1a.Objectives (from AD-416)
1) Begin to define the potential receptor-myokinins interaction sites by developing anti-receptor antibodies against extracellular receptor regions, and test them in the functional calcium assay. Verify cell surface recognition/binding by immunological techniques (immunocytochemistry or immunofluorescence and flow cytometry). Determine the receptor cellular distribution in different female tissues and at different days post-blood feeding.
2) Validate the myokinin receptor as a suitable target for tick disruption/ control through in vivo experiments.
A. Determine if the receptor antigen produces an immunological response in cattle and if this response provides protection against ticks.
B. Determine if excess agonist causes a detrimental effect on the tick (if time allows).
3) Clone from R. microplus other candidate GPCRs involved in water balance and begin immunolocalization (or in situ hybridization studies).
1b.Approach (from AD-416)
A controlled stall trial for vaccination of cattle and tick challenge will be conducted to determine if the receptor fragments can protect cattle against the tick. Determine if the receptor antigen produces an immunogical response in cattle as it did in rabbits. Cattle will be inoculated with a mixture of synthetic peptides encompassing receptor extracellular regions and part of the transmembrane regions; peptides will be linked to keyhole limpet hemocyanin. If cattle develop anti-receptor (peptide) antibodies, cattle will be challenged with ticks; tick survival, weight, egg laying capacity, and egg viability will be measured and statistically compared with controls.
Antibodies against the cloned receptors will be developed also in rabbits for the immunolocalization in different tick tissues, ideally also following a temporal-spatial analysis, depending on the availability of tick females at different ages.
For objective 2.B: active myokinins will be injected directly into the female tick.
The myokinin receptors were cloned by extracting and processing nucleic acids from tick tissues using standard molecular techniques. A mammalian expression vector is under construction to express the receptor in CHO-K1 cells in a stable manner for functional studies.
The ARS scientist monitored progress of the project primarily through teleconferences and exchanges of e-mail with the cooperator.