Effective Methods of Estimation of Pathogen Prevalence in Pooled Ticks.

Since tick-borne diseases (TBDs) incidence, both in human and animal populations, is increasing worldwide, there is the need to assess the presence, distribution and prevalence of tick-borne pathogens. Reliable estimates on tick-borne pathogens (TBPs) prevalence represent the public health foundation to create risk maps and take effective prevention and control actions against TBDs. Tick surveillance consists of collecting and testing (usually in pools) thousands of specimens. Construction and analysis of tick pools represent a challenge due to the complexity of tick-borne pathogens and tick-borne diseases ecology. The aim of this study is to provide a practical guideline on appropriate pooling strategies and statistical analysis of infection prevalence through: (i) reporting the different pooling strategies and statistical methodologies commonly used to calculate pathogen prevalence in tick populations and (ii) practical comparison between statistical methods utilising a real dataset of infection prevalence in ticks collected in Northern Italy. Reporting detailed information on tick pool composition and size is as important as the correct TBPs prevalence estimation. Among the prevalence indexes, we suggest using maximum-likelihood estimates of pooled prevalence instead of minimum infection rate or pool positivity rate given the merits of the method and availability of software.

Male mating preference in an ixodid tick.

BACKGROUND: Mate choice is a fundamental element of sexual selection and has the potential to shape the evolution of traits. Mate choice based on body size has been shown to be a common trait in several arthropod species. In hard ticks, a taxon of medical and veterinary importance, engorgement weight is positively correlated with reproductive output but it is unknown whether adult males show mate choice. Here, we experimentally investigated whether males (i) use chemical cues to choose their mating partner, (ii) consistently choose for the same female individual and (iii) prefer females with highest weight after feeding. METHODS: We used two experimental setups which allowed chemical communication between ticks: (i) a horizontal tube preventing physical contact with the female and (ii) an arena where tactile cues were allowed. In total, we tested 62 different triads in 124 tests (66 tests in the horizontal tube and 58 in the arena) composed of one male that could choose between two engorged females. Specifically, we tested 42 triads in the tube and 46 in the arena; 24 triads were repeatedly tested in the tube while 38 triads were tested in both setups. RESULTS: We found no preference for individual or heavier females in either setup. However, in the horizontal tube setup, males significantly preferred females that were not visited by them in the previous test. CONCLUSIONS: Our results suggest a lack of male mate choice despite heavier females having higher fecundity. However, future studies should take into account that males may recognize the potential mating partners they previously met.

Predictors of individual performance and evolutionary potential of life-history traits in a hematophagous ectoparasite.

Little is known about the intraspecific variation of parasite life-history traits and how this variation may affect parasite fitness and evolution. We investigated how life-history traits predict success of individual tree-hole ticks Ixodes arboricola and estimated their evolutionary potential, as well as genetic correlations within stages and phenotypic correlations within and across stages. Ticks were followed individually over two generations while allowed to feed on great tits Parus major. After accounting for host and tick maternal effects, we found that short feeding times and high engorgement weights strongly increased molting success. Molting time was also positively correlated with feeding success in adults. In larvae and nymphs, we found negative phenotypic correlations between engorgement weight and both feeding and molting time, the latter supported by a negative genetic correlation. We found sex-related differences in feeding time (longer in male nymphs) and molting time (longer in male larvae but shorter in male nymphs). Also, time since the last feeding event (set experimentally) reduced larval and nymphal fitness, whereas it increased adult female fitness. Furthermore, we found significant heritability and evolvability, that is, the potential to respond to selection, for engorgement weight and molting time across all stages but no significant heritability for feeding time. Our findings suggest that variation in tick fitness is shaped by consistent individual differences in tick quality, for which engorgement weight is a good proxy, rather than by life-history trade-offs.

Ineffective humoral anti-tick IgY-response in birds: reaction against pathogen constituents?

