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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.

This study was conducted in four districts (Malakand, Swat, Bajaur and Shangla) of Northern Pakistan to investigate the prevalence, associated risk factors and phylogenetic analyses of Theileria and Anaplasma species in small ruminants. A total of 800 blood samples, 200 from each district, were collected from apparently healthy animals. PCR assays were performed using generic primers for Anaplasma spp. and Theileria spp. as well as species specific primers for A. ovis and T. ovis. Overall infection prevalence was 361/800 (45.1%). Theileria spp. infection prevalence (187/800, 23.3%) was higher than Anaplasma spp. (174/800, 21.7%). Amplified partial 18S rRNA genes were sequenced and enrolled animals were found to be infected by T. ovis (115/800, 14.3%), and at least two more Theileria species (72/800, 9%) were present (T. lestoquardi and T. annulata). All blood samples that were found to be positive for Anaplasma spp. were also positive for A. ovis. Infection prevalence was higher in sheep (227/361, 28.3%) compared to goats (134/361, 16.6%) (p

The tick Rhipicephalus microplus is an obligate hematophagous with a great impact in the cattle industry due to its expoliative and vectorial capacity to transmit diseases such as Babesiosis and Anaplasmosis. The control of this tick is based on the use of ixodicides, however, their inadequate use has selected highly resistant populations to these components. For this reason, the search for new forms of biological control turns out to be the most viable alternative. Currently, one of the questions unsolved in the biology of the tick, is the elucidation of some mechanisms involved in the host-parasite relationship. In this regard, during the engorging tick process, the host hormones in the blood may be regulating mechanisms related with establishment, stage differentiation, and reproduction, and even with the resistance or susceptibility to ixodicides. Several working groups worldwide have described the relationship between host hormones and parasites, and the course of the infection. Hormones exert their effects through the interaction with nuclear and membrane receptors, that results in genomic and nongenomic effects. Prolactin (PRL) is a peptide hormone produced and secreted mostly by specialized cells located in the adenohypophysis of mammals. In this work, we describe, for the first time, a PRL receptor-like gene (r-prl) encoded in the genome of R. microplus. Our results revealed the presence of the gene and the relative expression by PCR assays. The structural modeling and the molecular dynamics analysis showed a high similarity with the Homo sapiens PRL receptor, and interaction with the PRL molecule. The elucidation of the mechanisms involved in the host-parasite relationship mediated by hormones opens the possibility of new targets for the control of pests. [ABSTRACT FROM AUTHOR]

Background: Theileria spp. are responsible for ovine and caprine theileriosis, leading to significant morbidity and mortality in small ruminants. The present study aims to investigate Theileria spp. infections in small ruminants from Southern Punjab in Pakistan, and genetic characterize revealed Theileria spp. isolates. Methods: A total of 93 sheep and 107 goats were sampled between May and August 2022. Blood smears were examined microscopically, and PCR amplification targeting the 18S rRNA gene was performed to detect Theileria spp. Additionally, specific PCR assays targeting 18S rRNA and ms1 partial sequences were used to identify Theileria ovis and T. lestoquardi , respectively.  Results: The prevalence of Theileria spp. was significantly higher using PCR (13.5%) compared to microscopic screening (5%). Sheep showed a higher prevalence rate (19.4%) compared to goats (8.4%) ( p = 0.024). Young sheep aged ≤ 1 year were more commonly infected with Theileria spp. (41%) compared to older sheep ( p = 0.006). The prevalence of Theileria spp. was higher in sheep-only herds (37.3%) compared to goat-only herds (18%) or mixed-species herds (8.1%) ( p = 0.015). The prevalence rates of T. ovis and T. lestoquardi were 9% and 2.5%, respectively, with four animals (2 goats and 2 sheep) showing co-infection. Phylogenetic analysis revealed that our T. ovis 18S rRNA sequence clustered with previously reported sequences from sheep in Turkey, China, Spain, and goats in Tanzania. The obtained T. lestoquardi ms1 partial sequence formed a distinct cluster from other T. lestoquardi isolates in Pakistan and neighboring countries.  Conclusion: Theileria spp. co-circulation in Pakistani small ruminants, particularly the presence of T. ovis and T. lestoquardi , highlights the need for attention from animal health decision-makers due to their financial and health impacts.

