Indoor and outdoor winter activity of Culicoides biting midges, vectors of bluetongue virus, in Italy

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1 Medical and Veterinary Entomology (2018) 32, doi: /mve Indoor and outdoor winter activity of Culicoides biting midges, vectors of bluetongue virus, in Italy A. MAGLIANO 1, P. SCARAMOZZINO 1, S. RAVAGNAN 2, F. MONTARSI 2, G. DA RO L D 2, G. CINCINELLI 3, A. MONI 4, P. SILVESTRI 5, A. CARVELLI 1 and C. D E L I B E R A T O 1 1 Istituto Zooprofilattico Sperimentale del Lazio e della Toscana M. Aleandri, Rome, Italy, 2 Istituto Zooprofilattico Sperimentale delle Venezie, Rome, Italy, 3 Azienda Unita Sanitaria Locale (USL) Toscana Sud Est, Arezzo, Italy, 4 Azienda USL Toscana Nord Ovest, Massa Carrara, Italy and 5 Azienda Sanitaria Locale (ASL) Rieti, Rieti, Italy Abstract. Indoor and outdoor winter activity of Culicoides spp. (Diptera: Ceratopogonidae) in central Italy was investigated in order to evaluate whether indoor activity might account for the overwintering of bluetongue virus, as has been hypothesized by some authors. Weekly Culicoides collections were performed at three farms over three consecutive winter seasons. At each farm, two black-light traps were operated simultaneously, indoors and outdoors. Culicoides were identified using both morphological and molecular means. The Culicoides obsoletus group accounted for 98.2% of sampled specimens. Within this group, C. obsoletus s.s. accounted for 56.8% and Culicoides scoticus for 43.2% of samples. Nulliparous, parous and engorged females were caught throughout the entire winter, both indoors and outdoors. At times, indoor catch sizes outnumbered outdoor collections. A significant inverse correlation was found between minimum temperature and the proportion of indoor Culicoides of the total midge catch, thus indicating that lower outdoor temperatures drive Culicoides midges indoors. High rates of engorged females were recorded indoors, possibly as the result of the propensity of C. obsoletus females to feed indoors. Higher proportions of parous females were found in indoor than in outdoor catches, indicating higher survival rates indoors and, consequently, higher vectorial capacities of midges sheltering indoors compared with those remaining outdoors. Key words. Culicoides, bluetongue, indoor, vectors, winter, Italy. Introduction Culicoides spp. are insect vectors of paramount importance in veterinary medicine (Mellor et al., 2000), particularly for their role in the transmission of the African horse sickness and bluetongue (BT) viruses (Reoviridae). Recently, in Europe Culicoides spp. were also identified as vectors of Schmallenberg virus (Bunyaviridae) (Rasmussen et al., 2012; Doceul et al., 2013). Bluetongue virus (BTV) is an Orbivirus of domestic and wild ruminants (Taylor, 1986). At present, 24 serotypes (denoted BTV1 24) have been recorded and are distributed on all continents except Antarctica (Rushton & Lyons, 2015). Clinical BT mainly affects sheep and is responsible for mortality rates of up to 75% (Mullen, 2009). Cattle are usually considered reservoir hosts of the virus (Carpenter et al., 2009). Bluetongue is among the more economically devastating animal diseases, mainly as a result of indirect causes such as movement and trade restrictions, as well as surveillance and vaccination costs. In 1998, the most extensive, prolonged and costly period of BT incursions ever recorded in Europe or worldwide started (Purse et al., 2005). After first-epidemic waves affecting only Mediterranean countries, BTV-8, until then never reported in Europe and considered to be of sub-saharan origin, appeared unexpectedly in 2006 in central Europe (Carpenter et al., 2009), Correspondence: Claudio De Liberato, Istituto Zooprofilattico Sperimentale del Lazio e della Toscana M. Aleandri, Via Appia Nuova 1411, Rome, Italy. Tel.: ; Fax: ; claudio.deliberato@izslt.it The Royal Entomological Society

