Phylogeographic Patterns of Haemoproteid Assemblages of Selected Avian Hosts: Ecological and Evolutionary Implications.

BACKGROUND: While the dynamics of disease emergence is driven by host-parasite interactions, the structure and dynamics of these interactions are still poorly understood. Here we study the phylogenetic and morphological clustering of haemosporidian parasite lineages in a local avian host community. Subsequently, we examine geographical patterns of parasite assemblages in selected avian hosts breeding in Europe. METHODS: We conduct phylogenetic and haplotype network analyses of Haemoproteus (Parahaemoproteus) lineages based on a short and an extended cytochrome b barcode region. Ordination analyses are used to examine changes in parasite assemblages with respect to climate type and geography. RESULTS: We reveal relatively low phylogenetic clustering of haemoproteid lineages in a local avian host community and identify a potentially new Haemoproteus morphospecies. Further, we find that climate is effectively capturing geographical changes in parasite assemblages in selected widespread avian hosts. Moreover, parasite assemblages are found to vary distinctly across the host's breeding range, even within a single avian host. CONCLUSIONS: This study suggests that a few keystone hosts can be important for the local phylogenetic and morphological clustering of haemoproteid parasites. Host spatio-temporal dynamics, both for partially and long-distance migratory birds, appear to explain geographical variation in haemoproteid parasite assemblages. This study also gives support to the idea that climate variation in terms of rainfall seasonality can be linked to the propensity for host switching in haemosporidians.

The Infection Rate of Bird-Feeding Ixodes ricinus Ticks with Borrelia garinii and B. valaisiana Varies with Host Haemosporidian Infection Status.

BACKGROUND: Birds are known to maintain and spread human pathogenic borreliae, but they are common hosts of diverse parasite communities, notably haemosporidians. Only a few studies examined whether tick infestation and/or Borrelia prevalences vary with hosts' haemosporidian infection status. METHODS: Here, we study whether Ixodes ricinus infestation rates and Borrelia infection rates in bird-feeding ticks vary according to haemosporidian infection status in a community of free-living avian tick hosts. RESULTS: Birds of six avian species harbored the majority of ticks. Both the tick infestation prevalence and the intensity peaked during spring and summer, but while bird-feeding nymphs prevailed in spring, bird-feeding larvae dominated in summer. Almost half of the bird-feeding ticks were found to be positive for B. burgdorferi s.l. Although the majority of infections involved bird-associated B. garinii and B. valaisiana, B. garinii appears to be the dominant Borrelia strain circulating in locally breeding avian species. We detected a negative link between the hosts' haemosporidian infection status and the Borrelia infection rate of bird-feeding ticks, but the association was dependent on the host's age. CONCLUSIONS: Our results on tick infestation intensity support the idea that more immunologically vulnerable hosts harbor more ticks but suggest that different mechanisms may be responsible for tick infestation rates among immunologically naïve and experienced avian hosts. The results on Borrelia infection rates in bird-feeding ticks are consistent with studies revealing that intracellular parasites, such as haemosporidians, can benefit from the host immune system prioritizing immune responses against extracellular parasites at the expense of immune responses against intracellular parasites. The findings of our study urge for a more robust design of parasitological studies to understand the ecology of interactions among hosts and their parasites.

Novel Genotypes of Nidicolous Argas Ticks and Their Associated Microorganisms From Spain.

