A new tool for studying waterfowl immune and metabolic responses: Molecular level analysis using kinome profiling.

Here, we describe the design of an Anas-specific kinome peptide array that can be used to study the immunometabolic responses of mallard and American black duck to pathogens, contaminants, and environmental stress. The peptide arrays contain 2,642 unique phosphorylate-able peptide sequences representing 1,900 proteins. These proteins cover a wide array of metabolic and immunological processes, and 758 Gene Ontology Biological processes are statistically significantly represented on the duck peptide array of those 164 contain the term "metabolic" and 25 "immune." In addition, we conducted a comparison of mallard to American black duck at a genetic and proteomic level. Our results show a significant genomic and proteomic overlap between these two duck species, so that we have designed a cross-reactive peptide array capable of studying both species. This is the first reported development of a wildlife species-specific kinome peptide array.

Maternal androgens in avian brood parasites and their hosts: Responses to parasitism and competition?

In the coevolutionary dynamic of avian brood parasites and their hosts, maternal (or transgenerational) effects have rarely been investigated. We examined the potential role of elevated yolk testosterone in eggs of the principal brood parasite in North America, the brown-headed cowbird, and three of its frequent host species. Elevated maternal androgens in eggs are a common maternal effect observed in many avian species when breeding conditions are unfavorable. These steroids accelerate embryo development, shorten incubation period, increase nestling growth rate, and enhance begging vigor, all traits that can increase the survival of offspring. We hypothesized that elevated maternal androgens in host eggs are a defense against brood parasitism. Our second hypothesis was that elevated maternal androgens in cowbird eggs are a defense against intra-specific competition. For host species, we found that elevated yolk testosterone was correlated with parasitized nests of small species, those whose nest success is most reduced by cowbird parasitism. For cowbirds, we found that elevated yolk testosterone was correlated with eggs in multiply-parasitized nests, which indicate intra-specific competition for nests due to high cowbird density. We propose experimental work to further examine the use of maternal effects by cowbirds and their hosts.

Enhanced innate immune responses in a brood parasitic cowbird species: Degranulation and oxidative burst.

We examined the relative effectiveness of two innate immune responses in two species of New World blackbirds (Passeriformes, Icteridae) that differ in resistance to West Nile virus (WNV). We measured degranulation and oxidative burst, two fundamental components of phagocytosis, and we predicted that the functional effectiveness of these innate immune responses would correspond to the species' relative resistance to WNV. The brown-headed cowbird (Molothrus ater), an obligate brood parasite, had previously shown greater resistance to infection with WNV, lower viremia and faster recovery when infected, and lower subsequent antibody titers than the red-winged blackbird (Agelaius phoeniceus), a close relative that is not a brood parasite. We found that cowbird leukocytes were significantly more functionally efficient than those of the blackbird leukocytes and 50% more effective at killing the challenge bacteria. These results suggest that further examination of innate immunity in the cowbird may provide insight into adaptations that underlie its greater resistance to WNV. These results support an eco-immunological interpretation that species like the cowbird, which inhabit ecological niches with heightened exposure to parasites, experience evolutionary selection for more effective immune responses.

Patterns of maternal yolk hormones in eastern screech owl eggs (Megascops asio).

Owl clutches typically hatch asynchronously, and brood size hierarchies develop. In this study, we describe intra-clutch variation of testosterone, androstenedione, estradiol, and corticosterone in Eastern screech owl egg yolks. In order to assess whether these hormones may have originated in the follicle, we also characterize variation of testosterone, androstenedione, and corticosterone within the exterior, intermediate, and interior regions of the yolk. Concentrations of testosterone and androstenedione were distributed relatively evenly across egg lay order with the exception of first-laid eggs that had significantly lower concentrations of both androgens than eggs later in the laying sequence. Corticosterone and estradiol did not vary with laying order. Our results suggest that when food is abundant, yolk hormones are deposited in patterns that minimize sibling differences except to reduce dominance by the first-hatching chick. Testosterone and androstenedione concentrations varied throughout the yolk, while corticosterone was evenly distributed throughout the yolk. This supports a follicular origin for both yolk androgens, and an adrenal origin for yolk corticosterone.

A simplified method for extracting androgens from avian egg yolks.