2 Culicoides indoor winter activity 71 where it found competent vectors that allowed its rapid spread to countries as far north as Norway. Another northern circulation of the virus, exceeding the traditional geographical limit of 35 N, was attributable to the BTV-1 epidemic in France in 2007 and At present, in Europe six BTV serotypes have been recorded: BTV-1, BTV-2, BTV-4, BTV-8, BTV-9 and BTV-16 (Maclachlan & Mayo, 2013). Since 2000, all six BTV serotypes circulating in Europe have been recorded in Italy (Calistri et al., 2010). Currently, virus circulation is regularly reported through the detection of seroconversions in sentinel cattle or of clinical outbreaks in sheep in many areas of the country, including northern regions, which had until recently been free from the disease (Animal Disease Notification System, 2017). Until the beginning of the Italian epidemic in , Culicoides imicola was considered to be the only Old World vector of BTV. After 2001, the epidemiology of BT was almost entirely rewritten with the identification of two new vectors, Culicoides obsoletus and Culicoides pulicaris, both species groups that are distributed across the whole of Europe (Caracappa et al., 2003; De Liberato et al., 2005; Baldet et al., 2008). Culicoides obsoletus in particular represents the dominant group of species (Clausen et al., 2009) almost everywhere, from Great Britain and Scandinavia to the southern regions of Italy (Jennings & Mellor, 1988; Rawlings & Mellor, 1994). The C. obsoletus group in Italy includes C. obsoletuss.s., Culicoides scoticus, Culicoides chiopterus, Culicoides montanus and Culicoides dewulfi (Goffredo et al., 2016). Only the males of this species group can be readily distinguished by morphology. Culicoides biting midges were traditionally considered to be exophilic and exophagic (Meiswinkel et al., 2000, 2008; Baylis et al., 2010). For this reason, during the central European BT epidemic, government authorities recommended that animals be housed at night as a way of protecting them from infection (Meiswinkel et al., 2008; Baylis et al., 2010; Viennet et al., 2012). However, over recent years, evidence has arisen that midges of the C. obsoletus group readily enter animal shelters when seeking a bloodmeal (Baldet et al., 2008; Meiswinkel et al., 2008; Baylis et al., 2010; Kameke et al., 2017). Indeed, many authors consider Culicoides indoor winter activity to represent a possible means of the overwintering of BTV in central and northern Europe (Baldet et al., 2008). In autumn 2006, an overwintering of BTV in central Europe was considered unlikely in view of the short BTV viraemia in ruminants and the supposed absence of adult Culicoides throughout the winter months (Mellor et al., 2000). However, virus reappeared unexpectedly and spread again in 2007 (Wilson & Mellor, 2009). Studies carried out during this epidemic highlighted the presence of active C. obsoletus adults in animal premises over the whole winter (Baldet et al., 2008). Thus at least two likely coexisting mechanisms by which the virus might overwinter were hypothesized: (a) the survival of infected adult midges, even if not active, during the cold months, which allows the virus to persist, and/or (b) activity in adult indoor midge populations that maintain continuous BTV circulation among susceptible animals throughout the winter, even if at very low rates (Clausen et al., 2009). Previous papers about endophily in Culicoides reported varying results (Baylis et al., 2010), with higher catches recorded indoors or outdoors depending on the study (Meiswinkel et al., 2008; Zimmer et al., 2008). Hence, it was considered advisable to acquire data on midge indoor activity, particularly during winter, in order to evaluate whether this activity might allow for the overwintering of BTV at latitudes equivalent to those of central Italy. With this purpose, a research project, aimed at investigating Culicoides indoor and outdoor winter activity in cow sheds, was carried out, focusing on those species and species groups with proven roles in the transmission of BTV. Materials and methods Study area The study was carried out on three livestock farms in central Italy: (a) a dairy farm with 25 head of cattle (site M) (Massa-Carrara Province, Toscana Region, N, E, 16 m a.s.l., 3 km inland); (b) a beef cattle farm with 100 head of cattle (site A) (Arezzo Province, Toscana Region, N, E, 178 m a.s.l., 60 km inland), and (c) a dairy farm with 200 head of cattle (site R) (Rieti Province, Lazio Region, N, E, 178 m a.s.l., 60 km inland). At all three farms, livestock buildings were well constructed and well maintained. The degree to which they were enclosed (Baylis et al., 2010) can be considered similar in sites A and R: as the study was carried out in winter, doors and windows were usually closed before sunset and kept closed until sunrise, and therefore possible routes of entry of midges at night were limited to cracks and crevices. At site M, the farm is located in a relatively warm geographic area and the openings of the livestock