The knowledge of the distribution, richness and epidemiological importance of soft ticks of the genus Argas is incomplete. In Spain, five Argas species have been recorded, including three ornitophilic nidicolous ticks, but their associated microorganisms remain unknown. This study aimed to investigate ticks from bird nests and their microorganisms. Ticks were collected extensively from natural cavities and nest-boxes used by European rollers (Coracias garrulus) and little owls (Athene noctua) in Southeastern and Central Spain. Ticks were morphologically and genetically identified and corresponding DNA/RNA tick extracts were analyzed [individually (n = 150) or pooled (n = 43)] using specific PCR assays for bacteria (Anaplasmataceae, Bartonella, Borrelia, Coxiella/Rickettsiella, and Rickettsia spp.), viruses (Flaviviruses, Orthonairoviruses, and Phenuiviruses), and protozoa (Babesia/Theileria spp.). Six Argas genotypes were identified, of which only those of Argas reflexus (n = 8) were identified to the species level. Two other genotypes were closely related to each other and to Argas vulgaris (n = 83) and Argas polonicus (n = 33), respectively. These two species have not been previously reported from Western Europe. Two additional genotypes (n = 4) clustered with Argas persicus, previously reported in Spain. The remaining genotype (n = 22) showed low sequence identity with any Argas species, being most similar to the African Argas africolumbae. The microbiological screening revealed infection with a rickettsial strain belonging to Rickettsia fournieri and Candidatus Rickettsia vini group in 74.7% of ticks, mainly comprising ticks genetically related to A. vulgaris and A. polonicus. Other tick endosymbionts belonging to Coxiella, Francisella and Rickettsiella species were detected in ten, one and one tick pools, respectively. In addition, one Babesia genotype, closely related to avian Babesia species, was found in one tick pool. Lastly, Anaplasmataceae, Bartonella, Borrelia, and viruses were not detected. In conclusion, five novel Argas genotypes and their associated microorganisms with unproven pathogenicity are reported for Spain. The re-use of nests between and within years by different bird species appears to be ideal for the transmission of tick-borne microorganisms in cavity-nesting birds of semiarid areas. Further work should be performed to clarify the taxonomy and the potential role of soft Argas ticks and their microorganisms in the epidemiology of zoonoses.

Local Population Structure and Seasonal Variability of Borrelia garinii Genotypes in Ixodes ricinus Ticks, Slovakia.

Lyme disease (LD) is the most common tick-borne human disease in Europe, and Borrelia garinii, which is associated with avian reservoirs, is one of the most genetically diverse and widespread human pathogenic genospecies from the B. burgdorferi sensu lato (s.l.) complex. The clinical manifestations of LD are known to vary between regions and depend on the genetic strain even within Borrelia genospecies. It is thus of importance to explore the genetic diversity of such pathogenic borreliae for the wide range of host and ecological contexts. In this study, multilocus sequence typing (MLST) was employed to investigate the local population structure of B. garinii in Ixodes ricinus ticks. The study took place in a natural wetland in Slovakia, temporally encompassing spring and autumn bird migration periods as well as the breeding period of resident birds. In total, we examined 369 and 255 ticks collected from 78 birds and local vegetation, respectively. B. burgdorferi s.l. was detected in 43.4% (160/369) of ticks recovered from birds and in 26.3% (67/255) of questing ticks, respectively. Considering the ticks from bird hosts, the highest prevalence was found for single infections with B. garinii (22.5%). Infection intensity of B. garinii in bird-feeding ticks was significantly higher than that in questing ticks. We identified ten B. garinii sequence types (STs) occurring exclusively in bird-feeding ticks, two STs occurring exclusively in questing ticks, and one ST (ST 244) occurring in both ticks from birds and questing ticks. Four B. garinii STs were detected for the first time herein. With the exception of ST 93, we detected different STs in spring and summer for bird-feeding ticks. Our results are consistent with previous studies of the low geographic structuring of B. garinii genotypes. However, our study reveals some consistency in local ST occurrence and a geographic signal for one of the clonal complexes.

Simultaneous Occurrence of Borrelia miyamotoi, Borrelia burgdorferi Sensu Lato, Anaplasma phagocytophilum and Rickettsia helvetica in Ixodes ricinus Ticks in Urban Foci in Bratislava, Slovakia.

BACKGROUND: Questing Ixodes ricinus ticks were collected in two urban parks (Zelezná studienka and Horský park) of the capital city of Slovakia, Bratislava, during two consecutive years in 2011 and 2012. A total of 932 ticks were analyzed for the presence of tick-borne agents: B. miyamotoi, B. burgdorferi s.l., A. phagocytophilum and R. helvetica. RESULTS: PCR analysis confirmed the presence of all pathogens at both localities. The overall infection prevalence of B. miyamotoi, B. burgdorferi s.l., A. phagocytophilum and R. helvetica was 0.75, 13.2, 5.6 and 8.9%, respectively. B. burgdorferi s.l. positive samples were represented by six genospecies. The most frequent one was B. afzelii followed by B. garinii and B. valaisiana. CONCLUSION: Our study confirms the presence of I. ricinus ticks and at least nine tick-borne bacterial agents in city forest parks, which are used for recreational purposes. Ordination analysis revealed significant differences in the composition of pathogens with respect to study site location, time of season and ambient temperature, despite the fact that both sites are located relatively close to one another within the city.

