Increased genetic diversity and immigration after West Nile virus emergence in American crows: No evidence for a genetic bottleneck.

Infectious diseases can cause steep declines in wildlife populations, leading to changes in genetic diversity that may affect the susceptibility of individuals to infection and the overall resilience of populations to pathogen outbreaks. Here, we examine evidence for a genetic bottleneck in a population of American crows (Corvus brachyrhynchos) before and after the emergence of West Nile virus (WNV). More than 50% of marked birds in this population were lost over the 2-year period of the epizootic, representing a 10-fold increase in adult mortality. Using analyses of single-nucleotide polymorphisms (SNPs) and microsatellite markers, we tested for evidence of a genetic bottleneck and compared levels of inbreeding and immigration in the pre- and post-WNV populations. Counter to expectations, genetic diversity (allelic diversity and the number of new alleles) increased after WNV emergence. This was likely due to increases in immigration, as the estimated membership coefficients were lower in the post-WNV population. Simultaneously, however, the frequency of inbreeding appeared to increase: Mean inbreeding coefficients were higher among SNP markers, and heterozygosity-heterozygosity correlations were stronger among microsatellite markers, in the post-WNV population. These results indicate that loss of genetic diversity at the population level is not an inevitable consequence of a population decline, particularly in the presence of gene flow. The changes observed in post-WNV crows could have very different implications for their response to future pathogen risks, potentially making the population as a whole more resilient to a changing pathogen community, while increasing the frequency of inbred individuals with elevated susceptibility to disease.

Self-Rehabilitation of a Captive American Crow at Binghamton Zoo.

The behavioral transition from an entirely unflighted-to-flighted, female yearling American crow (Corvus brachyrhynchos) in captivity in a specially designed exhibit was documented at the Binghamton Zoo at Ross Park in Binghamton, NY. Upon arrival, the focal crow had no complete primary feathers or retrices and had been in captivity since fledging. She apparently had never flown successfully and was using her legs and an abnormal body orientation to cushion her landing on the ground. In a social and physical environment with 3 flying companion crows and staggered perches, she developed and appeared to "practice" routines that ultimately resulted in her recovering normal body posture and flight ability. The crow's practice routine was recorded during daily observations using an ethogram of social and locomotor behaviors. Both enclosure design and the social environment may have provided an ideal setting for the self-motivation of practice and this recovery. Attention to the potential for such practice could facilitate rehabilitation in individuals for whom rehabilitation was not thought possible.

Plasmid-mediated resistance to cephalosporins and quinolones in Escherichia coli from American crows in the USA.

American crow (Corvus brachyrhynchos) faeces were tested for Escherichia coli with plasmid-mediated quinolone resistance (PMQR), extended-spectrum beta-lactamases (ESBL) and AmpC beta-lactamases. A total of 590 faecal samples were collected at four roosting sites in the USA and cultivated on selective media. Pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) were performed to assess clonality. Transferability of resistance genes was studied using conjugation and transformation bioassays. In total, 78 (13%, n = 590) cefotaxime-resistant isolates were obtained, of which 66 and 12 displayed AmpC and ESBL phenotypes, respectively. Fifty-four AmpC-producing isolates carried blaCMY-2 . Isolates producing ESBLs contained genes blaCTX-M-27 (5 isolates), blaCTX-M-15 (4), blaCTX-M-14 (2) and blaCTX-M-1 (1). Ninety isolates (15%, n = 590) with reduced susceptibility to ciprofloxacin were obtained, among which 14 harboured PMQR genes aac(6')-Ib-cr (4 isolates), qnrB19 (3), qnrS1 (2), qnrA1 (2), qnrB2 (1), qnrB6 (1) and qnrD3 (1). High genetic diversity was revealed by PFGE and MLST. Epidemiologically important E. coli clones (e.g., ST131, ST405) were identified. Plasmids carrying blaCMY-2 were assigned predominantly to IncA/C (8 plasmids), IncI1/ST23 (5) and IncI1/ST12 (3). The study demonstrates a widespread occurrence of E. coli with ESBL, AmpC and PMQR genes associated with clinically important multidrug-resistant clones and epidemic plasmids, in American crows.

Acoustic profiling in a complexly social species, the American crow: caws encode information on caller sex, identity, and behavioural context.

