| Summary: | <i>Haemaphysalis longicornis</i> is a tick and a vector of various pathogens, including the human pathogenetic <i>Babesia microti</i>. The objective of this study was to identify female <i>H. longicornis</i> genes differentially expressed in response to infection with <i>B. microti </i>Gray strain by using a suppression subtractive hybridization (SSH) procedure. A total of 302 randomly selected clones were sequenced and analyzed in the forward subtracted SSH cDNA library related to <i>Babesia</i> infection, and 110 clones in the reverse cDNA library. Gene ontology assignments and sequence analyses of tick sequences in the forward cDNA library showed that 14 genes were related to response to stimulus or/and immune system process, and 7 genes had the higher number of standardized sequences per kilobase (SPK). Subsequent real-time PCR detection showed that eight genes including those encoding for Obg-like ATPase 1 (ola1), Calreticulin (crt), vitellogenin 1 (Vg1) and Vg2 were up-regulated in fed ticks. Compared to uninfected ticks, infected ticks had six up-regulated genes, including <i>ola1</i>, <i>crt</i> and <i>Vg2</i>. Functional analysis of up-regulated genes in fed or<i> Babesia</i>-infected ticks by RNA interference showed that knockdown of<i> crt</i> and <i>Vg2 </i>in infected ticks and knockdown of<i> ola1</i> in uninfected ticks accelerated engorgement. In contrast, <i>Vg1</i> knockdown in infected ticks had delayed engorgement. Knockdown of<i> crt</i> and <i>Vg1 </i>in infected ticks decreased engorged female weight. <i>Vg2</i> knockdown reduced <i>B. microti</i> infection levels by 51% when compared with controls. The results reported here increase our understanding of roles of <i>H. longicornis</i> genes in blood feeding and <i>B. microti</i> infection.
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