Chemical Profiles Differ between Communal Breeding Groups in a Highly Social Bird.

AbstractGregarious species must distinguish group members from nongroup members. Olfaction is important for group recognition in social insects and mammals but rarely studied in birds, despite birds using olfaction in social contexts from species discrimination to kin recognition. Olfactory group recognition requires that groups have a signature odor, so we tested for preen oil and feather chemical similarity in group-living smooth-billed anis (Crotophaga ani). Physiology affects body chemistry, so we also tested for an effect of egg-laying competition, as a proxy for reproductive status, on female chemical similarity. Finally, the fermentation hypothesis for chemical recognition posits that host-associated microbes affect host odor, so we tested for covariation between chemicals and microbiota. Group members were more chemically similar across both body regions. We found no chemical differences between sexes, but females in groups with less egg-laying competition had more similar preen oil, suggesting that preen oil contains information about reproductive status. There was no overall covariation between chemicals and microbes; instead, subsets of microbes could mediate olfactory cues in birds. Preen oil and feather chemicals showed little overlap and may contain different information. This is the first demonstration of group chemical signatures in birds, a finding of particular interest given that smooth-billed anis live in nonkin breeding groups. Behavioral experiments are needed to test whether anis can distinguish group members from nongroup members using odor cues.

An evaluation of germline mutations and reproductive impacts in fathead minnow (Pimephales promelas) exposed to contaminated sediment.

Polycyclic aromatic hydrocarbons (PAHs) have become ubiquitous in the aquatic environment. Some PAHs are mutagenic, potentially causing germline mutations in fish that inhabit PAH contaminated waters. We evaluated the effect of exposure to sediment-borne PAHs on reproduction and germline mutation rates in fathead minnows (Pimephales promelas). Exposure to the contaminated sediment had no significant impact on the reproductive endpoints measured in this study. Germline mutations rates at three microsatellite DNA loci were 1.69 × 10-3 in fish exposed to PAH-contaminated sediment and 0.55 × 10-3 in control fish, with zero mutations being observed in fish exposed to sediment from a reference site. While the difference in mutation rates between treatments was not statistically significant for the sample size used (15-19 families per treatment), the observed mutations rates enabled us to estimate the sample size required to detect a significant effect. To our knowledge, this is the first report of germline mutation rates in fathead minnow exposed to an environmental contaminant, providing baseline data for use in the design of future experiments.

The effect of social group size on feather corticosterone in the co-operatively breeding Smooth-billed Ani (Crotophaga ani): An assay validation and analysis of extreme social living.

Living closely with others can provide a myriad of fitness benefits, from shared territory defense to co-operative resource acquisition. Costs of social aggregation are not absent, however, and likely influence optimal and observed groups' sizes in a social species. Here, we explored optimal group size in a joint-nesting cuckoo species (the Smooth-billed Ani, Crotophaga ani) using endocrine markers of stress physiology (corticosterone, or CORT). Smooth-billed Anis exhibit intense reproductive competition that is exacerbated in atypically large groups. We therefore hypothesized that intra-group competition (measured by social group size) mediates the desirability and physiological cost of social group membership in this species. To test this hypothesis, we captured 47 adult Smooth-billed Anis (31 males, 16 females) during the breeding seasons of 2012-2014 in south-western Puerto Rico, and documented social group sizes. Tail feathers were sampled and used to quantify CORT (pg/mg) in enzyme-linked immunosorbent assays (ELISAs) (n = 50). Our analyses show significant differences in feather-CORT of adults between categorical group sizes, with individuals from atypically large social groups (≥ x + 1SD) having highest mean concentrations (33.319 pg/mg), and individuals from atypically small social groups (≤ x - 1SD) having lowest mean concentrations (8.969 pg/mg). Whether reproductive competition or effort is responsible for elevated CORT in atypically large social groups, however, remains unclear. Our results suggest that living in atypically large groups is physiologically expensive and may represent an evolutionarily unstable strategy. To our knowledge, this is the first study to explore a correlation between stress physiology and group size in a joint-nesting species.