Previous research on inter-individual variation in the calls of corvids has largely been restricted to single call types, such as alarm or contact calls, and has rarely considered the effects of age on call structure. This study explores structural variation in a contextually diverse set of "caw" calls of the American crow (Corvus brachyrhynchos), including alarm, foraging recruitment and territorial calls, and searches for structural features that may be associated with behavioural context and caller sex, age, and identity. Automated pitch detection algorithms are used to generate 23 pitch-related and spectral parameters for a collection of caws from 18 wild, marked crows. Using principal component analysis and mixed models, we identify independent axes of acoustic variation associated with behavioural context and with caller sex, respectively. We also have moderate success predicting caller sex and identity from call structure. However, we do not find significant acoustic variation with respect to caller age.

American crows as carriers of vancomycin-resistant enterococci with vanA gene.

We studied the vanA-carrying vancomycin-resistant enterococci (VRE) isolated from American crows in the United States during the winter 2011/2012. Faecal samples from crows were cultured selectively for VRE and characterized. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to examine epidemiological relationships of vanA-containing VRE. Isolates were tested in vitro for their ability to horizontally transfer the vancomycin resistance trait. VRE with the vanA gene were found in 15 (2.5%) of 590 crows samples, from which we obtained 22 different isolates. Enterococcal species were Enterococcus faecium (14) and E. faecalis (8). One, two and 19 isolates originated from Kansas, New York State and Massachusetts, respectively. Based on MLST analysis, E. faecium isolates were grouped as ST18 (6 isolates), ST555 (2), and novel types ST749 (1), ST750 (3), ST751 (1), ST752 (1). Enterococcus faecalis isolates belonged to ST6 (1), ST16 (3) and ST179 (4). All isolates were able to transfer the vancomycin resistance trait via filter mating with very high transfer range. Clinically important enterococci with the vanA gene occur in faeces of wild American crows throughout the United States. These migrating birds may contribute to the dissemination of VRE in environment over large distances. [Correction added after first online publication on 06 August 2013: The number of E. faecium ST752 isolate is now amended to '1', consistent with that shown in the 'Results' section and Figure 2.].

Evidence for contagious behaviors in budgerigars (Melopsittacus undulatus): an observational study of yawning and stretching.

Yawning is contagious in humans and some non-human primates. If there are social functions to contagious behaviors, such as yawning, they might occur in other highly social vertebrates. To investigate this possibility, we conducted an observational study of yawning and an associated behavior, stretching, in budgerigars (Melopsittacus undulatus), a social, flock-living parrot. Flock-housed budgerigars were videotaped for 1.5h at three time-blocks during the day (early morning, afternoon and early evening), and the times of all yawns and stretches for each bird were recorded. Both yawning and stretching were temporally clumped within sessions, but were uniformly distributed across the trials of a particular time-block. This suggests that clumping was not a result of circadian patterning and that both behaviors could be contagious. There was additional evidence of contagion in stretching, which occurred in two forms - a posterior-dorsal extension of either one foot or both feet. Birds that could have observed a conspecific stretch, and that then stretched themselves within 20s, replicated the form of the earlier stretch significantly more often than expected by chance. This study provides the first detailed description of temporal patterns of yawning under social conditions in a flock-living species as well as the first support for contagious yawning and stretching in a non-primate species in a natural context. Experimental evidence will be necessary to confirm the extent of contagion in either behavior.

Auditory disturbances promote temporal clustering of yawning and stretching in small groups of budgerigars (Melopsittacus undulatus).

Yawning may serve both social and nonsocial functions. When budgerigars (Melopsittacus undulatus) are briefly held, simulating capture by a predator, the temporal pattern of yawning changes. When this species is observed in a naturalistic setting (undisturbed flock), yawning and also stretching, a related behavior, are mildly contagious. On the basis of these findings, we hypothesized that a stressful event would be followed by the clustering of these behaviors in a group of birds, which may be facilitated both by a standard pattern of responding to a startling stressor and also contagion. In this study, we measured yawning and stretching in 4-bird groups following a nonspecific stressor (loud white noise) for a period of 1 hr, determining whether auditory disturbances alter the timing and frequency of these behaviors. Our results show that stretching, and to a lesser degree yawning, were nonrandomly clumped in time following the auditory disturbances, indicating that the temporal clustering is sensitive to, and enhanced by, environmental stressors while in small groups. No decrease in yawning such as found after handling stress was observed immediately after the loud noise but a similar increase in yawning 20 min after was observed. Future research is required to tease apart the roles of behavioral contagion and a time-setting effect following a startle in this species. This research is of interest because of the potential role that temporal clumping of yawning and stretching could play in both the collective detection of, and response to, local disturbances or predation threats.

CHANGES IN AMBIENT TEMPERATURE TRIGGER YAWNING BUT NOT STRETCHING IN RATS.

