Differential gene expression between two deveopmental stages of the Gené\u27s organ in the cattle fever tick, Rhipicephalus (Boophilus) microplus

The southern cattle fever tick (SCFT), Rhipicephalus microplus, is considered the most economically important ectoparasite of livestock worldwide and ranks sixth among the most pesticide resistant arthropods globally. This single-host hard tick is a vector of the infectious agents causing bovine babesiosis and anaplasmosis. The estimated impact of these SCFT-borne diseases on the livestock industry in the world is $7 billion annually. This project focuses on the Gené’s organ and addresses the need to identify novel targets that can serve as the basis for sustainable SCFT control strategies. Wax secreted by the Gené’s organ protects tick eggs from desiccation, bacterial and fungal infections, inorganic chemicals, and pesticides. We identified 1,513 new transcripts to add to the R. microplus genome database. A comparison of the two developmental stages of the Gene’s organ through quantitative RNA-seq analysis revealed 5,454 differentially expressed transcripts. The RNA-seq data was confirmed through RT-PCR

Rhipicephalus microplus salivary gland molecules induce differential CD86 expression in murine macrophages

<p>Abstract</p> <p>Background</p> <p>Tick parasitism is a major impediment for cattle production in many parts of the world. The southern cattle tick, <it>Rhipicephalus </it>(<it>Boophilus</it>) <it>microplus</it>, is an obligate hematophagous parasite of domestic and wild animals that serves as vector of infectious agents lethal to cattle. Tick saliva contains molecules evolved to modulate host innate and adaptive immune responses which facilitates blood feeding and pathogen transmission. Tick feeding promotes CD4 T cell polarization to a Th2 profile usually accompanied by down-regulation of Th1 cytokines through as yet undefined mechanisms. Co-stimulatory molecules on antigen presenting cells are central to development of T cell responses including Th1 and Th2 responses. Tick induced changes to antigen presenting cell signal transduction pathways are largely unknown. Here we document the ability of <it>R</it>. <it>microplus </it>salivary gland extracts (SGE) to effect differential CD86 expression.</p> <p>Results</p> <p>We examined changes in co-stimulatory molecule expression in murine RAW 264.7 cells in response to <it>R</it>. <it>microplus </it>SGE exposure in the presence of the toll-like receptor 4 (TLR4) ligand, LPS. After 24 hrs, CD86, but not CD80, was preferentially up-regulated on mouse macrophage RAW 264.7 cells when treated with SGE and then LPS, but not SGE alone. CD80 and CD40 expression was increased with LPS, but the addition of SGE did not alter expression. Higher concentrations of SGE were less effective at increasing CD86 RNA expression. The addition of mitogen or extracellular kinase (MEK) inhibitor, PD98059, significantly reduced the ability for SGE to induce CD86 expression, indicating activation of MEK is necessary for SGE induced up-regulation.</p> <p>Conclusions</p> <p>Molecules in SGE of <it>R. microplus </it>have a concentration-dependent effect on differential up-regulation of CD86 in a macrophage cell line activated by the TLR4 ligand, LPS. This CD86 up-regulation is at least partially dependent on the ERK1/2 pathway and may serve to promote Th2 polarization of the immune response.</p

High-resolution melt (HRM) analysis for detection of SNPs associated with pyrethroid resistance in the southern cattle fever tick, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