Maternal corticosterone deposition in avian yolk: Influence of laying order and group size in a joint-nesting, cooperatively breeding species.

Glucocorticoid hormones play a key role in day-to-day adjustments to fluctuating metabolic needs. These hormones also mediate physiological and behavioral responses to stressful events, allowing individuals to cope with stressors. Various environmental insults, such as a food shortages, predation attempts, and agonistic encounters often elevate plasma glucocorticoid levels in vertebrates. Because exposure to maternally-derived (via circulation or egg) glucocorticoids may be detrimental to the developing embryo, maternal stress can have negative carryover effects on offspring fitness. We examined corticosterone, the primary avian glucocorticoid, concentrations in egg yolk in a plural-breeding, joint-nesting species, the smooth-billed ani (Crotophaga ani), in which females compete among themselves to lay eggs in the final incubated clutch. We investigated whether yolk corticosterone levels varied with laying order and group size. Because egg-laying competition leads to physiological and social stress that is intensified with group size and laying order, we predicted that yolk corticosterone levels should increase from the early to the late egg-laying period and from single female to multi-female groups. In this two-year field study, we found that yolk corticosterone levels of late-laid eggs within the communal clutch were higher in multi-female groups than in single female groups. Results from this study suggest that laying females experience higher levels of stress in multi-female groups and that this maternal stress influences yolk corticosterone concentrations. This study identifies a novel cost of group-living in plural-breeding cooperatively breeding birds, namely an increase in yolk corticosterone levels with group size that may result in detrimental effects on offspring development.

A subchronic in situ exposure method for evaluating effects in small-bodied fish at contaminated sites.

In situ fish-caging studies at contaminated sites can provide information that is more realistic compared to traditional laboratory-based studies. However, few methods have been developed for exposing sentinel fish species for subchronic durations, and fewer still are optimized for exposing small-bodied fish while maintaining fish health and growth throughout the caging trial. Those methods typically lack a feeding regimen during the fish caging trial. While that may be acceptable or even appropriate for typical short-term toxicity testing, it does limit the duration of the exposure, and may not be suitable when post-caging trials or observations are necessary. Returning healthy fish to the lab following the in situ exposure would be important, for example, in studies designed to examine long-term or multigenerational effects following an in situ exposure. In this article we describe a subchronic method for caging small fish at contaminated sites while maintaining growth and reproductive development. Fathead minnow (Pimephales promelas) were caged in situ for 6 weeks, after which time they were returned to the lab where they were evaluated for health and reproductive performance. Growth and reproductive endpoints revealed no adverse effect on fish due to fish caging and related handling, demonstrating the suitability of our caging and feeding method for long-term caging studies.

Manipulating the appearance of a badge of status causes changes in true badge expression.

Signals of dominance and fighting ability (i.e. status signals) are found in a wide range of taxa and are used to settle disputes between competitive rivals. Most previous research has considered status-signal phenotype as an attribute of the individual; however, it is more likely that signal expression is an emergent property that also incorporates aspects of the social environment. Furthermore, because an individual's signal phenotype is likely to influence its social interactions, the relationships between status signals, social environment and individual quality are probably much more complex than previously appreciated. Here, we explore the dynamic relationship between social interactions and signal expression in a previously undescribed status signal, the frontal shield of the pukeko (Porphyrio porphyrio melanotus: Aves). We demonstrate that frontal shield size is a strong predictor of dominance status within social groups, even after controlling for potentially confounding variables. Then, we evaluate the relationship between social interactions and signal expression by testing whether manipulating apparent shield size influences (i) dominance interactions and (ii) future signal expression. By showing that decreasing apparent shield size causes both an increase in the amount of aggression received and a decrease in an individual's true shield size, we provide the first evidence of dynamic feedback between signal expression and social interactions. Our study provides important insight into the role of receiver-dependent (i.e. social) costs in maintaining signal honesty and demonstrates a unique approach to studying status signalling applicable to future studies on dynamic morphological signals.