Yawning appears to be involved in arousal, state change, and activity across vertebrates. Recent research suggests that yawning may support effective changes in mental state or vigilance through cerebral cooling. To further investigate the relationship between yawning, state change, and thermoregulation, 12 Sprague-Dawley rats (Rattus norvegicus) were exposed to a total of two hours of ambient temperature manipulation over a period of 48 hours. Using a repeated measures design, each rat experienced a range of increasing (22→32°C), decreasing (32→22°C), and constant temperatures (22°C; 32°C). Yawning and locomotor activity occurred most frequently during initial changes in temperature, irrespective of direction, compared to more extended periods of temperature manipulation. The rate of yawning also diminished during constant high temperatures (32°C) compared to low temperatures (22°C). Unlike yawning, however, stretching was unaffected by ambient temperature variation. These findings are compared to recent work on budgerigars (Melopsittacus undulatus), and the ecological selective pressures for yawning in challenging thermal environments are discussed. The results support previous comparative research connecting yawning with arousal and state change, and contribute to refining the predictions of the thermoregulatory hypothesis across vertebrates.

Condition, innate immunity and disease mortality of inbred crows.

Cooperatively breeding American crows (Corvus brachyrhynchos) suffer a severe disease-mediated survival cost from inbreeding, but the proximate mechanisms linking inbreeding to disease are unknown. Here, we examine indices of nestling body condition and innate immunocompetence in relationship to inbreeding and disease mortality. Using an estimate of microsatellite heterozygosity that predicts inbreeding in this population, we show that inbred crows were in relatively poor condition as nestlings, and that body condition index measured in the first 2-33 days after hatching, in addition to inbreeding index, predicted disease probability in the first 34 months of life. Inbred nestlings also mounted a weaker response along one axis of innate immunity: the proportion of bacteria killed in a microbiocidal assay increased as heterozygosity index increased. Relatively poor body condition and low innate immunocompetence are two mechanisms that might predispose inbred crows to ultimate disease mortality. A better understanding of condition-mediated inbreeding depression can guide efforts to minimize disease costs of inbreeding in small populations.

The direction and range of ambient temperature change influences yawning in budgerigars (Melopsittacus undulatus).

Comparative research suggests that yawning is a thermoregulatory behavior in homeotherms. Our previous experiments revealed that yawning increased in budgerigars (Melopsittacus undulatus) as ambient temperature was raised toward body temperature (22-->34 degrees C). In this study, we identify the range of temperatures that triggers yawning to rule out the possible effect of changing temperature in any range. To corroborate its thermoregulatory function, we also related the incidence of yawning to other avian thermoregulatory behaviors in budgerigars (e.g., panting, wing venting). In a repeated measures design, 16 budgerigars were exposed to 4 separate 10-min periods of changing temperatures: (a) low-increasing (23-->27 degrees C), (b) high-increasing (27-->33 degrees C), (c) high-decreasing (34-->28 degrees C), and (d) low-decreasing (28-->24 degrees C). Birds yawned significantly more during the high-increasing temperature range, and yawning was positively correlated with ambient temperature across trials. Yawning was also positively correlated with other thermoregulatory behaviors. This research clarifies the previously demonstrated relationship between yawning rate and temperature by providing evidence that the physiological trigger for yawning is related to increasing body temperatures rather than the detection of changing external temperatures.

Non-West Nile virus-associated mortality in a population of American crows (Corvus brachyrhynchos): a gross and histopathologic study.

The American crow (Corvus brachyrhynchos) is a common urban and rural inhabitant of the Northeast and Midwest United States that is commonly infected with West Nile virus (WNV). The current study was initiated to determine non-WNV-associated causes of mortality in the American crow. All animals (40/40) tested negative for WNV infection via polymerase chain reaction and had no evidence of infection based on immunohistochemistry. Common gross necropsy findings included external trauma (6/40), hepatosplenomegaly (6/40), poxviral dermatitis (5/40), and pneumonia (3/40). Common histologic findings included endoparasitism (32/40), multifocal hepatic and splenic necrosis (7/40), pigment accumulation in the spleen (5/40), and disseminated bacterial infection (3/40). The most significant and debilitating diseases included fungal pneumonia and poxvirus-associated lesions. The present report increases the knowledge of diseases present in the American crow population.

Direct benefits and genetic costs of extrapair paternity for female American crows (Corvus brachyrhynchos).