Culicoides paolae and C. circumscriptus as potential vectors of avian haemosporidians in an arid ecosystem.

BACKGROUND: Haemosporidians are the most important vector-borne parasites due to their cosmopolitan distribution and their wide range of hosts, including humans. Identification of their vectors is critical to highlight ecologically and epidemiologically relevant features such as host specificity or transmission routes. Biting midges of the genus Culicoides are considered the main vectors of Haemoproteus spp., yet important information on aspects such as vector feeding preferences or vector-host specificity involving haemosporidian parasites is frequently missing. METHODS: We assessed the abundance of Culicoides circumscriptus and C. paolae and blood sources of the latter at the nests of cavity-nesting bird species (mainly the European roller Coracias garrulus) and in their surroundings. We also explored the prevalence and genetic diversity of avian haemosporidians in parous females of both species. RESULTS: Both C. circumscriptus and C. paolae were abundant in the study area and common at European roller nests. Culicoides paolae had a diverse ornithophilic diet, feeding on at least seven bird species. Human DNA was also detected in the blood meal of some individuals. Four Haemoproteus lineages, including a new one reported here for the first time, were isolated from parous females of both biting midges. CONCLUSIONS: Culicoides circumscriptus and C. paolae can play a locally important role in the transmission dynamics of Haemoproteus parasites in a community of cavity-nesting bird species in an arid ecosystem.

Seasonal Patterns in the Prevalence and Diversity of Tick-Borne Borrelia burgdorferi Sensu Lato, Anaplasma phagocytophilum and Rickettsia spp. in an Urban Temperate Forest in South Western Slovakia.

In Europe, Ixodes ricinus is the most important vector of tick-borne zoonotic bacteria. It transmits spirochaetes from the Borrelia burgdorferi sensu lato complex, Anaplasma phagocytophilum and Rickettsia spp. Although spatial differences in the prevalence of tick-borne pathogens have been intensively studied, seasonal (within-year) fluctuations in the prevalence of these pathogens within sites are often overlooked. We analyzed the occurrence and seasonal dynamics of Ixodes ricinus in an urban forest in Bratislava, Slovakia. Furthemore, we examined temporal trends in the community structure of B. burgdorferi s.l., A. phagocytophilum and Rickettsia spp. in questing and bird-feeding ticks. The total prevalence for B. burgdorferi s.l. in questing I. ricinus was 6.8%, involving six genospecies with the dominance of bird-associated B. garinii and B. valaisiana.A. phagocytophilum, R. helvetica and R. monacensis occurred in 5.9%, 5.0% and 0.2% of questing ticks, respectively. In total, 12.5% and 4.4% of bird-feeding I. ricinus ticks carried B. burgdorferi s.l. and R. helvetica. The total prevalence of B. burgdorferi s.l. in our study site was two times lower than the mean prevalence for Europe. In contrast, A. phagocytophilum prevalence was significantly higher compared to those in other habitats of Slovakia. Our results imply that tick propagation and the transmission, suppression and seasonal dynamics of tick-borne pathogens at the study site were primarily shaped by abundance and temporal population fluctuations in ruminant and bird hosts.

Nest ecology of blood parasites in the European roller and its ectoparasitic carnid fly.

Haemosporidian parasites are considered the most important vector-borne parasites. However, vector identity and ecology is unknown for most such host-vector-parasite systems. In this study, we employ microscopic and molecular analyses to examine haemosporidian prevalence in a migratory, cavity-nesting bird, European roller Coracias garrulus, and its nidicolous blood-feeding ectoparasite Carnus hemapterus. This system is unique in that the ectoparasite is confined to a near-closed environment, in contrast to the free-wandering system of haematophagous dipterans such as mosquitoes. Blood film analysis confirms previous works in that Haemoproteus parasites are widely prevalent in adult rollers and belong to a single species, Haemoproteus coraciae. Leucocytozoon sp. and Trypanosoma sp. also are detected in adult rollers at low intensities with this technique. By means of molecular analysis, we report for the first time Plasmodium sp. presence in C. garrulus. Based on PCR results, Plasmodium parasites are relatively less prevalent than Haemoproteus parasites (20% vs. 31%) in rollers. In contrast, haemosporidian prevalences show the opposite trend for Carnus flies: Plasmodium sp. occurrence (62%) clearly predominates over that of Haemoproteus sp. (5%). A comparison between roller and Carnus samples reveals a significantly higher prevalence of Plasmodium sp. in Carnus samples. Insect survey and phylogenetic analysis suggest Culicoides flies as Haemoproteus sp. vectors, which appear to readily transmit the parasite in southern Spain. This study does not find support for Carnus flies to serve as biological or mechanical vectors of haemosporidians. In spite of this, nidicolous blood-feeding ectoparasites, such as carnid flies, appear as a suitable model for studies on the occurrence and temporal dynamics of avian haemosporidians such as Plasmodium sp. present at low intensities.

