Evolution, expansion and expression of the Kunitz/BPTI gene family associated with long-term blood feeding in Ixodes Scapularis

In this study, the evolution, expansion and expression of Kunitz/BPTI family in Ixodes scapularis were investigated. Single- and multi-domain Kunitz/BPTI proteins have similar gene structures. Single-domain proteins were classified into three groups (groups I, II and III) based on their cysteine patterns. Group I represents the ancestral branch of the Kunitz/BPTI family, and members of this group function as serine protease inhibitors. The group I domain was used as a module to create multi-domain proteins in hard ticks after the split between hard and soft ticks. However, groups II and III, which evolved from group I, are only present and expanded in the genus Ixodes. These lineage-specific expanded genes exhibit significantly higher expression during long-term blood feeding in Ixodes scapularis. Interestingly, functional site analysis suggested that group II proteins lost the ability to inhibit serine proteases and evolved a new function of modulating ion channels. Finally, evolutionary analyses revealed that the expansion and diversification of the Kunitz/BPTI family in the genus Ixodes were driven by positive selection.These results suggest that the differences in the Kunitz/BPTI family between soft and hard ticks may be linked to the evolution of long-term blood feeding in hard ticks. In Ixodes, the lineage-specific expanded genes (Group II and III) lost the ancient function of inhibiting serine proteases and evolved new functions to adapt to long-term blood feeding. Therefore, these genes may play a profound role in the long-term blood feeding of hard ticks. Based our analysis, we propose that the six genes identified in our study may be candidate target genes for tick control.Ticks are classified into two major families: Ixodidae (hard ticks) and Argasidae (soft ticks) [1,2]. The family Ixodidae is further divided into two groups, Prostriata and Metastriata. Prostriata contains only a single genus, Ixodes. In contrast, Metastriata contains four subfamilies: A