Soft ticks pose significant health risks as vectors of various pathogens. This study explored the spatio-temporal distribution and genetic relationships of the soft tick species Argas persicus infesting domestic hens (Gallus gallus domesticus) across different districts in Pakistan. An examination of 778 hens revealed a notable tick infestation prevalence of 70.82%, with a total of 1299 ticks collected from 551 hens. The overall mean intensity was 2.19 soft ticks per infested chicken, and the overall mean abundance was 1.61 soft ticks per examined hen. Morphological identification confirmed all collected ticks (n = 1210) as A. persicus, comprising 719 males, 333 females, 121 nymphs, and 38 larvae. The Haveli, Muzaffarabad, and Kotli districts had the highest infestation rates, while Bagh had the lowest. Molecular analyses of tick DNA, focusing on 16S rDNA and 12S rDNA sequences, revealed genetic similarities among A. persicus soft ticks from Pakistan and other regions, providing insights into their evolutionary history. Importantly, no Babesia, Rickettsia, or Anaplasma infections were detected in the examined samples. These findings enhance the understanding of soft tick infestation patterns and the genetic diversity of A. persicus in the studied region., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). Published by IMR Press.)

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., (© 2024 The Author(s).)

Ticks and tick-borne diseases have gained increasing attention in recent years due to their impact on public health and significant losses in livestock production. The use of synthetic compounds for tick control is becoming problematic, mainly due to the resistance to commercially available products as well as their toxicity. Therefore, new alternative control methods are required. For this purpose, plant-derived extracts may be considered as effective repellents and/or acaricides. The present literature review focuses on studies evaluating the acaricidal and repellent activity of plant-derived extracts and plant secondary metabolites. We also noted recent advances in protein-ligand-docking simulation to examine the possible toxic effect of natural chemical compounds on ticks. In conclusion, plant-derived repellents/acaricides can be effective against ticks, especially in rural areas and livestock farms., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Ticks pose significant health risks to both wildlife and humans due to their role as vectors for various pathogens. In this study, we investigated tick infestation patterns, tick-associated pathogens, and genetic relationships within the tick species Amblyomma gervaisi, focusing on its prevalence in monitor lizards (Varanus bengalensis) across different districts in Pakistan. We examined 85 monitor lizards and identified an overall mean intensity of 19.59 ticks per infested lizard and an overall mean abundance of 11.98 ticks per examined lizard. All collected ticks (n = 1019) were morphologically identified as A. gervaisi, including 387 males, 258 females, 353 nymphs, and 21 larvae. The highest tick prevalence was observed in the Buner district, followed by Torghar and Shangla, with the lowest prevalence in Chitral. Lizard captures primarily occurred from May to October, correlating with the period of higher tick infestations. Molecular analysis was conducted on tick DNA, revealing genetic similarities among A. gervaisi ticks based on 16S rDNA and ITS2 sequences. Notably, we found the absence of A. gervaisi ITS2 sequences in the NCBI GenBank, highlighting a gap in existing genetic data. Moreover, our study identified the presence of pathogenic microorganisms, including Ehrlichia sp., Candidatus Ehrlichia dumleri, Anaplasma sp., Francisella sp., Rickettsia sp., and Coxiella sp., in these ticks. BLAST analysis revealed significant similarities between these pathogenic sequences and known strains, emphasizing the potential role of these ticks as vectors for zoonotic diseases. Phylogenetic analyses based on nuclear ITS2 and mitochondrial 16S rDNA genes illustrated the genetic relationships of A. gervaisi ticks from Pakistan with other Amblyomma species, providing insights into their evolutionary history. These findings contribute to our understanding of tick infestation patterns, and tick-borne pathogens in monitor lizards, which has implications for wildlife health, zoonotic disease transmission, and future conservation efforts. Further research in this area is crucial for a comprehensive assessment of the risks associated with tick-borne diseases in both wildlife and humans., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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 .

