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Puumala hantavirus infection alters the odour attractiveness of its reservoir host Nelika K. Hughes, Sanne Helsen, Katrien Tersago, Herwig Leirs Evolu8onary Ecology Group, Department of Biology, University of Antwerpen, Belgium INTRODUCTION METHODS Model assump,ons and the real world We used a Y-‐maze to test whether PUUV infec8on alters the aYrac8veness of male bank voles’ urine. Most epidemiological models are based on a random mixing assump8on, i.e. that the probability of encountering an infected host is equal among individuals. We presented healthy (i.e. PUUV-‐free), wild-‐
caught male and female bank voles with: This assump8on is only true, however, if infec8on does not alter the movements of infected individuals, or their interac8ons with healthy conspeciﬁcs. •  urine from PUUV-‐infected bank voles, A large body of evidence suggests that host behaviour such as social, parental and sexual interacBons, is modiﬁed following infecBon. Hosts may reduce transmission if healthy individuals can recognise, and consequently avoid, infected individuals on the basis of behavioural, visual, and/or olfactory cues. •  urine from PUUV-‐free bank voles, and a •  water control. All voles were in non-‐breeding condi8on. Urine came from adult male bank voles. Many mammals use urine, faeces and other secre8ons as olfactory signals, conveying ﬁxed informa8on (e.g. individual iden8ty), in addi8on to variable informa8on such as their social and reproduc8ve status, and their health. We measured the latency to ﬁrst visit, the number of visits, and the total 8me bank voles spent inves8ga8ng each treatment in the ﬁrst 10 minutes of a trial, and over the en8re 30 minutes. Some pathogens reduce the aYrac8veness of their hosts’ excre8ons, poten8ally altering contact rates and thus the predicted force of infec8on for pathogens transmissible by contact with excre8ons. Puumala hantavirus transmission dynamics – a role for odour? We calculated the voles’: Bank voles (Myodes glareolus) use excre8ons (urine and faeces) for social and sexual communica8on. •  rela,ve preferences: individuals’ responses to the two urine treatments rela8ve to their response to the control, and their Contact with contaminated urine is also an important route of transmission for Puumala hantavirus (PUUV), a zoono8c pathogen for which bank voles are the reservoir host. Strong seasonal shifs in transmission rates suggest that PUUV transmission might be more complex than a simple random-‐
mixing model would imply – could deliberate avoidance of infec8ous urine by suscep8ble individuals be the cause of this? •  absolute preferences: the latency, total number of visits, and the total 8me spent inves8ga8ng each treatment. We used a choice experiment to test whether PUUV infecBon changes the aJracBveness of bank voles’ urinary odours. Infected
Uninfected
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Rela,ve preferences (Figure 1a) 0.2
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•  Voles spent longer inves8ga8ng uninfected than infected urine (χ2 = 5.29, d.f. = 1, P = 0.021) −0.2
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•  Voles made more visits to uninfected urine than infected urine (χ2 = 6.61, d.f. = 1, P = 0.010) −0.8
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•  There were no eﬀects of sex, nor a sex by infec8on status interac8on (P > 0.2 for all tests). b) Absolute preferences
ln(value +1)
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Infected
Uninfected
Control
2.0
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Absolute preferences (Figure 1b) •  Voles visited the uninfected urine more frequently than the infected urine (χ2 = 7.06, d.f. = 0.5
2, P = 0.029, Tukey’s HSD infected vs. uninfected: P = 0.029) Time (sec)
Infected
Uninfected
Control
•  There was a strong trend for voles to spend longer inves8ga8ng the uninfected than infected urine (χ2 = 5.65, d.f. = 2, P = 0.059) Time (sec)
No. Visits
Response
Figure 1. In the ﬁrst 10 minutes of the trial, the mean (± S.E.) a) rela8ve preference for the infected (white) and uninfected (light grey) urine over the water control, and the b) absolute preference for the infected (white) and uninfected (light grey) urine and the water control (dark grey). •  There were no signiﬁcant eﬀects of sex, nor a sex by infec8on status interac8on (P > 0.2 for all). •  Voles spent longer inves8ga8ng uninfected than infected urine 3