Natural foci of Borrelia lusitaniae in a mountain region of Central Europe.

Lyme borreliosis is the most prevalent tick-borne disease in Europe. It is caused by spirochaetes of the Borrelia burgdorferi sensu lato (s.l.) complex and transmitted to humans by ticks of the genus Ixodes. Borrelia afzelii, Borrelia garinii, and Borrelia valaisiana are the most common genospecies in Central Europe. In contrast, Borrelia lusitaniae predominates in Mediterranean countries such as Portugal, Morocco, and Tunisia. In Slovakia, its prevalence is low and restricted to only a few sites. The aim of our research was to study the expansion of ticks into higher altitudes in the ecosystem of the Malá Fatra mountains (north Slovakia) and their infection with B. burgdorferi s.l. pathogens. Questing ticks were collected by flagging in seven years (2004, 2006-2011) at three different altitudes: low (630-660 m above sea level (ASL)), intermediate (720-750 m ASL), and high (1040-1070 m ASL). Tick abundance was highest at the lowest altitude and lowest at the highest altitude. The average infection prevalence of B. burgdorferi s.l. in nymphs and adults was 16.8% and 36.2%, respectively. The number of infected ticks decreased from 38.5% at the lowest altitude to 4.4% at the highest altitude. B. lusitaniae was the most frequently found genospecies (>60% of the ticks found positive for B. burgdorferi s.l.) in all sites in all the studied years with the exception of 2008 when B. afzelii predominated (62%). Our study confirms the spread of Ixodes ricinus ticks to higher altitudes in Slovakia. The discovery that our mountain study sites were a natural foci of B. lusitaniae was unexpected because this genospecies is usually associated with lizards and xerothermic habitats.

The effect of anthelmintic treatment on coccidia oocyst shedding in a wild mammal host with intermittent cestode infection.

While hosts are routinely exploited by a community of parasite species, the principles governing host responses towards parasites are unclear. Identifying the health outcomes of coinfections involving helminth macroparasites and microparasites is one area of importance for public and domestic animal health. For instance, it is controversial how deworming programmes affect incidence and severity of such important microparasite diseases as malaria. One problem is that most study systems involve domestic and laboratory animals with conditions hardly comparable to those of free-living animals. Here, we study the effect of anthelmintic treatment on coccidia infection intensity in wild Alpine marmots, M. marmota. Our results lend support to the hypothesis that helminth infection has a positive effect on concurrent microparasite infection. However, our work also points to the fact that within-host interactions between helminths and microparasites are context-dependent and can turn to negative ones once helminth burdens increase. Our study suggests that coccidia benefit from intermittent helminth infection in marmots due to the protective effects of helminth infection only during the early phase of the host's active season. Also, the marmot's response towards coccidia infection appears optimal only under no helminth infection when the host immune response towards coccidia would not be compromised, thereby pointing to the importance of regular intestinal helminth elimination by marmots just before hibernation.

Candidatus Neoehrlichia mikurensis and its co-circulation with Anaplasma phagocytophilum in Ixodes ricinus ticks across ecologically different habitats of Central Europe.

BACKGROUND: Candidatus Neoehrlichia mikurensis is a newly emerging tick-borne bacterium from the family Anaplasmataceae. Its presence in Ixodes ricinus ticks was reported from various European countries, however, it's ecology and co-circulation with another member of the same family, Anaplasma phagocytophilum has not been rigorously studied yet. FINDINGS: Candidatus N. mikurensis was detected in all sampling sites. In total, 4.5% of ticks were positive including larvae. The highest positivity was detected in Austria with a prevalence of 23.5%. The probability of Candidatus N. mikurensis occurrence increased with the proportion of ticks infected with Anaplasma phagocytophilum. CONCLUSION: A positive association between the occurrences of Candidatus N. mikurensis and A. phagocytophilum indicates that both bacteria share similar ecology for their natural foci in Central Europe.