Sarcoptes scabiei is an ectoparasite of humans and animals that causes scabies. The Rhipicephalus (Boophilus) microplus is a blood-sucking ectoparasite that transmits various pathogens. These two parasites have caused great losses to a country's dairy and agriculture sectors. The aim of this study was to determine the in vitro and in silico efficacy of Camellia sinensis plant extracts. Different concentrations of C. sinensis ethanolic plant extracts were prepared using the maceration method and were used against mites and ticks (in adult immersion test AIT and larval packet test LPT) to evaluate their in vitro acaricidal activity. Additionally, in silico molecular docking was performed to investigate the inhibitory interactions between the phytochemicals of the plant and S. scabiei and R. microplus glutathione transferase proteins (SsGST and RmGST). This study observed that the plant extract showed high efficacy in vitro against mites and different tick stages in adult immersion and larval packet tests. Additionally, the in silico study revealed a strong binding interaction between ellagic acid and SsGST protein, with a binding energy of -7.3 kcal/mol, with respect to permethrin (-6.7 kcal/mol), whereas quercetin and RmGST resulted in a docking score of -8.6 kcal/mol compared to deltamethrin (-8.2 kcal/mol). Overall, this study explored the potential of C. sinensis as a natural alternative for controlling tick and mite infestations and provided insights into the inhibitory mechanisms of its phytochemicals., Competing Interests: The authors declare no conflict of interest.

Anaplasma (A.) ovis is the most important cause of anaplasmosis in small ruminants. The current study was planned to estimate the molecular prevalence, risk factors, and phylogenetic analysis of A. ovis infection in sheep and goats from different agro-climatic regions of Central and Southern Punjab, Pakistan. A total of 400 jugular blood samples were collected from asymptomatic goats ( n = 200) and sheep ( n = 200) from the Jhang and Dera Ghazi Khan districts from January 2021 to February, 2023. Two hundred blood samples were collected from each district. Ten union councils (UC) were randomly chosen from each district, and 20 samples were collected from each UC based on the multistage cluster sampling technique. The samples were analyzed with PCR targeting the major surface protein ( msp4) gene of A. ovis . The overall molecular prevalence of anaplasmosis was 57.5%. The disease occurrence was higher in Dera Ghazi Khan (61.5%) than in the Jhang district (53.5%). Infection positivity was greater in goats (65.5%) than in sheep (49.5%). Multivariate logistic regression analysis indicated that host species [sheep; Odds Ratio (OR) = 3.212; p = 0.000, Confidence Interval (CI) = 1.968-5.242], age (adult; OR = 2.606; p = 0.003, CI = 1.398-4.858), and acaricide use (never; OR = 13.671; p = 0.000, CI = 6.414-26.283) were significantly higher risk for A. ovis in small ruminants ( p < 0.05; OR > 1). The sequencing and phylogenetic analysis of four representative isolates in the current study (Genbank numbers; Goats: OQ302202, OQ302203; Sheep: OQ319592, OQ319593) revealed novel strains of A. ovis with 97-100% similarity from different countries. The msp4 -based goat isolates showed greater genetic diversity, while sheep genotypes showed homology with isolates from Italy, Spain, Hungary, Cyprus, Spain, Iran, and China. The current surveillance study will help in devising prevention and control strategies regarding anaplasmosis in small ruminants. However, there is a need for further study on the clinicopathological and vector competence aspects of these genotypes.

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.

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 10 5 conidia/mL. At a concentration of 10 7 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 10 8 conidia/mL. Furthermore, the tick larval progeny that hatched from the infected tick egg masses showed evident symptoms of Aspergillus infection after incubation.

Pinus roxburghii , also known by the name "Himalayan chir pine," belongs to the Pinaceae family. Rhipicephalus ( Boophilus ) microplus tick is one of the most significant bovine ectoparasites, making it a major vector of economically important tick-borne diseases. The researchers conducted adult immersion tests (AIT) and larval packet tests (LPT) to investigate the acaricidal effect of P. roxburghii plant extract on R. ( B. ) microplus and its potential modulatory function when used with cypermethrin. Eggs were also assessed for their weight, egg-laying index (IE), hatchability rate, and control rate. After exposure to essential extract concentrations ranging from 2.5 to 40 mg/mL for 48 h, adult female ticks' oviposition inhibition and unfed R. ( B. ) microplus larvae's mortality rates were analyzed. Engorged females exposed to P. roxburghii at 40 mg/mL had reduced biological activity (oviposition, IE) compared to positive and negative controls. A concentration of 40 mg/mL of P. roxburghii caused 90% mortality in R. ( B. ) microplus larvae, whereas cypermethrin (the positive control) caused 98.3% mortality in LPT. In AIT, cypermethrin inhibited 81% of oviposition, compared to the 40 mg/mL concentration of P. roxburghii , which inhibited 40% of the ticks' oviposition. Moreover, this study assessed the binding capacity of selected phytocompounds with the targeted protein. Three servers (SWISS-MODEL, RoseTTAFold, and TrRosetta) recreated the target protein RmGABACl's 3D structure. The modeled 3D structure was validated using the online servers PROCHECK, ERRAT, and Prosa. Molecular docking using Auto Dock VINA predicted the binding mechanisms of 20 drug-like compounds against the target protein. Catechin and myricetin showed significant interactions with active site residues of the target protein, with docking scores of -7.7 kcal/mol and -7.6 kcal/mol, respectively. In conclusion, this study demonstrated the acaricidal activity of P. roxburghii extract, suggesting its potential as an alternative natural acaricide for controlling R. ( B .) microplus.