Female birds deposit significant amounts of steroid hormones into the yolks of their eggs. Studies have demonstrated that these hormones, particularly androgens, affect nestling growth and development. In order to measure androgen concentrations in avian egg yolks, most authors follow the extraction methods outlined by Schwabl (1993. Proc. Nat. Acad. Sci. USA 90:11446-11450). We describe a simplified method for extracting androgens from avian egg yolks. Our method, which has been validated through recovery and linearity experiments, consists of a single ethanol precipitation that produces substantially higher recoveries than those reported by Schwabl (1993. Proc. Nat. Acad. Sci. USA 90:11446-11450). Zoo Biol 28:137-143, 2009.

Comparison of immune responses of brown-headed cowbird and related blackbirds to west Nile and other mosquito-borne encephalitis viruses.

The rapid geographic spread of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) across the United States has stimulated interest in comparative host infection studies to delineate competent avian hosts critical for viral amplification. We compared the host competence of four taxonomically related blackbird species (Icteridae) after experimental infection with WNV and with two endemic, mosquito-borne encephalitis viruses, western equine encephalomyelitis virus (family Togaviridae, genus Alphavirus, WEEV), and St. Louis encephalitis virus (family Flaviviridae, genus Flavivirus, SLEV). We predicted differences in disease resistance among the blackbird species based on differences in life history, because they differ in geographic range and life history traits that include mating and breeding systems. Differences were observed among the response of these hosts to all three viruses. Red-winged Blackbirds were more susceptible to SLEV than Brewer's Blackbirds, whereas Brewer's Blackbirds were more susceptible to WEEV than Red-winged Blackbirds. In response to WNV infection, cowbirds showed the lowest mean viremias, cleared their infections faster, and showed lower antibody levels than concurrently infected species. Brown-headed Cowbirds also exhibited significantly lower viremia responses after infection with SLEV and WEEV as well as coinfection with WEEV and WNV than concurrently infected icterids. We concluded that cowbirds may be more resistant to infection to both native and introduced viruses because they experience heightened exposure to a variety of pathogens of parenting birds during the course of their parasitic life style.

Experimental West Nile virus infection in Eastern Screech Owls (Megascops asio).

Eastern Screech Owls (EASOs) were experimentally infected with the pathogenic New York 1999 strain of West Nile virus (WNV) by subcutaneous injection or per os. Two of nine subcutaneously inoculated birds died or were euthanatized on 8 or 9 days postinfection (DPI) after <24 hr of lethargy and recumbency. All subcutaneously inoculated birds developed levels of viremia that are likely infectious to mosquitoes, with peak viremia levels ranging from 10(5.0) to 10(9.6) plaque-forming units/ml. Despite the viremia, the remaining seven birds did not display signs of illness. All birds alive beyond 5 DPI seroconverted, although the morbid birds demonstrated significantly lower antibody titers than the clinically normal birds. Cagemates of infected birds did not become infected. One of five orally exposed EASOs became viremic and seroconverted, whereas WNV infection in the remaining four birds was not evident. All infected birds shed virus via the oral and cloacal route. Early during infection, WNV targeted skin, spleen, esophagus, and skeletal muscle. The two morbid owls had myocardial and skeletal muscle necrosis and mild encephalitis and nephritis, whereas some of the clinically healthy birds that were sacrificed on 14 DPI had myocardial arteritis and renal phlebitis. WNV is a significant pathogen of EASOs, causing pathologic lesions with varying clinical outcomes.

Auditory brainstem responses in the Eastern Screech Owl: an estimate of auditory thresholds.

The auditory brainstem response (ABR), a measure of neural synchrony, was used to estimate auditory sensitivity in the eastern screech owl (Megascops asio). The typical screech owl ABR waveform showed two to three prominent peaks occurring within 5 ms of stimulus onset. As sound pressure levels increased, the ABR peak amplitude increased and latency decreased. With an increasing stimulus presentation rate, ABR peak amplitude decreased and latency increased. Generally, changes in the ABR waveform to stimulus intensity and repetition rate are consistent with the pattern found in several avian families. The ABR audiogram shows that screech owls hear best between 1.5 and 6.4 kHz with the most acute sensitivity between 4-5.7 kHz. The shape of the average screech owl ABR audiogram is similar to the shape of the behaviorally measured audiogram of the barn owl, except at the highest frequencies. Our data also show differences in overall auditory sensitivity between the color morphs of screech owls.