS: RmMAPRC relative expression in salivary glands, ovaries, and embryonic cells showed overexpression of 3%, 13%, and 24%, respectively. Bioinformatic analysis revealed that RmMAPRC corresponded to a Progesterone Receptor Membrane Component 1 (RmPGRMC1) of ~23.7 kDa, with an N-terminal transmembrane domain and a C-terminal Cytochrome b5-like heme/steroid binding domain. The docking results suggest that RmPGRMC1 could bind to progesterone (P4), some progestins, and P4 antagonists. The phylogenetic reconstruction showed that Rhipicephalus spp. MAPRC receptors were clustered in a clade that includes R. appendiculatus, R. sanguineus, and R. microplus (RmMAPRC), and mammals and helminths MAPRC receptors clustered in two separated clades away from ticks. CONCLUSIONS: The presence of RmPGRMC1 highlights the importance of transregulation as a conserved adaptive mechanism that has succeeded for arthropod parasites, making it a target for tick control.

Assuntos

RESUMO

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.

RESUMO

The expression of the Fasciola hepatica carboxylesterase type B (CestB) gene is known to be induced upon exposure to the anthelmintic triclabendazole (TCBZ), leading to a substantial rise in enzyme-specific activity. Furthermore, the nucleotide sequence of the CestB gene displays variations that can potentially result in radical amino acid substitutions at the ligand binding site. These substitutions hold the potential to impact both the ligand-protein interaction and the catalytic properties of the enzyme. Thus, the objective of our study was to identify novel CestB polymorphisms in TCBZ-resistant parasites and field isolates obtained from a highly endemic region in Central Mexico. Additionally, we aimed to assess these amino acid polymorphisms using 3D modeling against the metabolically oxidized form of the anthelmintic TCBZSOX. Our goal was to observe the formation of TCBZSOX-specific binding pockets that might provide insights into the role of CestB in the mechanism of anthelmintic resistance. We identified polymorphisms in TCBZ-resistant parasites that exhibited three radical amino acid substitutions at positions 147, 215, and 263. These substitutions resulted in the formation of a TCBZSOX-affinity pocket with the potential to bind the anthelmintic drug. Furthermore, our 3D modeling analysis revealed that these amino acid substitutions also influenced the configuration of the CestB catalytic site, leading to alterations in the enzyme's interaction with chromogenic carboxylic ester substrates and potentially affecting its catalytic properties. However, it is important to note that the TCBZSOX-binding pocket, while significant for drug binding, was located separate from the enzyme's catalytic site, rendering enzymatic hydrolysis of TCBZSOX impossible. Nonetheless, the observed increased affinity for the anthelmintic may provide an explanation for a drug sequestration type of anthelmintic resistance. These findings lay the groundwork for the future development of a molecular diagnostic tool to identify anthelmintic resistance in F. hepatica.

RESUMO

The search for targets to control ticks and tick-borne diseases has been an ongoing problem, and so far, we still need efficient, non-chemical alternatives for this purpose. This search must consider new alternatives. For example genomics analysis is a widely applied tool in veterinary health studies to control pathogens. On the other hand, we propose that regulation of endocrine mechanisms represents a feasible alternative to biologically controlling tick infestations. Thus, we performed the molecular identification of an estrogen-related receptor gene of Rhipicephalus microplus called RmERR by RT-PCR in tick ovaries, embryonic cells, and hemolymph, which allowed us to analyze its expression and propose potential functions in endocrine mechanisms and developmental stages. In addition, we performed an in silico characterization to explore the molecular interactions of RmERR with different estrogens, estrogenic antagonists, and endocrine disruptor Bisphenol A (BPA), finding potential interactions predicted by docking analysis and supported by negative values of ΔG (which suggests the potential interaction of RmERR with the molecules evaluated). Additionally, phylogenetic reconstruction revealed that RmERR is grouped with other tick species but is phylogenetically distant from host vertebrates' ERRs. In summary, this study allowed for the identification of an ERR in cattle tick R. microplus for the first time and suggested its interaction with different estrogens, supporting the idea of a probable transregulation process in ticks. The elucidation of this interaction and its mechanisms unveiled its potential as a target to develop tick control strategies.