Rhipicephalus microplus is a major bovine ectoparasite that negatively impacts the cattle industry. The acaricidal activity of Datura innoxia ethanolic plant extract against R. microplus , compared with trichlorfon, was examined using the adult immersion test (AIT), and larval packet test (LPT). In vitro acaricidal activity of the selected plant extract against R. microplus engorged females was evaluated at different concentrations (2.5, 5, 10, 20, and 40 mg/mL), and was the same for AIT and LPT. It was further supported by in silico molecular docking of D. innoxia 's 21 phytochemicals against the R. microplus Glutathione S-transferases (RmGST) protein's three-dimensional (3D) structure predicted by the trRosetta server. The modeled 3D structure was then evaluated and confirmed with PROCHECK, ERRAT, and Verify3D online servers. To predict the binding mechanisms of these compounds, molecular docking was performed using Auto dock Vina software, and molecular dynamic (MD) simulations were used to investigate the protein atom's dynamic motion. D. innoxia has a relatively higher inhibitory effect on oviposition (from 9.81% to 45.37%) and total larval mortality (42.33% at 24 h and 93.67% at 48 h) at 40 mg/mL. Moreover, the docking results showed that the chemicals norapoatropine and 7-Hydroxyhyoscyamine have strong interactions with active site residues of the target protein, with a docking score of -7.3 and -7.0 Kcal/mol, respectively. The current work also provided a computational basis for the inhibitors of Glutathione S-transferases that were studied in this research work, and this new knowledge should aid in creating new and effective acaricidal chemicals. Furthermore, this plant extract's acaricide activity and its effect on oviposition and larval mortality were established in this work for the first time, indicating the possible use of this extract in the management of ticks.

Ticks are hematophagous ectoparasites that infest a diverse number of vertebrate hosts. The tick immunobiology plays a significant role in establishing and transmitting many pathogens to their hosts. To control tick infestations, the acaricide application is a commonly used method with severe environmental consequences and the selection of tick-resistant populations. With these drawbacks, new tick control methods need to be developed, and the immune system of ticks contains a plethora of potential candidates for vaccine design. Additionally, tick immunity is based on an orchestrated action of humoral and cellular immune responses. Therefore, the actors of these responses are the object of our study in this review since they are new targets in anti-tick vaccine design. We present their role in the immune response that positions them as feasible targets that can be blocked, inhibited, interfered with, and overexpressed, and then elucidate a new method to control tick infestations through the development of vaccines. We also propose Extracellular Traps Formation (ETosis) in ticks as a process to eliminate their natural enemies and those pathogens they transmit (vectorial capacity), which results attractive since they are a source of acting molecules with potential use as vaccines.