building were kept open permanently during the day and night. At each sampling site, temperatures (minimum, mean and maximum) were collected using a digital thermometer located outside the animal shelter. Culicoides spp. sampling and identification Catches were performed one night per week from November to March over three consecutive winter seasons (2010/2011, 2011/2012 and 2012/2013) using Onderstepoort black light traps. At each farm, two traps were run simultaneously from dusk to dawn, one inside and one outside the cow shed. Both traps were located in close proximity to the animals, in exactly the same positions over the three sampling seasons. For the whole sampling period and at all three farms, cattle were present both indoors and outdoors, which avoided any possible bias arising as a result of the greater proximity of animals to one trap compared with the other. Insecticides were not used either inside or outside during the whole study period. Culicoides spp. were morphologically identified according to female wing patterns and male genital morphology (Delécolle, 1985; Mathieu et al., 2012). Females were classified according to their physiological stage as nulliparous, parous or engorged (Dyce, 1969). The majority of the data analysis was performed considering the C. obsoletus group as a whole. However, to determine the presence, abundance and relative endophily of the different species of this group, a significant subsample of females

3 72 A. Magliano et al. Fig. 1. Arezzo (site A): Culicoides obsoletus group indoor (I) and outdoor (O) catches in the three winter seasons and minimum (TMIN), mean (TMEAN) and maximum (TMAX) temperatures. was identified to species level using molecular tools. Based on the results of males morphological identification and of previous data from the same study area (De Liberato et al., 2010), attention was focused on C. obsoletus s.s. and C. scoticus. To avoid the bias caused by the different seasonal dynamics of the two species, females for molecular identification were randomly selected from catches performed throughout the whole study period in each of the three sampling seasons. Groups of 100 females for each of the associations Site/indoor and Site/outdoor, for a total of 600 specimens, were identified as follows. Pools of two specimens were homogenized after the addition of 300 μl of phosphate-buffered saline (PBS), using a TissueLyser (Qiagen GmbH, Hilden, Germany) with one 4.5-mm steel bead. DNA was extracted from 150 μl of homogenate using the automated liquid-handling workstation Microlab Starlet [Hamilton (Bonaduz) AG, Bonaduz, Switzerland] with the MagMAX Pathogen RNA/DNA Kit (Life Technologies, Inc., Rockville, MD, U.S.A.) according to the manufacturer s instructions, to obtain a final volume of 200 μl of DNA, which was stored at 20 C until use. A negative control (PBS) was used in parallel with the extraction of each set of 75 samples. Samples were amplified using a duplex real-time polymerase chain reaction (PCR), with specific primers and TaqMan probes, able to concurrently detect C. obsoletus s.s. and C. scoticus in a single tube, as previously described (Mathieu et al., 2011). Amplifications were performed in a StepOnePlus instrument (Applied Biosystems, Inc., Foster City, CA, U.S.A.). Negative (sterile water) and positive (DNA extracted from C. obsoletus and C. scoticus specimens) controls were included in each run. Data and statistical analysis The following entomological parameters were calculated for the C. obsoletus group as both cumulative data and by sampling season and farm site: indoor and outdoor catch sizes of nulliparous, parous and engorged females (Baldet et al., 2008); indoor (%ParI) and outdoor (%ParO) parous rates (number of parous females/female total catch); indoor (%EngI) and outdoor (%EngO) engorged rates (number of engorged females/female total catch), and indoor trapping rates (ITRs) [indoor catch/total (indoor + outdoor) catch] (Baldet et al., 2008) of, respectively, nulliparous, parous and engorged females. Data were tested for normality using the Shapiro Wilk test. Differences between indoor and outdoor counts of midges were tested with the non-parametric Wilcoxon test for matched pairs. Indoor and outdoor total counts for each physiological stage and site were compared using two- or three-variable chi-squared tests. The association between ITR and minimum temperature was evaluated using the Kendall rank test. A P-value of < 0.05 (two-tailed) was considered to indicate statistical significance. All statistical analyses were performed in Stata SE Version 12.0 (StataCorp LP, College Station, TX, U.S.A.). Results Overall, 336 light trap collections were performed (168 indoors and 168 outdoors) and Culicoides were caught ( outdoors and 5901 indoors). The C. obsoletus group accounted for 98.2% of sampled specimens; the remaining 1.8% consisted