RESUMO

Aspergillus flavus has been found to be an effective entomopathogenic fungus for various arthropods, including ticks. In particular, natural fungal infections in cattle ticks show promise for biocontrol of the Rhipicephalus (Boophilus) microplus tick, which is a major ectoparasite affecting cattle worldwide. Our study aimed to elucidate the specific entomopathogenic virulence factors encoded in the genome of an A. flavus strain isolated from naturally infected cattle ticks. We performed morphological and biochemical phenotyping alongside complete genome sequencing, which revealed that the isolated fungus was A. flavus related to the L morphotype, capable of producing a range of gene-coded entomopathogenic virulence factors, including ribotoxin, aflatoxin, kojic acid, chitinases, killer toxin, and satratoxin. To evaluate the efficacy of this A. flavus strain against ticks, we conducted experimental bioassays using healthy engorged female ticks. A morbidity rate of 90% was observed, starting at a concentration of 105 conidia/mL. At a concentration of 107 conidia/mL, we observed a 50% mortality rate and a 21.5% inhibition of oviposition. The highest levels of hatch inhibition (30.8%) and estimated reproduction inhibition (34.64%) were achieved at a concentration of 108 conidia/mL. Furthermore, the tick larval progeny that hatched from the infected tick egg masses showed evident symptoms of Aspergillus infection after incubation.

RESUMO

As the most important bovine ectoparasite, the southern cattle tick Rhipicephalus microplus transmits lethal cattle diseases such as babesiosis and anaplasmosis, costing the global livestock industry billions of dollars annually. To control cattle ticks, preventive treatment of cattle with pesticides is a common practice; however, after decades of chemical treatment, pesticide resistance has arisen in cattle ticks, rendering most formulations ineffective over time. Facing the perspective of running out of effective chemical treatments against R. microplus, research on biocontrol alternatives is necessary. Acaro-pathogenic microorganisms isolated from different developmental stages of R. microplus offer potential as biocontrol agents. Aspergillus flavus strain INIFAP-2021, isolated from naturally infected cattle ticks, produced high levels of mobility and mortality in the tick population during experimental infections. The whole genome of the fungi was sequenced using the DNBSEQ platform by BGI. The genome was assembled using SOAPaligner, and A. flavus NRRL3357 was used as the reference genome; the complete genome contained eight pairs of chromosomes and 36.9 Mb with a GC content of 48.03%, exhibiting 11482 protein-coding genes. The final genome assembly was deposited at GenBank as a bio project under accession number PRJNA758689, and supplementary material is accessible through Mendeley DOI: 10.17632/mt8yxch6mz.1.

RESUMO

Fasciola hepatica anthelmintic resistance may be associated with the catalytic activity of xenobiotic metabolizing enzymes. The gene expression of one of these enzymes, identified as carboxylesterase B (CestB), was previously described as inducible in adult parasites under anthelmintic treatment and exhibited a single nucleotide polymorphism at position 643 that translates into a radical amino acid substitution at position 215 from Glutamic acid to Lysine. Alphafold 3D models of both allelic sequences exhibited a significant affinity pocket rearrangement and different ligand-docking modeling results. Further bioinformatics analysis confirmed that the radical amino acid substitution is located at the ligand affinity site of the enzyme, affecting its affinity to serine hydrolase inhibitors and preferences for ester ligands. A field genotyping survey from parasite samples obtained from two developmental stages isolated from different host species from Argentina and Mexico exhibited a 37% allele distribution for 215E and a 29% allele distribution for 215K as well as a 34% E/K heterozygous distribution. No linkage to host species or geographic origin was found in any of the allele variants.

Assuntos

RESUMO

Ticks are hematophagous ectoparasites with importance to animal and human health. In recent years, the study of ticks has had significant development, including immune response, vector-host interactions, physiological and multi-omics approaches. However, one of the main impediments is obtaining a significant amount of high-quality hemolymph. For this reason, we developed a protocol that allows obtaining up to 100 µl of hemolymph free of host blood per engorged tick. The technique consists of continuous hipocuticular punctures of the tick dorsum and an anticoagulant buffer that impedes hemolymph coagulation, allowing constant extravasation and ensuring high yields. Additionally, the hemocytes recovered with this protocol are intact and can be used for further analysis. The high-quality hemolymph obtained using this protocol and its applications will help to better understand the processes involving the hemolymph and its components. Although there are other hemolymph extraction protocols, the method developed here is very well suited for Rhipicephalus microplus, and in our experience, results in better yields and high-quality samples.

RESUMO

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.