Acute stress during ontogeny suppresses innate, but not acquired immunity in a semi-precocial bird (Larus delawarensis).

Wild animals often encounter adverse conditions, and in response, activate their hypothalamic-pituitary-adrenal (HPA) axis. To date, work examining the development of the stress response has focused on altricial species, with little work focusing on species with other developmental patterns. Additionally, the effects of acute stress on indices of innate and adaptive immunity have been little studied in birds, particularly during development. We examined the ontogeny of the stress response in the semi-precocial ring-billed gull (Larus delawarensis). At hatch, 10, and 20days post-hatching, chicks underwent a standardized handling stress protocol, with blood samples taken within 3min of, and 30min after, initial disturbance. Levels of corticosterone (CORT), natural antibodies (NAb), complement activity, and immunoglobulins (IgY) were assessed in plasma samples. In contrast with altricial species, ring-billed gull chicks had detectible CORT levels at hatch, and were able to mount a stress response. At all ages, acute handling stress depressed NAb levels and complement-mediated lysis, but not IgY levels. IgY levels were higher in two chick broods than three chick broods, suggesting levels are determined in part by resource dependence. Our data provide insight into the development of the stress response and immune function in a colonial waterbird species, in which chicks are mobile shortly post hatch, and subject to aggression and possible injury from nearby adults.

Factors affecting germline mutations in a hypervariable microsatellite: a comparative analysis of six species of swallows (Aves: Hirundinidae).

Microsatellites mutate frequently by replication slippage. Empirical evidence shows that the probability of such slippage mutations may increase with the length of the repeat region as well as exposure to environmental mutagens, but the mutation rate can also differ between the male and female germline. It has been hypothesized that more intense sexual selection or sperm competition can also lead to elevated mutation rates, but the empirical evidence is inconclusive. Here, we analyzed the occurrence of germline slippage mutations in the hypervariable pentanucleotide microsatellite locus HrU10 across six species of swallow (Aves: Hirundinidae). These species exhibit marked differences in the length range of the microsatellite, as well as differences in the intensity of sperm competition. We found a strong effect of microsatellite length on the probability of mutation, but no residual effect of species or their level of sperm competition when the length effect was accounted for. Neither could we detect any difference in mutation rate between tree swallows (Tachycineta bicolor) breeding in Hamilton Harbour, Ontario, an industrial site with previous documentation of elevated mutation rates for minisatellite DNA, and a rural reference population. However, our cross-species analysis revealed two significant patterns of sex differences in HrU10 germline mutations: (1) mutations in longer alleles occurred typically in the male germline, those in shorter alleles in the female germline, and (2) male germline mutations were more often expansions than contractions, whereas no directional bias was evident in the female germline. These results indicate some fundamental differences in male and female gametogenesis affecting the probability of slippage mutations. Our study also reflects the value of a comparative, multi-species approach for locus-specific mutation analyses, through which a wider range of influential factors can be assessed than in single-species studies.

Tetracycline-resistant coliforms in the effluent of the main sewage treatment plant in Hamilton, Ontario - do they have a common ancestral strain?

Sewage, a major source of bacterial contamination of the environment, can be an important health hazard. The presence of antibiotic-resistant bacteria in sewage can exacerbate this problem. The sources of antibiotic-resistant bacteria in sewage are, for this reason, worth identifying and addressing. The bacterial flora in the effluent of the Woodward Avenue Wastewater Treatment Plant (WAWTP) in Hamilton, Ontario, Canada, contains many antibiotic-resistant coliforms. Here we ask, are the antibiotic resistance genes in the coliforms in the effluent of WAWTP descended from a recent common ancestor strain? If so, the source could be identified and eliminated. If, on the other hand, the antibiotic resistance genes in the bacterial flora of the WAWTP have more than one origin, identification and elimination of the source(s) could be difficult. There was considerable diversity of antibiotic resistance patterns and antibiotic resistance genes among the effluent and influent coliform isolates of the WAWTP, suggesting multiple genetic ancestry. The patterns of horizontal transmissibility and sequence differences in the genes tetA and tetE among these coliform isolates also suggest that they have no one predominant ancestral strain. Using the same logic, the evidence presented here is not compatible with a single ancestral origin of the antibiotic resistance genes in the isolates described herein.

