Silencing expression of the defensin, varisin, in male Dermacentor variabilis by RNA interference results in reduced Anaplasma marginale infections

Abstract

Antimicrobial peptides, including defensins, are components of the innate immune system in ticks that have been shown to provide protection against both gram-negative and gram-positive bacteria. Varisin, one of the defensins identified in Dermacentor variabilis, was shown to be produced primarily in hemocytes but transcript levels were also upregulated in midguts and other tick cells. In this research, we studied the role of varisin in the immunity of ticks to the gram-negative cattle pathogen, Anaplasma marginale. Expression of the varisin gene was silenced by RNA interference (RNAi) in which male ticks were injected with varisin dsRNA and then allowed to feed and acquire A. marginale infection on an experimentallyinfected calf. Silencing expression of varisin in hemocytes, midguts and salivary glands was confirmed by real time RT-PCR. We expected that silencing of varisin would increase A. marginale infections in ticks, but the results demonstrated that bacterial numbers, as determined by an A. marginale msp4 quantitative PCR, were significantly reduced in the varisin-silenced ticks. Furthermore, colonies of A. marginale in ticks •used for RNAi were morphologically abnormal from those seen in elution buffer injected control ticks. The colony. .. . shape was irregular and in some cases the A.' marginale appeared to be free in the cytoplasm of midgut cells. Some ticks were found to be systemically infected with a microbe that may have been related to the silencing of varisin. This appears to be the first. report of the silencing of expression of a defensin in ticks by RNAi that resulted in reduced A. marginale infections. Response to Reviewers: We have revised the manuscript extensively in accordance with the recommendations of the reviewers. In most cases, we accepted the reviewer's suggestions for rewriting the text and substituted those revisions as recommended. There is not sufficient space in this comment box to detail every change that was made to every specific comment, but we did attempt to address all comments from the several reviewers and incorporate them.

RNAi in Agriculturally-Important Arthropods

“…R. microplus is an economically significant tick ectoparasite that transmits a variety of pathogens, such as Anaplasma marginale, thereby increasing cattle exposure to vector-borne infectious diseases. Silencing of a defensin gene, varisin, by injection of dsRNA into male ticks reduced their ability to infect calves with A. marginale (Kocan et al, 2008). Additional RNAi targets, such as those affecting tick mortality and fecundity, have been identified through RNAi screens in R. microplus (Kurscheid et al, 2009).…”

Defensins from the tick Ixodes scapularis are effective against phytopathogenic fungi and the human bacterial pathogen Listeria grayi

“…A functional domain of one I. scapularis defensin, Scapularisin-20, was functionally characterised and found to show activity against Gramnegative (Gram-) and Gram-positive (Gram+) bacteria [5]. However, tick defensins have a wide spectrum of antimicrobial activity, including antibacterial, antifungal, antiprotozoal and antiviral activities; thus defensins are excellent candidates to tackle the increasing problem of drug resistance seen in microbes and fungi [9][10][11].…”

Tick Innate Immunity

“…This result indicates that varisin FRQWULEXWHV VLJQL¿FDQWO\ EXW QRW H[FOXVLYHO\ WR WKH RYHUDOO DQWLPLFURELDO DFWLYLW\ LQ WKH D. variabilis hemolymph 61. A study of the effect of varisin silencing on the transmission of a relevant pathogen, the rickettsia Anaplasma marginale SUHVHQWHG D VXUSULVLQJ ¿QGLQJ In opposite to authors' expectations, the A. marginale LQIHFWLRQ ZDV VLJQL¿FDQWO\ UHGXFHG in the varisin-KD ticks for yet unknown reasons 62. …”