2
1
Time (sec)
•  Voles visited the uninfected urine before the infected urine No. Visits Latency (sec)
b) Absolute preferences
5
1.5
0
Rela,ve preferences (Figure 1a) (χ2 = 4.61, d.f. = 1, P = 0.032) 4
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a) Relative preferences
ln(No. visits +1)
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Urine donor PUUV infecBon status also aﬀected vole behaviour over the 30-‐
minute trial, although to a lesser extent. 1.0
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(χ2 = 4.90, d.f. = 1, P = 0.027) •  The number of visits did not diﬀer between treatments, and there were no eﬀects of sex, nor a signiﬁcant interac8on (P > 0.09 for all tests). 1000
Latency to first visit (sec)
The PUUV infecBon status of bank vole urine donors signiﬁcantly aﬀected conspeciﬁcs’ behaviour in the short term (ﬁrst 10 minutes). a) Relative preferences
ln(sec +1)
Relative preference for urine
0.8
Relative preference for urine
RESULTS No. Visits
800
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Absolute preferences (Figure 2b) 200
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Latency (sec)
Response
Figure 2. Over the 30-‐minute trial, the mean (± S.E.) a) rela8ve preference for the infected (white) and uninfected (light grey) urine over the water control, and b) absolute preference for the infected urine (white), uninfected urine (light grey) and the water control (dark grey). The strong aversive response to urine from PUUV infected voles declined over the 30-‐minute trial, and no responses diﬀered signiﬁcantly between treatments (P > 0.09 for all tests). DISCUSSION Our results suggest that PUUV infecBon alters the odour proﬁle of their bank vole host, and that voles are able to detect and diﬀerenBally respond to the urine of infected and healthy (PUUV-‐free) conspeciﬁcs. This suggests that random mixing assumpBons within PUUV transmission models may not be valid. These ﬁndings generalise previous research, which has largely focussed on the consequences of infec8on for mate choice. Our results have par8cular relevance for social interac8ons and transmission during the non-‐breeding winter months, when territoriality declines and bank voles nest communally, and when the survival of PUUV in the environment is probably extended. In contrast to most previous studies, the voles’ percep8on of infec8on risk in our study was in response to natural, rather than experimental, infec8ons. Thus, while correla8onal, our study demonstrates that the natural variability in infec8on status and immune response found in wild popula8ons s8ll translates into percep8ble eﬀects on individual behaviour. Bank voles in our experiment did not inspect uninfected conspeciﬁc urine at higher rates than the water controls. Although unexpected, all animals were in non-‐breeding condi8on, so there were probably few social or sexual incen8ves to inspect conspeciﬁc scents at a high rate, especially given the perceived risk of preda8on whilst doing so. The majority of urine donors in our study were chronically infected, a state associated with rela8vely lower rates of virus shedding than during acute infec8ons. As the strength of the aversive response to infected excreta increases with the stage of infecBvity, it is possible that healthy bank voles would show an even stronger aversion towards the urine of acutely infected animals than the response we have reported here. If so, this process would further confound assumpBons of random mixing and reduce the predicted force of infecBon. For more details, see: Hughes NK, Helsen S, Tersago K & Leirs H (2014) Puumala hantavirus infec,on alters the odour a<rac,veness of its reservoir host. Oecologia 176: 955-‐963 For correspondence: Nelika Hughes Email: [email protected] or [email protected] Acknowledgements: This study was par8ally funded by EU grant FP7-‐261504 EDENext and is catalogued by the EDENext Steering CommiYee as EDENext212. We gratefully acknowledge the University of Antwerp for ﬁnancial support (BOF-‐GOA project FFB3567), the Agency for Nature and Forest (ANB) for permits and assistance, and Olli Vapalah8 for providing IFA slides. We also thank S Baeckens, I Jacobs and S Puls for ﬁeld assistance, and B Borremans, J Reijniers, S Vandongen, L De Bruyn and N Van HouYe for laboratory and sta8s8cal assistance. This experiment was conducted with the approval of the University of Antwerp’s Animal Ethics CommiYee (approval number 2012-‐14).