Influence of a contaminated fish diet on germline expanded-simple-tandem-repeat mutation frequency in mice.

Herring gulls (Larus argentatus) in polluted areas on the North American Great Lakes were previously shown to have elevated germline mutation frequencies at minisatellite DNA loci. Airborne or dietary contaminants likely caused induced mutations, but the importance of each exposure type was unknown. Follow-up experiments with lab mice determined that air pollution significantly induced germline mutations; however, an evaluation of mutations induced by the diet of herring gulls has not yet been conducted. To address this issue, we fed mice a high-fish diet (58% wet mass) of the most common prey species for herring gulls nesting in Hamilton Harbour, a polluted industrial area on Lake Ontario. We bred the mice and screened pedigrees for germline mutations at expanded-simple-tandem-repeat (ESTR) DNA loci. Mutation frequencies were compared to those in a reference group that was fed fish from Atlantic Canada, and a control group that was fed commercial chow. Germline mutation frequencies were highest in mice fed contaminated fish, but were only marginally or not significantly affected by diet treatment. Statistical power to detect differences among treatment groups was low, and the effect of diet may have more clearly emerged if larger sample sizes were available. Levels of organic pollutants in the fish from Hamilton Harbour were higher than those from Atlantic Canada, but their ability to induce ESTR mutations is unknown. Our findings suggest that a contaminated fish diet may contribute to the elevated germline mutation frequencies observed previously in gulls at this site, but air pollution is likely a more important route of exposure.

Do group size and laying order influence maternal deposition of testosterone in smooth-billed ani eggs?

The avian egg contains maternal hormones that affect behavior, growth, morphology, and offspring survival. Evidence to date suggests that patterns of yolk androgen deposition could provide females with a means to manipulate sibling competition and, thereby, increase their fitness. We examined yolk testosterone (T) concentrations in eggs of the smooth-billed ani (Crotophaga ani) to understand patterns of androgen deposition in eggs of this plural-breeding joint-nesting cooperatively breeding species. We tested the hatching asynchrony adjustment hypothesis, which states that increases in yolk androgen levels over the laying sequence function to mitigate the disadvantage of being a later-hatched chick in species without adaptive brood reduction. We also investigated the effect of group size on yolk T deposition to test the hypothesis that females in multi-female groups could give a competitive edge to their own chicks by depositing higher T levels in their eggs. Predictions of the hatching asynchrony adjustment hypothesis were supported in both single- and multi-female groups as yolk testosterone levels increased from early- to late-laid eggs. This suggests that ani females can influence nestling competition and chick survival by within-clutch differential T allocation. Unexpectedly, we did not observe an effect of group size on yolk T deposition. Yolk testosterone concentrations may not be a mere reflection of a female's hormonal status as female plasma circulating levels of T did not vary in the same direction as yolk T levels. Results of this study therefore support the idea that females may adaptively manipulate chick behavior through hormonal deposition in eggs.

An approach to feeding high-percentage fish diets to mice for human and wildlife toxicology studies.

Experimental feeding of sport fish to rodents has been an important tool for the study of biological effects induced by a contaminated fish diet. Most rodent feeding studies have used low-to-moderate levels of tissue from large fish species incorporated into diets fed to rats and have given little consideration to issues of diet palatability or nutrition. There are currently no rodent diet models suitable for assessing the risk to human populations of diets very high in daily fish content or to wildlife species consuming high percentages of whole, small-bodied fish. In this study, we describe an approach to feeding mice high percentages (up to 50%) of homogenized, whole fish using Atlantic herring (Clupea harengus) as a test species. We created a novel gel diet medium for mice that contains a variety of nutritional supplements and is flexible in terms of the fish percentage that can be incorporated. In choice trials, mice preferred 30 and 35% fish gels to their regular commercial dry chow, indicating that the gel diet medium was palatable. In a longer feeding trial, mice ate 35% fish gel for 12 days and 50% fish gel for 12 days (total of 24 consecutive days) and did not differ in body mass compared to age- and sex-matched controls. We conclude that our fish-based gel diet is suitable for rodent feeding trials in toxicology studies that examine dose responses to fish consumption and risk in human and wildlife populations among which daily fish intake is very high. Our general approach may also be applicable for feeding mice materials other than fish.