The southern cattle fever tick, Rhipicephalus (Boophilus) microplus, is the most economically important ectoparasite of cattle worldwide. A limitation for sustainable control and eradication is the emergence of acaricide resistance among tick populations. Molecular diagnostic tools offer the opportunity to detect resistance rapidly, which can be complemented with confirmatory bioassays with larvae and adult ticks that are more resource and time consuming to generate. Synthetic pyrethroid resistance is one of the most prevalent and well-studied forms of resistance in arthropods, being linked with target site alterations in the sodium ion channel gene. Here, we report research on a novel molecular method to detect mutations in the para-sodium channel gene of R. microplus associated with acaricide resistance that is based on quantitative PCR high-resolution melt (HRM) analysis. Genomic DNA fragments of domains II and III of the para-sodium channel gene were amplified by real-time PCR in the presence of EVA®Green dye to test resistant and susceptible reference ticks from the U.S., Brazil, and Mexico. Larval packet tests with discriminating doses and a modified lethal time analysis were performed to confirm resistance to permethrin, cypermethrin, deltamethrin, and flumethrin in laboratory strains. Tick specimens collected from cattle that were inspected at the United States Port-of-Entry at the Texas-Mexico border were also genotyped. Previously described mutations associated with pyrethroid resistance (T170C, C190A, G184C, and T2134A) were successfully detected by qPCR-HRM in different genotypes and confirmed by sequencing. A novel non-synonymous SNP located at domain III (C2136A) and the G215T mutation in domain II, previously described only in Asian R. microplus and R. australis, were also detected with the HRM and confirmed by sequencing. This technique could be adapted for high-throughput screening, detection, and discovery of allele-specific mutations in cattle tick outbreak populations to inform eradication strategies in the USA. This knowledge could also be applied to integrated control programs in other parts of the world where R. microplus is endemic and where similar SNPs have been identified associated with pyrethroid resistance. This study highlights the existence of several mutations in the para-sodium channel gene in different combinations in field populations of R. microplus from Mexico

The 13th Southern Hemisphere Conference on the Teaching and Learning of Undergraduate Mathematics and Statistics

Ngā mihi aroha ki ngā tangata katoa and warm greetings to you all. Welcome to Herenga Delta 2021, the Thirteenth Southern Hemisphere Conference on the Teaching and Learning of Undergraduate Mathematics and Statistics. It has been ten years since the Volcanic Delta Conference in Rotorua, and we are excited to have the Delta community return to Aotearoa New Zealand, if not in person, then by virtual means. Although the limits imposed by the pandemic mean that most of this year’s 2021 participants are unable to set foot in Tāmaki Makaurau Auckland, this has certainly not stopped interest in this event. Participants have been invited to draw on the concept of herenga, in Te Reo Māori usually a mooring place where people from afar come to share their knowledge and experiences. Although many of the participants are still some distance away, the submissions that have been sent in will continue to stimulate discussion on mathematics and statistics undergraduate education in the Delta tradition. The conference invited papers, abstracts and posters, working within the initial themes of Values and Variables. The range of submissions is diverse, and will provide participants with many opportunities to engage, discuss, and network with colleagues across the Delta community. The publications for this thirteenth Delta Conference include publications in the International Journal of Mathematical Education in Science and Technology, iJMEST, (available at https://www.tandfonline.com/journals/tmes20/collections/Herenga-Delta-2021), the Conference Proceedings, and the Programme (which has created some interesting challenges around time-zones), by the Local Organizing Committee. Papers in the iJMEST issue and the Proceedings were peer reviewed by at least two reviewers per paper. Of the ten submissions to the Proceedings, three were accepted. We are pleased to now be at the business end of the conference and hope that this event will carry on the special atmosphere of the many Deltas which have preceded this one. We hope that you will enjoy this conference, the virtual and social experiences that accompany it, and take the opportunity to contribute to further enhancing mathematics and statistics undergraduate education. Ngā manaakitanga, Phil Kane (The University of Auckland | Waipapa Taumata Rau) on behalf of the Local Organising Committ

High-resolution melt (HRM) analysis for detection of SNPs associated with pyrethroid resistance in the southern cattle fever tick, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