4 Culicoides indoor winter activity 73 Fig. 2. Massa-Carrara (site M): Culicoides obsoletus group indoor (I) and outdoor (O) catches in the three winter seasons and minimum (TMIN), mean (TMEAN) and maximum (TMAX) temperatures. of members of the C. pulicaris group and C. imicola, although the latter species was caught only at the sea-level sampling site (sitem) (n = 8 specimens). Given these low abundances, the C.pulicaris group and C. imicola were not considered in the statistical analysis. Males of the C. obsoletus group were caught both indoors and outdoors at all sampling sites and were identified as C. obsoletus s.s.and C. scoticus (77 and 15 specimens, respectively). Overall, a total of 592 females were identified by PCR. Of these, C.obsoletus s.s. accounted for 56.8% and C. scoticus for 43.2%. Culicoides obsoletus s.s. was dominant indoors (62.9% of caught specimens), whereas the two species accounted for exactly 50% each of outdoor catches. At sampling sites A and M, C. obsoletus s.s. was more abundant than C. scoticus, both indoors (57.0% and 62.9%, respectively) and outdoors (72.6% and 57.0%, respectively). Collections at site R indicated the opposite: C. scoticus made up 51.1% of the indoor and 71.0% of the outdoor subsamples (P < 0.001). No DNA amplification was obtained for eight females (1.3%), indicating that they should be ascribed to the other species of the group, namely C. chiopterus, C. montanus or C. dewulfi. The C. obsoletus group was active at the study sites throughout the whole study period. Smaller catch sizes were recorded during January and the first half of February, when catches at site A were at times negative (Fig. 1). At the beginning and at the end of the sampling period, maximum catch sizes were recorded. At site M, secondary peaks in midge abundance were observed throughout the season (Fig. 2). The temporal trends of indoor and outdoor catch sizes at the three study sites are shown in Figs 1 3, in relation to minimum, maximum and mean temperatures. At site M (Fig. 2), outdoor catch sizes always outnumbered indoor ones; at site R (Fig. 3), indoor catch sizes were at times higher than outdoor ones. At site A (Fig. 1), this inversion was almost constant and the largest recorded catch was an indoor one, with 959 C. obsoletus caught in November At this site, the total C. obsoletus group indoor mean catch size (30.1 specimens/catch) was larger than that outdoors (14.4 specimens/catch). Figure 4 shows the ITRs recorded at the three sampling sites for each of the three sampling seasons. Whereas the ITR at site M was always < 50%, at sites A and R it varied according to sampling season. Farm A was the only sampling site at which the ITR over the whole study period remained > 50%. Culicoides obsoletus group females of all physiological stages were sampled both outdoors and indoors at all sampling sites. In outdoor catches, nulliparous females were dominant at all sampling sites. In indoor catches, parous females were more abundant at site A (P < 0.001). Engorged females were more abundant indoors than outdoors at sites A and M (P < 0.001). Table 1 reports rates (%) of parous and engorged females in outdoor and indoor catches. Over the whole study period, %ParO was highest at site A (34.3%), as was %ParI, which showed a value of 68.4% over the whole study period and peaked to 70.8% in the last sampling season (Table 1). With regard to engorged females, %EngO was highest at site M (9.2%), as was %EngI. Table 2 shows ITRs of females in the different physiological stages according to sampling season and sampling site. Nulliparous females were in general more abundant outdoors (except at site R during the second sampling season), whereas engorged females were usually more abundant indoors, with ITRs for sites R and A reaching 80.0% and 100%, respectively, in the second sampling season. With respect to parous specimens,