A marine fish diet reduces spontaneous lymphoma in outbred Swiss-Webster mice.

Diets rich in marine organisms or their oils are known to suppress solid tumor development in humans and rodents, but the potential for marine foods to affect hematopoietic system cancers is not well understood. As part of a toxicology study, we fed groups of mice three different diets for 10 weeks: marine fish, 58% homogenized Atlantic smelt and herring; freshwater fish, 58% smelt and alewife from the North American Great Lakes, and commercial dry rodent chow. Between 1 and 15 weeks following dietary treatment, 20 of 103 (19.4%) mice unexpectedly developed spontaneous lymphoma. Disease incidence peaked when the mice were 7-8 months old, and was not distributed equally across treatment groups. Mice in the control (30%) and fresh water fish (27.5%) groups had significantly higher incidences of lymphoma than those fed Atlantic fish species (5%). Although our experiment was not originally designed for this purpose, our results indicate that consumption of fat-rich Atlantic smelt and herring protected mice against hematopoietic tumor development.

Reduction of particulate air pollution lowers the risk of heritable mutations in mice.

Urban and industrial air pollution can cause elevated heritable mutation rates in birds and rodents. The relative importance of airborne particulate matter versus gas-phase substances in causing these genetic effects under ambient conditions has been unclear. Here we show that high-efficiency particulate-air (HEPA) filtration of ambient air significantly reduced heritable mutation rates at repetitive DNA loci in laboratory mice housed outdoors near a major highway and two integrated steel mills. These findings implicate exposure to airborne particulate matter as a principal factor contributing to elevated mutation rates in sentinel mice and add to accumulating evidence that air pollution may pose genetic risks to humans and wildlife.

Particulate air pollution and inheritable mutations in mice: Possible health effects?

Extract: In a recent study we showed that the particulate fraction of air pollution was capable of increasing the rate at which DNA changes were passed to the next generation (germline mutations) in mice. Here we briefly describe the research that brought us to this experiment, followed by a description of the recent study and its implications. Herring gulls (Larus argentatus) are fish-eating water birds that breed throughout the Laurentian Great Lakes and in many other parts of the Northern Hemisphere. In the early to mid 1990's, these birds were used as a sentinel wildlife species in our studies of germline mutations as a function of distance from integrated steel mills. The assay used involved comparison of the DNA profiles (minisatellite DNA -- non-coding sequences) of gull offspring with those of their parents, and identification of novel bands, or mutations, in the DNA profiles of offspring.

Air pollution induces heritable DNA mutations.

Hundreds of thousands of people worldwide live or work in close proximity to steel mills. Integrated steel production generates chemical pollution containing compounds that can induce genetic damage (1, 2). Previous investigations of herring gulls in the Great Lakes demonstrated elevated DNA mutation rates near steel mills (3, 4) but could not determine the importance of airborne or aquatic routes of contaminant exposure, or eliminate possible confounding factors such as nutritional status and disease burden. To address these issues experimentally, we exposed laboratory mice in situ to ambient air in a polluted industrial area near steel mills. Heritable mutation frequency at tandem-repeat DNA loci in mice exposed 1 km downwind from two integrated steel mills was 1.5- to 2.0-fold elevated compared with those at a reference site 30 km away. This statistically significant elevation was due primarily to an increase in mutations inherited through the paternal germline. Our results indicate that human and wildlife populations in proximity to integrated steel mills may be at risk of developing germline mutations more frequently because of the inhalation of airborne chemical mutagens.