Longistatin, a novel EF-hand protein from the ixodid tick Haemaphysalis longicornis, is required for acquisition of host blood-meals.

Calcium and the EF-hand Ca(++)-binding proteins have been undisputedly recognised as the key players in almost all aspect of cell functions, starting from the cell's birth, during mitosis to its end with apoptosis. But in a few exceptional cases the EF-hand proteins are secreted from the cells and play their crucial roles extracellularly. Here, to our knowledge for the first time, we have identified and characterised an EF-hand Ca(++)-binding protein from the salivary glands of the ixodid tick, Haemaphysalis longicornis, herein called longistatin. Longistatin possesses two EF-hand domains which conserve canonical structure and bind with Ca(++). Both the recombinant and endogenous proteins were stained with Rutheninum red. Reverse-transcription PCR data showed that longistatin-specific transcript was expressed in all life-cycle stages of H. longicornis and was up-regulated only in blood-fed ticks. Organ-specific transcription analysis revealed a salivary gland-specific expression of the gene which peaked at 96-120 h of feeding when ticks acquired full blood-meals and become engorged but its expression declined sharply as they detached and dropped off the host. Consistently, endogenous protein was localised in the salivary glands of adult ticks and in the lumen of the functional acini of the salivary glands. Furthermore, longistatin was detected in feeding lesions at the site of attachment of ticks on the host. These results suggest that longistatin is synthesised in, and is secreted from, the salivary glands and may have functional roles in the feeding process of ixodid ticks.