Background: Variation in parasite burdens among hosts is typically related to differences in adaptive immunity. Comprehension of underlying mechanisms is hence necessary to gain better insights into endemic transmission cycles. Here we investigate whether wild songbirds that have never been exposed to ticks develop adaptive humoral immunity against endemic Ixodes ricinus ticks. Methods: Blue tits were exposed three times in succession to wild Ixodes ricinus ticks. For each infestation, serum samples were obtained. An enzyme-linked immunosorbent assay was developed, using tick salivary antigens, in order to quantify the bird's IgY response against ticks. In addition, at every sampling occasion the birds' body weight (corrected for body size) and haematocrit level was determined. Results: Individual IgY levels against the ticks' salivary proteins increased over three consecutive tick infestations, and large among-individual variation was observed. The responses were specifically directed against I. ricinus; cross-reactivity against the congeneric tree-hole tick Ixodes arboricola was negligibly low. IgY responses did not impinge on tick feeding success (engorgement weight and attachment success). Yet, those birds with the highest immune responses were more capable to reduce the acute harm (blood depletions) by compensating erythrocyte loss. Furthermore, at the end of the experiment, these birds had gained more body weight than birds with lower IgY levels. Conclusions: Latter observations can be considered as an effect of host quality and/or tolerance mechanisms. Birds anticipate the (future) costs of the activation of the immune system by ticks and/or ongoing tick-borne pathogen infections. Furthermore, although unsuccessful against tick feeding, the IgY responses may indirectly protect birds against tick-borne disease by acting against salivary protein secretions on which pathogens rely for transmission.

Isolation of Candidatus Rickettsia vini from Belgian Ixodes arboricola ticks and propagation in tick cell lines.

Candidatus Rickettsia vini was originally detected in Ixodes arboricola ticks from Spain, and subsequently reported from several other Western Palearctic countries including Belgium. Recently, the bacterium was isolated in mammalian (Vero) cell culture from macerated male I. arboricola from Czech Republic, but there have been no reports of propagation in tick cells. Here we report isolation in a tick cell line of three strains of Ca. R. vini from I. arboricola collected from nests of great tits (Parus major) in Belgium. Internal organs of one male and two engorged female ticks were dissected aseptically, added to cultures of the Rhipicephalus microplus cell line BME/CTVM23 and incubated at 28 °C. Rickettsia-like bacteria were first seen in Giemsa-stained cytocentrifuge smears between 2 and 15 weeks later. Two of the isolates grew rapidly, destroying the tick cells within 2-4 weeks of onward passage in BME/CTVM23 cells, while the third isolate grew much more slowly, only requiring subculture at 4-5-month intervals. PCR amplification of bacterial 16S rRNA and Rickettsia gltA, sca4, ompB, ompA and 17-kDa genes revealed that all three isolates were Ca. R. vini, with 100 % identity to each other and to published Ca. R. vini sequences from other geographical locations. Transmission electron microscopy revealed typical single Rickettsia bacteria in the cytoplasm of BME/CTVM23 cells. The Ca. R. vini strain isolated from the male I. arboricola tick, designated Boshoek1, was tested for ability to grow in a panel of Ixodes ricinus, Ixodes scapularis and R. microplus cell lines and in Vero cells. The Boshoek1 strain grew rapidly, causing severe cytopathic effect, in the R. microplus line BME26, the I. ricinus line IRE11 and Vero cells, more slowly in the I. ricinus line IRE/CTVM19, possibly established a low-level infection in the I. ricinus line IRE/CTVM20, and failed to infect cells of any of four I. scapularis lines over a 12-week observation period. This study confirmed the applicability of the simple tick organ-cell line co-cultivation technique for isolation of tick-borne Rickettsia spp. using BME/CTVM23 cells.

Experimental study of micro-habitat selection by ixodid ticks feeding on avian hosts.

Mechanisms of on-host habitat selection of parasites are important to the understanding of host-parasite interactions and evolution. To this end, it is important to separate the factors driving parasite micro-habitat selection from those resulting from host anti-parasite behaviour. We experimentally investigated whether tick infestation patterns on songbirds are the result of an active choice by the ticks themselves, or the outcome of songbird grooming behaviour. Attachment patterns of three ixodid tick species with different ecologies and host specificities were studied on avian hosts. Ixodes arboricola, Ixodes ricinus and Ixodes frontalis were put on the head, belly and back of adult great tits (Parus major) and adult domestic canaries (Serinus canaria domestica) which were either restricted or not in their grooming capabilities. Without exception, ticks were eventually found on a bird's head. When we gave ticks full opportunities to attach on other body parts - in the absence of host grooming - they showed lower attachment success. Moreover, ticks moved from these other body parts to the host's head when given the opportunity. This study provides evidence that the commonly observed pattern of ticks feeding on songbirds' heads is the result of an adaptive behavioural strategy. Experimental data on a novel host species, the domestic canary, and a consistent number of published field observations, strongly support this hypothesis. We address some proximate and ultimate causes that may explain parasite preference for this body part in songbirds. The link found between parasite micro-habitat preference and host anti-parasite behaviour provides further insight into the mechanisms driving ectoparasite aggregation, which is important for the population dynamics of hosts, ectoparasites and the micro-pathogens for which they are vectors.