The effect of herbivore faeces on the edaphic mite community: implications for tapeworm transmission.

Oribatid mites may be of epidemiological and medical importance because several species have been shown to serve as intermediate hosts for anoplocephalid tapeworms of wild and domestic animals. Despite their economic and conservation significance, relatively few studies examined factors influencing the effective number of oribatid mites that can serve as intermediate hosts. We examined variation in the structure of the edaphic arthropod community in functionally different territory parts of the Alpine marmot (Marmota marmota latirostris), a known definitive host of a prevalent anoplocephalid tapeworm, Ctenotaenia marmotae. We used a field experiment to test whether the abundance of oribatid mites in marmot pastures is affected by the presence of fresh herbivore faeces. We found that the abundance of soil and litter dwelling oribatid mites in marmot pastures did not change shortly after faeces addition. In contrast, numbers of other predominant soil-litter and phoretic microarthropods increased after faeces addition. The abundance of the two predominant phoretic mites colonizing the faeces was inversely related to the abundance of oribatid mites. In contrast, the abundance of a ubiquitous soil-litter mesostigmatid mite was a positive function of oribatid numbers. Although absolute numbers of oribatid mites did not change after faeces addition, our study suggests that, depending on soil quality or type, the probability of tapeworm egg ingestion by oribatid mites can be reduced due to increased interspecific prey-predatory and trophic interactions. Latrine site selection in Alpine marmots is consistent with a reduced probability of tapeworm transmission by oribatids.

Assessing the effects of climate on host-parasite interactions: a comparative study of European birds and their parasites.

BACKGROUND: Climate change potentially has important effects on distribution, abundance, transmission and virulence of parasites in wild populations of animals. METHODOLOGY/PRINCIPAL FINDING: Here we analyzed paired information on 89 parasite populations for 24 species of bird hosts some years ago and again in 2010 with an average interval of 10 years. The parasite taxa included protozoa, feather parasites, diptera, ticks, mites and fleas. We investigated whether change in abundance and prevalence of parasites was related to change in body condition, reproduction and population size of hosts. We conducted analyses based on the entire dataset, but also on a restricted dataset with intervals between study years being 5-15 years. Parasite abundance increased over time when restricting the analyses to datasets with an interval of 5-15 years, with no significant effect of changes in temperature at the time of breeding among study sites. Changes in host body condition and clutch size were related to change in temperature between first and second study year. In addition, changes in clutch size, brood size and body condition of hosts were correlated with change in abundance of parasites. Finally, changes in population size of hosts were not significantly related to changes in abundance of parasites or their prevalence. CONCLUSIONS/SIGNIFICANCE: Climate change is associated with a general increase in parasite abundance. Variation in laying date depended on locality and was associated with latitude while body condition of hosts was associated with a change in temperature. Because clutch size, brood size and body condition were associated with change in parasitism, these results suggest that parasites, perhaps mediated through the indirect effects of temperature, may affect fecundity and condition of their hosts. The conclusions were particularly in accordance with predictions when the restricted dataset with intervals of 5-15 years was used, suggesting that short intervals may bias findings.

Molecular detection of murine herpesvirus 68 in ticks feeding on free-living reptiles.

The MHV-68 (designed as Murid herpesvirus 4 (MuHV 4) strain 68) isolated from two rodents, Myodes glareolus and Apodemus flavicollis, is considered as a natural pathogen of free-living murid rodents. Recently, the detection of MHV antibodies in the blood of animals living in the same biotope as MHV-infected mice has suggested that ticks may have a role in the transmission of this pathogen. Ixodes ricinus is one the most abundant tick species in Europe known to transmit multiple pathogens causing human and animal diseases. In this study, nymphs and larvae feeding on 116 individuals of a temperate lizard species-the green lizard Lacerta viridis captured in the Slovak Karst National Park, were examined for MHV-68. The specific sequence of virion glycoprotein 150 was amplified in DNA individually isolated from I. ricinus ticks using single-copy sensitive nested polymerase chain reaction. MHV-68 was detected in ten of 649 nymphs and in five of 150 larvae, respectively. We found that 9.6% of green lizards fed at least one MHV-68-infected immature tick. Occurrence of MHV-68 within all ticks tested was 1.8%. This study is first to show that immature I. ricinus ticks feeding on free-living lizards in a Central European region could be infected with gammaherpesvirus (MHV-68), naturally infecting free-living murid rodents. Our results provide evidence supporting the hypothesis that ticks may play a mediating role in circulation of MHV-68 in nature.