The southern cattle fever tick, Rhipicephalus (Boophilus) microplus, is the most economically important ectoparasite of cattle worldwide. A limitation for sustainable control and eradication is the emergence of acaricide resistance among tick populations. Molecular diagnostic tools offer the opportunity to detect resistance rapidly, which can be complemented with confirmatory bioassays with larvae and adult ticks that are more resource and time consuming to generate. Synthetic pyrethroid resistance is one of the most prevalent and well-studied forms of resistance in arthropods, being linked with target site alterations in the sodium ion channel gene. Here, we report research on a novel molecular method to detect mutations in the para-sodium channel gene of R. microplus associated with acaricide resistance that is based on quantitative PCR high-resolution melt (HRM) analysis. Genomic DNA fragments of domains II and III of the para-sodium channel gene were amplified by real-time PCR in the presence of EVA®Green dye to test resistant and susceptible reference ticks from the U.S., Brazil, and Mexico. Larval packet tests with discriminating doses and a modified lethal time analysis were performed to confirm resistance to permethrin, cypermethrin, deltamethrin, and flumethrin in laboratory strains. Tick specimens collected from cattle that were inspected at the United States Port-of-Entry at the Texas-Mexico border were also genotyped. Previously described mutations associated with pyrethroid resistance (T170C, C190A, G184C, and T2134A) were successfully detected by qPCR-HRM in different genotypes and confirmed by sequencing. A novel non-synonymous SNP located at domain III (C2136A) and the G215T mutation in domain II, previously described only in Asian R. microplus and R. australis, were also detected with the HRM and confirmed by sequencing. This technique could be adapted for high-throughput screening, detection, and discovery of allele-specific mutations in cattle tick outbreak populations to inform eradication strategies in the USA. This knowledge could also be applied to integrated control programs in other parts of the world where R. microplus is endemic and where similar SNPs have been identified associated with pyrethroid resistance. This study highlights the existence of several mutations in the para-sodium channel gene in different combinations in field populations of R. microplus from Mexico. Keywords: Southern cattle fever tick, Pyrethroid resistance, Molecular diagnosis, High-resolution melt analysis, Sodium channe

Additional file 1 of Periviscerokinin (Cap2b; CAPA) receptor silencing in females of Rhipicephalus microplus reduces survival, weight and reproductive output

Additional file 1: Data S1. Nucleotide sequences of the Rhimi-CAP2bR (KC614697.1), two Rhimi-CAP2bR clones (clones #4 and #6) both identical to the original KC614697.1 cDNA, and a 5’-RACE PCR sequence obtained to extend the 5’-UTR region of the R. microplus periviscerokinin receptor. An alignment between the R. microplus genome (isolate Rmic-2018 chromosome 3, ASM1333972v1, NC_051167.1) and the R. microplus periviscerokinin receptor extended RACE-5’-UTR fragment (Accession number OP191701) is provided

Additional file 2 of Periviscerokinin (Cap2b; CAPA) receptor silencing in females of Rhipicephalus microplus reduces survival, weight and reproductive output

Additional file 2: Data S2. Nucleotide sequences of the Rhimi-CAP2bR (KC614697.1) and the extended Rhimi-CAP2bR 5’-UTR sequence. The dsRNAs used for Rhimi-CAP2bR silencing are displayed on these sequences. Further, an EMBO-Clustal Omega 1.2.4 multiple sequence alignment between KC614697.1 sequence and R. microplus periviscerokinin receptor RACE 5’-UTR fragment (R-5’CAP2bR) (Accession number OP191701) is provided

Additional file 5 of Periviscerokinin (Cap2b; CAPA) receptor silencing in females of Rhipicephalus microplus reduces survival, weight and reproductive output

Additional file 5: Table S2. Pictures of ticks showing feeding progression from days 6 to 11 (December 2019), or from days 5 to 14 (July 2021) for all treatments

Additional file 4 of Periviscerokinin (Cap2b; CAPA) receptor silencing in females of Rhipicephalus microplus reduces survival, weight and reproductive output

Additional file 4: Table S1. dsRNA treatments and respective concentrations used in each replicate of the RNAi experiment

Additional file 3 of Periviscerokinin (Cap2b; CAPA) receptor silencing in females of Rhipicephalus microplus reduces survival, weight and reproductive output

Additional file 3: Data S3. NCBI-BLASTn searches to check for possible off-target effects of the Rhimi-CAP2bR dsRNA sequences. Only two short identical sequences to ds956-1109 were found to be ≤ 15 nt in length, which is not sufficient to cause off-target RNAi effects