Background: Acaricide resistance is a central problem for the control of the cattle tick Rhipicephalus microplus. The physiological effects and phenotypes of the mutations that cause acaricide resistance are not always well understood or characterized. Single nucleotide polymorphisms (SNPs) that confer cypermethrin knockdown resistance (kdr) have been reported in R. microplus. These SNPs have been associated and correlated with pyrethroid resistance although there is no direct physiological evidence that their presence does confer kdr in this organism., Methods: Resistant and susceptible strain resistance profiles were obtained using the larval packet discriminating dose assay. The relevant genomic regions of the para-sodium channel were amplified using standard PCR; SNPs were detected by sequencing the corresponding amplicons. Ovary response to cypermethrin exposure/treatment was evaluated using videometrical analysis., Results: We found that the pyrethroid resistance trait is stable in a resistant reference strain after years without selection, suggesting that the resistance conferring mutations are fixed in the population. In this strain, a change in the structure of the pre-synaptic para-sodium channel caused by the G184C, the C190A and the T2134A SNPs appears to confer resistance. These mutations are absent in the susceptible strain used as control. We demonstrate that cypermethrin blocks ovary contraction in cypermethrin-susceptible ticks. We also show that ovaries from organisms that carry the kdr associated SNPs still contract at cypermethrin concentrations that completely block ovary contraction in the susceptible strain. The configuration of the experimental system excludes a xenobiotic detoxification mechanism., Conclusions: This is the first report that presents physiological evidence that the presence of the G184C, the C190A, and the T2134A mutations in the para-sodium channel correlates with maintaining muscle contractility in R. microplus exposed to cypermethrin. These SNPs may confer cypermethrin resistance in this organism by avoiding presynaptic blockage, inhibiting the flaccid muscle paralysis characteristic of this acaricide. The videometric assay that we previously validated can be used to detect more rapidly than other assays that involve larval mortality kdr-like cypermethrin resistant tick strains, permitting to directly assay adult pre-engorged females after they are collected on the field without waiting until eggs are laid and larvae eclose.

The purpose of this work was to contribute to the understanding of the mechanism of action of two new ixodicides. The histological and ultrastructural alterations of Rhipicephalus microplus oocytes (San Alfonso strain) treated with two new ethyl-carbamates (ethyl-4-bromophenyl carbamate and ethyl-4-chlorophenyl carbamate) by the adult immersion test were evaluated by light microscopy and transmission electron microscopy. The effects of the carbamates on embryogenesis in eggs were evaluated by fluorescence microscopy using DAPI staining. Both ethyl-carbamates inhibited the maturation of most oocytes and induced a concentration-dependent decrease (r 2   = 0.5, p < 0.05) in the embryonation percentage in the small number of eggs oviposited by treated ticks. Evident ultrastructural alterations were observed in the oocytes from ticks exposed to the ethyl-carbamates, including modification of the chorion structure, myelinic bodies and autophagic vacuoles that were associated with degenerated organelles (mitochondria, endoplasmic reticulum and yolk granules), nucleolus fragmentation and chromatin clumping in germinal vesicles. In conclusion, these ethyl-carbamates affect the reproductive potential of R. microplus due to their negative effects on oogenesis and their repercussions for embryonic development., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Although genome sequencing has become a very promising approach to conduct microbial taxonomy, few labs have the resources to afford this especially when dealing with data sets of hundreds to thousands of isolates. The goal of this study was to identify the most adequate loci for inferring the phylogeny of the species within the genus Staphylococcus ; with the idea that those who cannot afford whole genome sequencing can use these loci to carry out species assignation confidently. We retrieved 177 orthologous groups (OGs) by using a genome-based phylogeny and an average nucleotide identity analysis. The top 26 OGs showed topologies similar to the species tree and the concatenation of them yielded a topology almost identical to that of the species tree. Furthermore, a phylogeny of just the top seven OGs could be used for species assignment. We sequenced four staphylococcus isolates to test the 26 OGs and found that these OGs were far superior to commonly used markers for this genus. On the whole, our procedure allowed identification of the most adequate markers for inferring the phylogeny within the genus Staphylococcus . We anticipate that this approach will be employed for the identification of the most suitable markers for other bacterial genera and can be very helpful to sort out poorly classified genera., Competing Interests: The authors declare there are no competing interests.

