Genetics: Published Articles Ahead of Print, published on February 21, 2011 as 10.1534/genetics.111.127696 1 Rhabdoviruses in two species of Drosophila: vertical transmission and a recent 2 sweep 3 4 5 Ben Longdon1*, Lena Wilfert2, Darren J Obbard1 and Francis M Jiggins2 6 7 1 Institute of Evolutionary Biology, and Centre for Immunity, Infection and Evolution, 8 University of Edinburgh, 9 Ashworth Labs, 10 Kings Buildings, 11 West Mains Road, 12 Edinburgh, 13 EH9 3JT, 14 UK 15 2 Department of Genetics, 16 University of Cambridge, 17 Cambridge, 18 CB2 3EH, 19 UK 20 21 Running title: Sigma virus transmission and sweep 22 23 24 * [email protected] 25 Phone: +61(0)434935946 26 27 Key words 28 vertical biparental transmission, paternal transmission, maternal transmission, sperm, 29 sigma virus 30 31 Word count Abstract: 252 Word Count text: 6997 32 33 Sequences in this study are deposited in genbank under the following accession 34 numbers: HQ149099 to HQ149304 1 Copyright 2011. 35 Abstract 36 37 Insects are host to a diverse range of vertically transmitted micro-organisms, but while 38 their bacterial symbionts are well-studied, little is known about their vertically 39 transmitted viruses. We have found that two sigma viruses (Rhabdoviridae) recently 40 discovered in Drosophila affinis and Drosophila obscura are both vertically 41 transmitted. As is the case for the sigma virus of Drosophila melanogaster, we find 42 that both males and females can transmit these viruses to their offspring. Males 43 transmit lower viral titres through sperm than females transmit through eggs, and a 44 lower proportion of their offspring become infected. In natural populations of D. 45 obscura in the UK we found that 39% of flies were infected and the viral population 46 shows clear evidence of a recent expansion, with extremely low genetic diversity and 47 a large excess of rare polymorphisms. Using sequence data we estimate that the virus 48 has swept across the UK within the last ~11 years, during which time the viral 49 population size doubled approximately every 9 months. Using simulations based on 50 our lab estimates of transmission rates, we show that the biparental mode of 51 transmission allows the virus to invade and rapidly spread through populations, at 52 rates consistent with those measured in the field. Therefore, as predicted by our 53 simulations, the virus has undergone an extremely rapid and recent increase in 54 population size. In light of this and earlier studies of a related virus in D. 55 melanogaster, we conclude that vertically transmitted rhabdoviruses may be common 56 in insects, and that these host-parasite interactions can be highly dynamic. 57 58 59 Introduction 60 61 Insects have a diverse range of vertically transmitted symbionts (BUCHNER 1965). Of 62 these the best studied are bacteria, which are usually transmitted exclusively by 63 females and have evolved a range of strategies to spread through host populations 64 (such as distorting the sex ratio towards females or providing a metabolic benefit to 65 their hosts (DOUGLAS 1989; HURST et al. 1993)). Far less is known about vertically 66 transmitted viruses in insects. Some viruses are both horizontally and vertically 67 transmitted (BEZIER et al. 2009; MIMS 1981). Other species contain endogenous 68 retroviruses or polydnaviruses which have integrated into the germline and are 2 69 inherited with the host genome (BEZIER et al. 2009; FLEMING and SUMMERS 1991; 70 HEREDIA et al. 2007). However, very few free living and purely vertically transmitted 71 viruses have been described in insects. 72 73 One such virus is the Drosophila melanogaster sigma virus (DMelSV), which infects 74 ~4 % of wild flies (BRUN and PLUS 1980; CARPENTER et al. 2007). DMelSV is a 75 negative-sense RNA virus in the family Rhabdoviridae that is found in the cytoplasm 76 of infected cells. Unlike bacterial symbionts, this virus is transmitted vertically 77 through both sperm and eggs (FLEURIET 1988), so it is able to spread through 78 populations despite being costly to infected flies (FLEURIET 1981; L'HERITIER 1970; 79 SEECOF 1964). The pattern of DMelSV transmission differs between the sexes, with 80 male flies transmitting at a lower rate than females (BRUN and PLUS 1980). 81 Additionally, the transmission rate is reduced when the fly is infected by its father 82 rather than its mother—if a female is infected by her father, her average transmission 83 rate drops from ~100% to a much lower rate (BRUN and PLUS 1980), and if a male is 84 infected by his father, he does not transmit the virus at all. Therefore, the virus cannot 85 be transmitted through males for two successive generations. 