5 74 A. Magliano et al. Fig. 3. Rieti (site R): Culicoides obsoletus group indoor (I) and outdoor (O) catches in the three winter seasons and minimum (TMIN), mean (TMEAN) and maximum (TMAX) temperatures. Fig. 4. Culicoides obsoletus group indoor trapping rates) at the three study sites across the three sampling seasons (1, 2 and 3). Sampling seasons: 1, 2010/2011; 2, 2011/2012; 3, 2012/2013. Trapping sites: M, Massa; A, Arezzo; R, Rieti. high relative (> 50%) ITR values were recorded at sites A and R. Over the whole study period, ITRs exceeding 50% were recorded for parous and engorged females at site A (Table 2) and for engorged females at site M. No significant difference (Wilcoxon test, P > 0.05) was found when comparing paired data for C. obsoletus outdoor and indoor catches collected at the different sites over the whole study period. When the study sites and sampling seasons were considered separately, significant differences were detected in the following comparisons: (a) at site A in 2011/2012, engorged females were more abundant indoors than outdoors (P = ); (b) at site R in 2010/2011, engorged females were more abundant indoors than outdoors (P = ); (c) at site R in 2011/2012, total Culicoides spp. and C. obsoletuss.l. catches were higher indoors than outdoors (P = and P = , respectively), and (d) at site R in 2011/2012, nulliparous females were more abundant indoors than outdoors (P = ). There was a significant inverse correlation between minimum temperature and the proportion of indoor Culicoides over the total number of Culicoides caught at the site (ITR) (Kendall tau b = , P < 0.01).

6 Culicoides indoor winter activity 75 Table 1. Culicoides obsoletus group parous (Par) and engorged (Eng) rates (%) in outdoor (O) and indoor (I) catches at the three sampling sites over the whole study period. Site ParO ParI EngO EngI M 23.7% 28.0% 9.2% 26.1% A 34.3% 68.4% 3.3% 19.1% R 28.1% 31.1% 1.5% 2.9% Trapping sites: M, Massa; A, Arezzo; R, Rieti. Table 2. Culicoides obsoletus group indoor trapping rates (ITRs) of females in different physiological stages at the three sampling sites across the three sampling seasons. ITR, % Site Nul Par Eng 2010/2011 M 29.0% 43.8% 59.8% A 15.8% 25.0% 0% R 48.5% 48.4% 89.5% 2011/2012 M 23.2% 15.7% 39.7% A 41.5% 54.3% 100% R 82.4% 80.0% 0% 2012/2013 M 20.9% 23.4% 56.6% A 47.8% 80.4% 97.0% R 12.3% 15.1% 20.5% Total M 26.2% 30.7% 56.8% A 45.5% 77.6% 92.3% R 20.4% 22.8% 34.4% Trapping sites: M, Massa; A, Arezzo; R, Rieti. Nul, nulliparous; Par, parous; Eng, engorged. Discussion The present study is the first to report on Culicoides spp. indoor winter activity in a Mediterranean country. The quantitative dominance of the C. obsoletus group, known to be a dominant, ubiquitous species group in central Italy (Conte et al., 2007; De Liberato et al., 2010), as well as in almost all other European countries (Baldet et al., 2008; Baylis et al., 2010), was expected. Similarly expected was the dominance of the species C. obsoletus s.s. and C. scoticus within the C. obsoletus group (De Liberato et al., 2010); it should be noted, however, that these data originate from the molecular identification of a small subset of specimens. These two species seem to enter animal shelters proportionally to their overall abundance, without showing a species-based preferential attitude to indoor activity. Less expected was the presence of C. imicola in winter, both indoors and outdoors. This species is considered strictly exophilic and its adults are not usually on the wing in the cold season in temperate regions (Baylis et al., 2010). Collections included very few specimens of C. imicola and only at the beginning of the sampling period and in the coastal sampling site at sea level, on nights with mean temperatures of > 10 C. Culicoides imicola is known to have a mainly coastal distribution in central Italy and adults on the wing in winter on days of relative warmth have already been reported (De Liberato et al., 2003). As expected, lower mean catch sizes of the C. obsoletus group were recorded at the sampling site at which lower temperatures were reported (site A). Nevertheless, midges were active throughout almost the entire winter and outdoor activity was reported even on nights at which minimum temperatures were close to 0 C. The high level of variability in C. obsoletus group abundance observed in successive catches (Figs 1 3), also reported for other Culicoides species (Baylis et al., 2010; Kirkeby et al., 2013), is likely to reflect a steep increase in adults on the wing following relatively warm days during the cold season, and then a steep decrease in midge activity when temperatures fall. These fluctuations usually followed similar trends in both outdoor