The idea that extrapair paternity (EPP) in birds is part of a mixed reproductive strategy driven primarily by females is controversial. In cooperatively breeding American crows, we compared predictions of four female benefits hypotheses-the genetic diversity, good genes, genetic compatibility, and direct benefits hypotheses-to our predictions if EPP was primarily male driven. We found that genetically diverse broods were not more successful, extrapair young were not in better condition and did not have a higher survival probability, and, contrary to prediction, offspring sired by within-group extrapair males were more inbred than within-pair offspring. There was evidence of direct benefits, however: provisioning rate and number of surviving offspring were higher in groups containing within-group extrapair sires. Females therefore derived no apparent benefits from extragroup extrapair males but both direct benefits and genetic costs from within-group extrapair males. We suggest that males and females both influence the distribution of EPP in this system.

Growth and nutritional state of American Crow nestlings vary between urban and rural habitats.

In urbanized areas, many adult birds find sufficient foods to survive, but the anthropogenic foods that are abundant there may be detrimental to nestling growth. In fact, American Crow (Corvus brachyrhynchos) nestlings are smaller in suburban than rural areas, possibly because of nutrient limitation. Here, we seek to identify possible causes of size differences by comparing both size and blood chemistry measures in rural and suburban crow nestlings. We quantified land use in known crow territories and distinguished three distinct environments: suburban-residential, suburban-managed (e.g., golf courses), and rural. We measured nestlings near fledging age in each environment and bled them for determination of unbound plasma calcium, total protein, and corticosterone. We supplemented a subset of broods in suburban-residential and rural areas with a food high in protein and calcium. Rural nestlings were significantly larger than suburban-residential crows and had higher total serum protein. Nestlings in suburban-managed areas were intermediate in size and serum protein but had the lowest plasma calcium levels. Nestling corticosterone levels did not differ significantly among habitats, indicating that, although suburban nestlings may be food-limited, they were not starving. Supplemented nestlings in suburban-residential areas were significantly larger in some growth measures than their unsupplemented counterparts. Unexpectedly, supplemented rural nestlings were significantly smaller than unsupplemented rural ones, suggesting that parents use easily accessible food even when it is nutritionally suboptimal. Our results indicate that nestlings in suburban areas are nutrient restricted, rather than calorie restricted.

Disease-mediated inbreeding depression in a large, open population of cooperative crows.

Disease-mediated inbreeding depression is a potential cost of living in groups with kin, but its general magnitude in wild populations is unclear. We examined the relationships between inbreeding, survival and disease for 312 offspring, produced by 35 parental pairs, in a large, open population of cooperatively breeding American crows (Corvus brachyrhynchos). Genetic analyses of parentage, parental relatedness coefficients and pedigree information suggested that 23 per cent of parental dyads were first- or second-order kin. Heterozygosity-heterozygosity correlations suggested that a microsatellite-based index of individual heterozygosity predicted individual genome-wide heterozygosity in this population. After excluding birds that died traumatically, survival probability was lower for relatively inbred birds during the 2-50 months after banding: the hazard rate for the most inbred birds was 170 per cent higher than that for the least inbred birds across the range of inbreeding index values. Birds that died with disease symptoms had higher inbreeding indices than birds with other fates. Our results suggest that avoidance of close inbreeding and the absence of inbreeding depression in large, open populations should not be assumed in taxa with kin-based social systems, and that microsatellite-based indices of individual heterozygosity can be an appropriate tool for examining the inbreeding depression in populations where incest and close inbreeding occur.

Reproductive partitioning and the assumptions of reproductive skew models in the cooperatively breeding American crow.

Understanding the benefits of cooperative breeding for group members of different social and demographic classes requires knowledge of their reproductive partitioning and genetic relatedness. From 2004-2007, we examined parentage as a function of relatedness and social interactions among members of 21 American crow (Corvus brachyrhynchos) family groups. Paired female breeders monopolized maternity of all offspring in their broods, whereas paired male breeders sired 82.7% of offspring, within-group auxiliary males sired 6.9% of offspring, and extragroup males sired 10.4% of offspring. Although adult females had fewer opportunities for direct reproduction as auxiliaries than males, they appeared to have earlier opportunities for independent breeding. These different opportunities for direct reproduction probably contributed to the male biased adult auxiliary sex ratio. Patterns of reproductive partitioning and conflict among males were most consistent with a synthetic reproductive skew model, in which auxiliaries struggled with breeders for a limited reproductive share, beyond which breeders could evict them. Counter to a frequent assumption of reproductive skew models, female breeders appeared to influence paternity, although their interests might have agreed with the interests of their paired males. Unusual among cooperative breeders, close inbreeding and incest occurred in this population. Incest avoidance between potential breeders did not significantly affect reproductive skew.