Location: Livestock Arthropod Pests ResearchTitle: The leucokinin-like peptide receptor from the cattle fever tick, Rhipicephalus microplus, is localized in the midgut periphery and receptor silencing with validated dsRNAs causes a reproductive fitness cost
|BROCK, CHRISTINA - Texas A&M University|
|YANG, YUNLONG - Texas A&M University|
|BLANDON, MARIA - Texas A&M University|
|CARREON-CAMACHO, DIANA - University Of Tamaulipas|
|LONGNECKER, MICHAEL - Texas A&M University|
|ALMAZAN, CONSUELO - University Of Tamaulipas|
|Perez De Leon, Adalberto - Beto|
|PIETRANTONIO, PATRICIA - Texas A&M University|
Submitted to: International Journal for Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/4/2018
Publication Date: 3/1/2019
Citation: Brock, C.M., Temeyer, K.B., Tidwell, J.P., Yang, Y., Blandon, M.A., Carreon-Camacho, D., Longnecker, M.T., Almazan, C., Perez De Leon, A.A., Pietrantonio, P.V. 2019. The leucokinin-like peptide receptor from the cattle fever tick, Rhipicephalus microplus, is localized in the midgut periphery and receptor silencing with validated dsRNAs causes a reproductive fitness cost. International Journal for Parasitology. 49(3-4):287-299. https://doi.org/10.1016/j.ijpara.2018.11.006.
Interpretive Summary: The southern cattle tick, Rhipicephalus microplus, is important because it transmits deadly cattle diseases for which effective protective vaccines are not available. Although R. microplus was eradicated in the USA, tick populations in Mexico and South America have developed resistance to many acaricides, making them ineffective for tick control. Recent detection of acaricide-resistant ticks within the U.S. underscore the need to develop new tick control methods. Amitraz is an acaricide thought to target specific cellular receptors that are members of a very large class of proteins known as G-Protein Coupled Receptors, or GPCRs. GPCR proteins are embedded in cellular membranes and allow cells to detect and respond to hormones, neurotransmitters, and other environmental signals. Some GPCR proteins are specific to invertebrates, and targeting such GPCRs may be feasible for discovery of novel control targets, and new anti-tick compounds. The R. microplus leucokinin-like peptide receptor (LKR), also known as myokinin- or kinin receptor, is one such GPCR, however, function of the leucokinin signaling system in ticks is unknown. In this work, specific antibodies that bind to the LKR were used to show that the LKR was present surrounding the midgut in female adult ticks. The function of the LKR was further investigated by gene silencing, using RNA interference, or RNAi, to reduce LKR production and function in adult female ticks. A dual-luciferase system was developed and used to optimize selection of LKR-double stranded RNA (dsRNA) silencing constructs prior to testing them in ticks placed on cattle. This assay identified two effective dsRNAs. Silencing of the LKR with the two validated dsRNA constructs was verified by quantitative Reverse Transcription-PCR in dissected tick midgut and carcasses. Silencing of the LKR in female ticks resulted in decreased egg production and egg hatching, as well as increased time to lay eggs and for eggs to hatch. It was apparent from these results that the kinin receptor exerts effects on tick reproduction, suggesting possible adaptation for use tick control.
Technical Abstract: The southern cattle tick, Rhipicephalus microplus (Canestrini) (Acari: Ixodidae), is a one-host tick that infests primarily cattle in Central and South America, Africa, and Australia. This species transmits deadly cattle pathogens, especially Babesia spp., for which a recombinant vaccine is not available. Therefore, disease control depends on tick vector control. Although R. microplus was eradicated in the USA, tick populations in Mexico and South America have acquired resistance to many of the applied acaricides. Recent acaricide-resistant tick reintroductions detected in the U.S. underscore the need for novel tick control methods. The octopamine and tyramine/octopamine receptors, both G protein-coupled receptors (GPCR), are believed to be the main molecular targets of the acaricide amitraz. This provides the proof of principle that investigating tick GPCRs, especially those that are invertebrate specific, may be a feasible strategy for discovering novel targets and subsequently new anti-tick compounds. The R. microplus leucokinin-like peptide receptor (LKR), also known as myokinin- or kinin receptor, is such a GPCR. While the receptor was previously characterized in vitro, the function of the leucokinin signaling system in ticks remains unknown. In this work, the LKR was immunolocalized to the periphery of the female midgut and silenced through RNAi in females. To optimize RNAi experiments, a dual-luciferase system was developed to determine the silencing efficiency of LKR-double stranded RNA (dsRNA) constructs prior to testing them in ticks placed on cattle. This assay identified two effective dsRNAs. Silencing of the LKR with these two validated dsRNA constructs was verified by qRT-PCR of female tick dissected tissues. Silencing was significant in midgut and carcasses. Silencing caused decreases in weight of egg masses and in the percentages of eggs hatched per egg mass, as well as delays in time to oviposition and egg hatching. A role of the kinin receptor in tick reproduction is apparent.