and indoor catches and were too rapid to be explained by a sudden emergence of new adults. Rather, they indicate rapid activation and eventual blood feeding of overwintering adults during relatively warm days. Relative indoor and outdoor abundances differed according to sampling site and temperature. An overall ITR of > 50% was recorded at the coldest sampling site (site A); ITR was significantly correlated with low outdoor temperatures, thus supporting the hypothesis that higher relative indoor activity is induced by colder climates, as reported by other authors (Baldet et al., 2008; Sarvasova et al., 2016). This finding has two possible explanations: (a) lower outdoor temperatures may drive more Culicoides to shelter indoors, or (b) lower temperatures may provoke higher rates of mortality in midges remaining outdoors, thereby increasing the relative ITR. Indeed, these two factors may coexist. A similar study carried out in late winter in Germany (Kameke et al., 2017) found an overall higher abundance of Culicoides spp. indoors compared with outdoors. In the present study, the finding that females of all physiological stages were active both indoors and outdoors during the cold season was remarkable. The presence and high percentages of nulliparous females throughout the winter may be explained by the emergence of new adults. With few exceptions, nulliparous females were more abundant outdoors than indoors because of the presence of larval breeding sites outside the structures. Nevertheless, high percentages of nulliparous females recorded indoors at times would confirm a tendency of newly emerged individuals to enter buildings, even if suitable hosts are present outdoors. That higher abundances of engorged females in indoor rather than outdoor catches were recorded at two of the three sampling sites, as in Meiswinkel et al. (2008) and Sarvasova et al. (2016), may be attributable to two possible factors that may coexist: (a) nulliparous females may enter sheds to avoid outdoor low temperatures and to blood feed indoors, and (b) females may enter sheds immediately after blood feeding outdoors. The present study suggests that the tendency of C. obsoletus to shelter indoors may play a possible role in winter BTV circulation and in BTV overwintering. In Culicoides, a population with a low parous rate is a population in which few individuals survive a gonotrophic cycle and hence a population with low vectorial capacity (Baylis et al., 1998), whereas a population with a high parous rate is a population in which a high number of females survive at least the first gonotrophic cycle,

7 76 A. Magliano et al. hence becoming potential vectors of BTV. At all three sites, parous rates were higher indoors than outdoors. Midges sheltering indoors will have higher rates of survival than those that remain outdoors and consequently higher theoretical vectorial capacities (Braverman et al., 1985; Meiswinkel et al., 2008). Some authors consider the degree to which animal accommodation is enclosed as a factor influencing the number of Culicoides present indoors (Baylis et al., 2010). In the present study, the sampling site at which the relative indoor catch sizes were lower was the only site at which the windows of the cow shed were kept open over the entire study period, which theoretically facilitated the entry of Culicoides from the outside. This finding seems to suggest that outdoor temperature is more relevant than the degree to which a building is enclosed in influencing Culicoides to move indoors. The present study shows that, in central Italy, the C. obsoletus group produces continuous generations during winter, sometimes mixing outdoor (reproduction and larval breeding) and indoor (sheltering and blood feeding) activities. In this scenario, high parous rates, mainly recorded in indoor catches, would validate the hypothesis that Culicoides winter populations may facilitate the circulation of BTV during the cold season and may play a role in the overwintering of the virus. Acknowledgements The authors are grateful to Dr Gioia Capelli, Istituto Zooprofilattico Sperimentale delle Venezie, for the revision of this manuscript. This research was supported by a grant from the Italian Ministry of Health under Project Code IZSLT 07/11 RC. All authors declare they have no competing interests. References Animal Disease Notification System (2017) Animal disease notification system. [accessed on 1 February 2017]. 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