1 Introduction Boophilus microplus Estrada-Pena et al., 2006 Boophilus B. microplus Rhipicephalus Murrell and Barker, 2003 B. microplus Babesia bovis Babesia bigemina Anaplasma marginale B. microplus Willadsen, 2004 Samish et al., 2004 de la Fuente and Kocan, 2003; Willadsen, 2004 ® ® Gough and Kemp, 1993 Boophilus annulatus Fragoso et al., 1998; Pipano et al., 2003 Boophilus decoloratus Hyalomma anatolicum anatolicum Hyalomma dromedarii Amblyomma variegatum, Amblyomma cajennense Rhipicephalus appendiculatus de Vos et al., 2001 B. microplus Willadsen et al., 1996 B. microplus Riding et al., 1994 Jarmey et al., 1995 Ixodes scapularis subolesin I. scapularis Almazan et al., 2003, 2005a,b I. scapularis Amblyomma americanum Rhipicephalus sanguineus Dermacentor variabilis Dermacentor marginatus subA subolesin de la Fuente et al., 2006a subA Rs86 Bm86 R. sanguineus de la Fuente et al., 2006c I. scapularis Anaplasma phagocytophilum Borrelia burgdorferi Pal et al., 2004; Ramamoorthi et al., 2005; Sukumaran et al., 2006 A. americanum Aljamali et al., 2003; Karim et al., 2004 Haemaphysalis longicornis Miyoshi et al., 2004 I. scapularis Narasimhan et al., 2004 Soares et al., 2005 Aljamali et al., 2003; Karim et al., 2005 Boophilus B. microplus Boophilus 2 Materials and methods 2.1 Experimental animals Three Holstein–Friesian calves, 5 months of age (#7793, #7794 and #7799), were used. All animals had no previous exposure to ticks. All tick feedings were approved by the Animal Experiments Committee (DEC) of the Faculty of Veterinary Medicine, Utrecht University (DEC No. 0111.0807). 2.2 Ticks and tick feeding Boophilus microplus ® Bm86 Bm91 2.3 RNA extraction and synthesis of tick cDNA for dsRNA preparations B. microplus 2.4 Cloning and sequencing of the B. microplus subolesin gene subA B. microplus de la Fuente et al., 2006a DQ923495 2.5 dsRNA synthesis B. microplus Bm86 Bm91 subolesin Table 1 2.6 Injection of ticks with dsRNA Bm86 Bm91 subolesin Bm86 subolesin 11 B. microplus Bm86 Bm91 subolesin 12 2.7 Analysis to confirm gene silencing by quantitative RT-PCR Bm86 Bm91 subolesin 2.8 Analysis to check for the presence of dsRNA in eggs Bm86 Bm91 subolesin Bm86, Bm91 subolesin Bm86 Bm91 subolesin 2.9 Statistical analysis t χ 2 P 3 Results 3.1 Cloning and sequencing of the subolesin gene from B. microplus subolesin subA) B. microplus subA B. microplus de la Fuente et al., 2006a 3.2 RNAi in freshly molted B. microplus females B. microplus Bm86 Bm91 subolesin Bm86 subolesin B. microplus Table 2 subolesin P Bm86- Bm91- subolesin Fig. 1 Bm86 Bm86 Bm86 subolesin Fig. 1 Bm91 Bm91 Bm91 subolesin P Bm86 subolesin Fig. 1 subolesin subolesin Bm86 subolesin Fig. 1 Bm86 Bm91 Bm86 Bm91 3.3 RNAi in engorged B. microplus females Bm86 Bm91 subolesin Bm91 Table 3 subolesin Fig. 2 subolesin subolesin Fig. 3 Bm86 Bm91 Figs. 3 Bm86 Bm86 Fig. 3 Bm91 Bm91 Fig. 3 Table 1 Bm86, Bm91 subolesin Bm86, Bm91 subolesin Fig. 4 Bm86 Bm91 Bm86 Bm91 Figs. 5 Subolesin subolesin Bm86 Bm91 Figs. 5 Bm86- Bm91 Bm91 4 Discussion subolesin B. microplus subolesin de la Fuente et al., 2005, 2006a,c Bm86 subolesin R. sanguineus de la Fuente et al., 2006c B. microplus Table 2 Bm86 Bm91 Willadsen et al., 1989 A. americanum. cystatin Karim et al., 2005 Bm86 Bm91 subolesin subolesin Bm91 Bm86 subolesin Bm91 Riding et al., 1994 Bm91 de la Fuente et al., 2006a B. microplus Bechara et al., 1988 Bm86 Bm91 subolesin Bm91 Bm86 Bm91 subolesin subolesin Bm86 Bm91 subolesin Saito et al., 2005 subolesin Diehl et al., 1982 B. microplus Winston et al., 2002; Feinberg and Hunter, 2003 B. microplus sid-1 sid-1 Schistocerca americana Dong and Friedrich, 2005 B. microplus Guerrero et al., 2005 Drosophila Liu et al., 2004; Miyoshi et al., 2005 B. microplus B. microplus de la Fuente et al., 2005 de la Fuente et al., 2006b B. microplus Guerrero et al., 2006 B. microplus A. marginale Brayton et al., 2005 B. bigemina B. bovis B. microplus B. microplus vitellin degrading cysteine endopeptidase Seixas et al., 2003 Babesia Li et al., 2003 B. microplus Wargelius et al., 1999 Tavernarakis et al., 2000 Grabarek et al., 2002