86 87 We have recently discovered two new sigma viruses in Drosophila obscura and 88 Drosophila affinis — DObsSV and DAffSV (LONGDON et al. 2010). Along with 89 DMelSV, these viruses form a deep-branching clade in the Rhabdoviridae, which we 90 have suggested be recognised as a new genus. However, important questions about 91 their biology remain unanswered, including whether these new viruses are vertically 92 transmitted. There is some evidence that DAffSV is; Williamson, (1961) found that 93 CO2-sensitivity was vertically transmitted in some lines of D. affinis in a way similar 94 to that seen in DMelSV-infected flies (CO2 paralysis is a symptom of sigma viruses 95 on their hosts (LONGDON et al. 2010)). We also do not know anything about the 96 prevalence, population dynamics or population genetics of these viruses. This paper 97 aims to address these questions by examining the transmission of these viruses in the 98 lab and the dynamics of DObsSV in natural populations. 99 100 Methods 101 102 Vertical transmission of viruses 3 103 104 To test the mode of transmission of these newly discovered viruses, we carried out 105 crosses between infected and uninfected virgin flies. The crosses used infected 106 isofemale lines of Drosophila affinis and Drosophila obscura that were collected from 107 Raleigh, North Carolina (U.S.A) and Essex (UK), respectively, as described in 108 (LONGDON et al. 2010). The crosses began with infected flies that had both an 109 infected mother and father (from a stock that was close to 100% infected). When both 110 parents are infected, it has been shown for DMelSV that the viral type in the offspring 111 is that of the mother (BRUN and PLUS 1980). The uninfected D. affinis isofemale lines 112 were collected from the same location at the same time as the infected lines, and the 113 uninfected D. obscura were isofemale lines collected during the present study (see 114 below). D. affinis were reared on a banana-malt based Drosophila medium (see 115 supplementary materials), whilst D. obscura were reared on a cornmeal medium 116 (LEWIS 1960) with a piece of peeled mushroom (Agaricus bisporus) on the surface. 117 118 To test whether flies were infected with sigma virus, we exposed them to CO2 at 12°C 119 for 15mins, and recorded flies dead or paralysed 30mins later as infected. To confirm 120 that CO2 sensitivity was linked to viral infection, we crossed infected males to 121 uninfected females, carried out the CO2 assay on their offspring, and tested 15 122 paralysed and 15 non paralysed/recovered offspring for sigma virus infection by 123 quantitative real time PCR (qRT-PCR ) (40 cycles: 950C 15 sec, 600C 1min) on an 124 Applied Biosystems StepOnePlus system using a Power SYBR Green PCR Master- 125 Mix (Applied Biosystems, CA, USA). Three technical replicates were carried out for 126 each sample and primer pair, and samples were run in a blocked design across plates. 127 The amount of virus was standardised to a housekeeping gene RpL32 (Rp49) to 128 account for RNA extraction and reverse transcription efficiencies using the delta delta 129 CT (critical threshold) method. Viral primers were designed to cross gene boundaries 130 so only viral genomes were quantified (rather than mRNA), primer sequences are 131 shown in table S2. RpL32 endogenous control primers also crossed an intron-exon 132 boundary so should not amplify gDNA contamination. 133 134 The following crosses were used to measure vertical transmission (diagram in Figure 135 1A) and to determine whether horizontal transmission occurred. In cross 1, infected 136 females were crossed to uninfected males. Cross 2 took the daughters from cross 1 4 137 and crossed them to uninfected males. In cross 3 infected males were crossed to 138 uninfected females. Cross 4 mated the daughters from cross 3 with uninfected males. 139 Cross 5 mated the sons from cross 3 to uninfected females. Uninfected partners were 140 assayed for infection to determine whether horizontal transmission had occurred. 141 142 For D. affinis, multiple flies were placed in each vial, as the flies appear more likely to 143 lay eggs when maintained at a higher stocking density. In cross 1, one to three females 144 were placed in a vial with two to three males, and allowed to lay eggs. For cross 3, 145 two or three infected males were placed in a vial with one to three uninfected females. 146 For crosses 4 and 5, the cross 3 offspring were placed individually in a vial with one 147 or two uninfected flies of the opposite sex. Once eggs or larvae were visible, the 148 adults were exposed to CO2 to confirm their infection status. In all crosses uninfected 149 partners were assayed for infection to test whether horizontal transmission had 150 occurred. 151 152 For D. obscura, all crosses were carried out with a single pair of flies in each vial.