Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila

Table 3 Effect of tick in vivo dsRNA gene knockdown treatments on female tick survival and subsequent reproduction fecundity by targeting Drosophila homologues described in Table 2

dsRNA treatment			Average (5 replicates per treatment)					qRT-PCR % knockdown (average 3 replicate reactions)
Treatment	R. microplus target BmiGI2 ID	D. mel homologue	Tick survival (days)	Egg output (g)	Egg morphology (see Figure 6)	Days from laying to larval hatch	% larval hatch	Viscera (adult ticks)	Eggs
dsRNA control (MEGAScript)	NA*	NA*	16.4	0.118	normal	26.9	69.8
PBS injection	NA*	NA*	15.0	0.107	normal	26.5	62.0
No injection	NA*	NA*	14.8	0.128	normal	27.1	64.9
Actin	TC12168	NA*	16.2	0.134	normal	26.3	65.8
Proteasome and ubiquitin	TC6372	Ubiquitin-63E	10.0	0.010	deformed	NL^±	0.0	95.6	ND^§
	TC9852	Proteasome 26S subunit ATPase	17.0	0.116	deformed	NL^±	0.0	98.0	76.0
	TC10417	Rpt 1	16.4	0.130	normal	26.2	65.1	94.0	ND^§
	TC13930	Tat-binding protein-1 ^¶	16.4	0.127	slow development	32.9	0.4	99.0	76.0
Ribosome and protein synthesis	TC5762	string of perls ^¶	17.6	0.129	deformed	NL^±	0.0	99.9	100.0
	TC9037	Ribosomal Protein L11	14.4	0.110	deformed	NL	0.0	21.3	98.9
	TC12306	Ribosomal protein L8	17.0	0.094	deformed	NL^±	0.0	99.2	ND^§
	TC12393	Ribosomal protein S13	16.6	0.135	deformed	NL^±	0.0	99.0	ND^§
DNA binding	TC6116	Histone H3.3A	15.8	0.114	slow development	30.7	3.1	97.6	100.0
	TC12182	Histone H3.3A	16.4	0.121	normal	27.8	35.2	99.2	94.7
^#LSD (P = 0.05)			2.1	0.029		1.3	17.7

*NA (not applicable) – control injections (no dsRNA treatment and R. microplus actin control)
^± NL (no larvae) indicates no larvae hatched
^§ ND (not done) indicates insufficient total RNA to undertake RT-PCR due to poor egg output.
^¶ Tat-binding protein 1 had a nil z score in the D. melanogaster study and string of perls had a z score <3 [43]
^#LSD = least significant difference values

ISSN: 1471-2199