The goal of the present study was to assess the gene expression of xenobiotic metabolizing enzymes (XMEs) Cytochrome P-450 (CYP) and carboxylesterase (CE) related to detoxification of synthetic pyrethroids, plus acetylcholinesterase (AChE), in field isolates of acaricide-resistant Rhipicephalus microplus. The XMEs expression levels were assessed by mRNA measurement using quantitative reverse transcription PCR. The XME expression levels of field-isolated acaricide-resistant ticks were compared against acaricide-susceptible reference ticks used in this study as a gene expression baseline and represented as relative expression units (REU). Field isolates were subjected to toxicological bioassays and determined resistant to all the Pyr acaricides (Pyr), whereas most of them were found susceptible to organophosphorous acaricides (OP), with the exception of three isolates, which exhibited moderate resistance to Diazinon. Significantly higher levels of CYP were detected in pyrethroid-resistance ticks when compared to Su ticks (P<0.01). A linear regression analysis showed that pyrethroid acaricide resistance levels of R. microplus were proportional to the CYP expression levels (correlation coefficient (R):0.85; P<0.05). Analysis on CE expression levels showed only one isolate resistant to Pyr and OP with a statistically significant increase (P<0.01). AChE expression levels showed statistically significant (P<0.01) subexpression in all tick isolates when compared to the susceptible reference. Our results suggest that pyrethroid acaricide resistance in the cattle tick may be diagnosed by measuring the CYP expression levels using quantitative PCR.

The tyraminergic/octopaminergic system is central for the control of arthropod oviposition. Previous works demonstrated that the pharmacological perturbation of this system inhibits oviposition in the cattle tick Rhipicephalus microplus. In this work, we describe a physiologically active whole-mount preparation of the contractile tick ovary that allows the quantitative videometrical analysis of ovary contraction in response to different compounds. Eight adrenergic ligands known to inhibit oviposition, including octopamine and tyramine were tested. These compounds exhibited antagonistic effects; octopamine relaxes the ovary preparation while tyramine induces a very strong contraction. The other adrenergic compounds tested were classified as able to contract or relax ovary muscle tissue. Isoprotenerol has a stronger relaxative effect than octopamine. Tyramine induces the biggest contraction observed of all the compounds tested, followed, in descending amount of contraction, by salbutamol, prazosin, epinastine, clonidine and the acaricide amitraz. The effect of these adrenergic ligands on the ovary preparation, explains why these molecules inhibit tick oviposition and suggest a regulatory mechanism for ovary contraction and relaxation during oviposition. Our results also provide a physiological explanation of the egg-laying inhibition effect of amitraz when used on the cattle tick.

The cattle tick Rhipicephalus microplus, is one of the most damaging livestock ectoparasites. Tropical tick infestation limits the introduction of high-yield bovine varieties because they do not have immunity to the diseases transmitted by these ectoparasites. This tick is usually controlled with chemical acaricides but their indiscriminate use has created resistant populations. The discovery of new molecules that can be used for tick control is urgent. Based on the knowledge that octopamine, a biogenic amine analog to epinephrine, is central to the regulation of oviposition in several studied arthropods and that an imbalance in octopamine release causes sterility in a Drosophila model. Tyramine, octopamine and epinastine and 83 adrenergic compounds classified by their effect in the vertebrate systems were screened for their ability to block oviposition in Rhipicephalus microplus. Of these molecules, we found that 10 alpha-agonists, 3 alpha-antagonists, 5 beta-adrenergic agonists, 7 beta-antagonists and Norepinephrine were able to inhibit oviposition in this tick at pharmacological concentrations. Surprisingly, tyramine appears to be more potent than octopamine. The probable physiological causes of this inhibition are discussed. Our results suggest that although there are alpha adrenergic-like receptors in the tick, they do not behave in a manner completely analogous to their vertebrate counterparts., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Phospholipid-hydroperoxide glutathione peroxidase (PHGPx) enzymes are associated with cellular protection by the role they play in reducing hydroperoxides of phospholipids, thereby preventing membrane lipoperoxidation. As part of their toxic effect, some pesticides stimulate peroxidation of cellular membranes. We isolated and sequenced a PHGPx gene from the cattle tick Boophilus microplus that encodes a protein of 169 amino acids, including a TGA-encoded selenocysteine at residue 46 and active site residues Gln(82) and Trp(135) that interact with the selenocysteine. The motif that directs the insertion of selenocysteine at the opal codon is found in the 3'-untranslated region. PHGPx sequences from pesticide-resistant and susceptible B. microplus ticks show nucleotide differences at eight positions among the strains, with five resulting in amino acid substitutions in the deduced protein sequence. Two distinct PHGPx alleles were identified in an organophosphate-resistant tick strain. Real-time PCR quantification of gene expression revealed increased PHGPx in two strains resistant to a single acaricide class. Strains resistant to two or more classes showed a reduction in PHGPx.