The Adaptive Evolution Of Blood-feeding Ralated Multigene Families In Tick Saliva

There exist many gene families in the salivary glands of ticks. They play key roles in the process of tick blood feeding. Study on the evolution of these gene families can help us understand the genetic basis and molecular mechanism of blood feeding, thus help to the development of effective measures for tick control. Furthermore, it provides a new case for the evolution of protein structure and function.Kunitz/BPTI proteins are abundant in the salivary glands of ticks and perform multiple functions in blood feeding by inhibiting the Xa and VIIa factors during platelet aggregation or coagulation of blood. Here, we have systematically examined the evolution, expansion and expression of Kunitz/BPTI family in Ixodes scapularis. We found the existence of three groups (groupⅠ,ⅡandⅢwith different cysteine patterns(CX(8)CX(15)CX(7)CX(12)CX(3)C,CX(8)CX(18)CX(5)CX(12)CX(3)C,CX(5,6)CX(15)CX(8)CX(11)CX(3)C) in Kunitz/BPTI family. GroupⅠ, which is present widely in ticks, represents the ancestor branch of Kunitz/BPTI family and functions as serine protease inhibitor. However, groupⅡandⅢ, which evolved from groupⅠ, are only present in the genus Ixodes and lack the motif for inhibiting serine protease. Interestingly, groupⅡevolved the new function of modulating ion channels suggested by structural alignment and functional sites analysis. Evolutionary analyses revealed that the expansion and diversification of Kunitz/BPTI family in the genus Ixodes was driven by positive selection.We summarized the evolution scenario of Kunitz/BPTI family in ticks: the common ancestor of ticks had Kunitz/BPTI genes orthologous to groupⅠ, as groupⅠgenes present in both soft and hard ticks. Hard and soft ticks were divergent between 120 and 92 MYA and whole genome duplication occurred in this process. After the split of the Prostriata and Metastriata, the events that groupⅡandⅢtype indels occurred in group I genes followed by multiple gene duplication gave rise to group ⅡandⅢ. The expression analysis show that GroupⅡandⅢgenes display significant higher expression level at 6–12 hours or longer time post host attachment in Ixodes scapularis, which suggests that these genes are functionally linked to long-term blood feeding. We suppose that the new function of modulating blood flow volume increases feeding efficiency at mid-and late phase of blood feeding. This profound role of Kunitz/BPTI family in long-term blood feeding may explain the reasons for different blood-feeding strategies in hard and soft ticks. Finally, we proposed that the six genes (Isc.218, Isc.190, Isc.255, Isc.196, Isc.180 and Isc.179) identified in our study may be candidate target genes for tick control because of functional importance and fewer host autoimmune responses.