Can house sparrows recognize familiar or kin-related individuals by scent?

In the last decades, higher attention has been paid to olfactory perception in birds. As a consequence, a handful of avian species have been discovered to use olfaction in different contexts. Nevertheless, we still have a very limited knowledge about the use of odor cues in avian social life, particularly, in the case of songbirds. Here, we investigate if female house sparrows Passer domesticus show any preference for the odor of kin and nonkin conspecifics and we also test a possible role of familiarity based on male scent in female choice. We performed the experiment with captive birds twice, during the nonbreeding and breeding seasons. Our results show that female house sparrows strongly avoided the odor of unrelated familiar (UF) males, both in the breeding and nonbreeding seasons. Our results suggest recognition for olfactory stimuli related to familiarity and kinship. We suggest that avoidance for UF males is associated with previous experience in this species. Also, we provided further evidence to the use of olfaction in passerine species by using a new experimental setup.

House sparrows' (Passer domesticus) behaviour in a novel environment is modulated by social context and familiarity in a sex-specific manner.

BACKGROUND: Exploratory behaviour is one of the best-investigated behavioural traits. However, little is known about how differences in familiarity, i.e. in the knowledge and previous experience with a companion can influence the exploration of a novel environment. However, to our knowledge, such a critical feature of the social environment has never been the target of a study relating it to exploratory behaviour in birds. Here we examined if familiarity with a conspecific could affect behavioural responses of individuals confronted with a novel environment. We recorded the latency to land on the ground, latency to feed, time spent feeding and number of sectors visited of 48 female and 48 male house sparrows (Passer domesticus) in an indoor aviary in three contexts: alone (individual context), with an unfamiliar and with a familiar same-sex companion. RESULTS: House sparrows landed sooner on the ground when in the familiar context than when in the individual context. Birds in unfamiliar pairs followed each other less than familiar birds, but this difference diminished with time spent exploring. Moreover, males and females differed in their behavioural responses in the unfamiliar context. Females with a familiar companion landed sooner than when they were paired with an unfamiliar conspecific, whereas only the presence of a companion but not familiarity reduced males latency to land on the ground. Finally, when considering the unfamiliar context males had shorter latencies to forage and thus spent more time eating than females. CONCLUSIONS: The presence or absence of a companion and its familiarity with the focal individual influenced differently the behavioural responses of male and female house sparrows in a novel environment. As house sparrows are strongly sociable, the influence of the social environment is likely to be of paramount importance to understand the selective pressures acting on them, particularly in recently colonized areas with ephemeral food sources. Our results shed light on the complex influence that the social environment has on the behavioural responses of a cosmopolitan bird.

Olfactory Assessment of Competitors to the Nest Site: An Experiment on a Passerine Species.

Since most avian species have been considered anosmic or microsmatic, olfaction and associated behavioural patterns have hardly been investigated. Most importantly, empirical data on avian olfaction is not equally distributed among species. Initial investigations focused on species with relatively big olfactory bulbs because they were thought to have better olfactory capabilities. Hence, in this study we tested the ability of house sparrows (Passer domesticus) to use chemical cues as parameters to estimate nest features. House sparrows are a commonly used model species, but their olfactory capabilities have not been studied so far. We offered two different odours to males and females, namely the scent of mouse urine (Mus musculus domesticus), representing a possible competitor and a threat to eggs and hatchlings, and the odour of hay, representing a familiar and innocuous odour. The experiment was performed at the sunset to simulate a first inspection to new possible roosting or nesting sites. Interestingly, males but not females preferred to spend significantly more time in front of the hay odour, than in front of the scent of mouse urine. Our results strengthen the hypothesis that oscines can not only perceive odours but also use olfaction to assess the environment and estimate nest site quality.