Social information in nest colonisation and occupancy in a long-lived, solitary breeding bird.

Recent work increasingly reveals the importance of social information in individual dispersal decisions, population dynamics and conservation. Much of the knowledge gained to date comes from studies on short-lived and/or densely breeding species. In contrast, our understanding of the processes involved in nest-site selection for long-lived, solitary breeding species is insufficient. We increased nest-site availability by nest-box supplementation over a 5-year period in a population of a long-lived, solitary, secondary-cavity nesting bird, the European roller Coracias garrulus, breeding in natural cavities and human constructions. We tested the nest limitation and the inadvertent conspecific social information hypothesis in order to study the dynamics and mechanisms of abandonment of previously used nests and the colonisation of new ones. Our data lend support to the nest-limitation hypothesis both in terms of quantity-population and the size of breeding clusters increased, and suitability--the majority of pairs used and re-occupied nest-boxes. Nevertheless, the use of natural cavities did not decrease after 5 years. At the between-patch scale, rollers were revealed to colonise nest-boxes based on conspecific social attraction, namely distance to the nearest neighbour in the same season. Despite the unpredictability of patch productivity, at the within--patch scale, the selection of previously unoccupied cavities was consistent with the performance-based conspecific attraction hypothesis. Philopatry could account for the repeated use of cavities, because nests that were used for two successive years were more likely to also be reused in the subsequent season.

Associations between coinfection prevalence of Borrelia lusitaniae, Anaplasma sp., and Rickettsia sp. in hard ticks feeding on reptile hosts.

An increasing number of studies reveal that ticks and their hosts are infected with multiple pathogens, suggesting that coinfection might be frequent for both vectors and wild reservoir hosts. Whereas the examination of associations between coinfecting pathogen agents in natural host-vector-pathogen systems is a prerequisite for a better understanding of disease maintenance and transmission, the associations between pathogens within vectors or hosts are seldom explicitly examined. We examined the prevalence of pathogen agents and the patterns of associations between them under natural conditions, using a previously unexamined host-vector-pathogen system--green lizards Lacerta viridis, hard ticks Ixodes ricinus, and Borrelia, Anaplasma, and Rickettsia pathogens. We found that immature ticks infesting a temperate lizard species in Central Europe were infected with multiple pathogens. Considering I. ricinus nymphs and larvae, the prevalence of Anaplasma, Borrelia, and Rickettsia was 13.1% and 8.7%, 12.8% and 1.3%, and 4.5% and 2.7%, respectively. The patterns of pathogen prevalence and observed coinfection rates suggest that the risk of tick infection with one pathogen is not independent of other pathogens. Our results indicate that Anaplasma can play a role in suppressing the transmission of Borrelia to tick vectors. Overall, however, positive effects of Borrelia on Anaplasma seem to prevail as judged by higher-than-expected Borrelia-Anaplasma coinfection rates.

Within-male melanin-based plumage and bill elaboration in male house sparrows.

If there is a cost to producing a dark color patch, the size of a patch may not correspond with its pigment concentration. The plumage of male house sparrows represents a case of dark, melanin-based ornamentation, but also a case of neglecting the composite nature of dark signals in birds. Here, I investigated what kind of associations exist between the brightness, chroma, and hue of dark integumentary patches and the size of a secondary sexual trait, the bib, in male house sparrows. I found that males with a larger bib also had a darker bib and bill, and a more saturated bib, bill, epaulets, head crown, and breast than small-bibbed males. Male bib coloration in terms of brightness and chroma was more strongly related to bib size than the coloration of other integumentary patches. However, with respect to hue, only the hue of the bill and cheeks was related to bib size. My results indicate that size, brightness, and chroma of the bib, but also chroma of other deeply colored patches, convey redundant information about the signaler's quality in male house sparrows.