Weathered MC252 crude oil-induced anemia and abnormal erythroid morphology in double-crested cormorants (Phalacrocorax auritus) with light microscopic and ultrastructural description of Heinz bodies.

Injury assessment of birds following the Deepwater Horizon (DWH) oil spill in 2010 was part of the Natural Resource Damage Assessment. One reported effect was hemolytic anemia with the presence of Heinz bodies (HB) in birds, however, the role of route and magnitude of exposure to oil is unknown. The purpose of the present study was to determine if double-crested cormorants (Phalacocorax auritis; DCCO) exposed orally and dermally to artificially weathered crude oil would develop hemolytic anemia including HB and reticulocytosis. In the oral experiment, sub-adult, mixed-sex DCCOs were fed control (n = 8) or oil-injected fish with a daily target dose of 5 (n = 9) or 10 (n = 9) ml oil/kg for 21 days. Then, subadult control (n = 12) and treated (n = 13) cormorant groups of similar sex-ratio were dermally treated with approximately 13ml of water or weathered MC252 crude oil, respectively, every 3 days for 6 dosages approximating 20% surface coverage. Collected whole blood samples were analyzed by light (new methylene blue) and transmission electron microscopy. Both oral and dermal treatment with weathered DWH MC252 crude oil induced regenerative, but inadequately compensated, anemia due to hemolysis and hematochezia as indicated by decreased packed cell volume, relative increase in reticulocytes with lack of difference in corrected reticulocyte count, and morphologic evidence of oxidant damage at the ultrastructural level. Hemoglobin precipitation, HB formation, degenerate organelles, and systemic oxidant damage were documented. Heinz bodies were typically <2µm in length and smaller than in mammals. These oblong cytoplasmic inclusions were difficult to see upon routine blood smear evaluation and lacked the classic button appearance found in mammalian red blood cells. They could be found as light, homogeneous blue inclusions upon new methylene blue staining. Ultrastructurally, HB appeared as homogeneous, electron-dense structures within the cytosol and lacked membranous structure. Oxidant damage in avian red blood cells results in degenerate organelles and precipitated hemoglobin or HB with different morphology than that found in mammalian red blood cells. Ultrastructural evaluation is needed to definitively identify HB and damaged organelles to confirm oxidant damage. The best field technique based on the data in this study is assessment of PCV with storage of blood in glutaraldehyde for possible TEM analysis.

Organ weights and histopathology of double-crested cormorants (Phalacrocorax auritus) dosed orally or dermally with artificially weathered Mississippi Canyon 252 crude oil.

A series of toxicity tests were conducted to assess the effects of low to moderate exposure to artificially weathered Deepwater Horizon Mississippi Canyon 252 crude oil on representative avian species as part of the Natural Resource Damage Assessment. The present report summarizes effects of oral exposure (n=26) of double-crested cormorants (DCCO; Phalacrocorax auritus) to 5 or 10ml oil kg-1 day-1 for up to 21 days or dermal application (n=25) of 13ml oil to breast and back feathers every three days totaling 6 applications in 21 days on organ weights and histopathology. Absolute and relative kidney and liver weights were increased in birds exposed to oil. Additionally, gross and/or histopathologic lesions occurred in the kidney, heart, pancreas and thyroid. Clinically significant renal lesions in the orally dosed birds included squamous metaplasia and increased epithelial hypertrophy of the collecting ducts and renal tubules and mineralization in comparison to controls. Gross cardiac lesions including thin walls and flaccid musculature were documented in both orally and dermally dosed birds and myocardial fibrosis was found in low numbers of dermally dosed birds only. Cytoplasmic vacuolation of the exocrine pancreas was noted in orally dosed birds only. Thyroid follicular hyperplasia was increased in dermally dosed birds only possibly due to increased metabolism required to compensate damaged feather integrity and thermoregulate. Gastrointestinal ulceration was found in orally dosed birds only. There were no significant hepatic histopathologic lesions induced by either exposure route. Therefore, hepatic histopathology is likely not a good representation of oil-induced damage. Taken together, the results suggest that oral or dermal exposure of DCCOs to artificially weathered MC252 crude oil induced organ damage that could potentially affect survivability.

Long term changes in aquaculture influence migration, regional abundance, and distribution of an avian species.

Agricultural development has been causing changes to the environment and the abundance and distribution of avian species. Agriculture is dynamic with changes in products occurring at large scales over relatively short time periods. The catfish aquaculture industry is one such agriculture industry that has undergone dramatic changes over the last 25 years. The double-crested cormorant (Nannopterum auritum) is a piscivorous bird that has an extensive history with the aquaculture industry of Mississippi due to its depredation of cultured catfish. A large-scale monitoring program began in 1989 to estimate the abundance and location of cormorants at every known roost in the primary catfish producing region of the state, regionally known as the Delta. We used this data set to address hypotheses pertaining to cormorant ecology within the Delta over time, particularly in relation to aquaculture. We found that, although the Midwest breeding population of cormorants has been increasing, the abundance of cormorants wintering in the Delta has been decreasing, closely following the decline of aquaculture, suggesting aquaculture area is the primary reason for cormorant inhabitation of the region. We also modeled cormorant presence and abundance at all roost sites to determine what factors most influenced cormorant distribution. Aquaculture area around roosts was a significant predictor of both cormorant presence and abundance. However, the influence of aquaculture area was seasonally dependent, with greater positive influences occurring prior to migration. Lastly, we found peak cormorant abundance in the Delta is occurring 2.14 days earlier each year, which may be indicative of changes to migration phenology. Information gained using this large dataset aids in cormorant damage mitigation and to further our understanding of cormorant ecology. Data indicate changes in agriculture, and potentially climate change, can influence phenology, distribution, and abundance of avian species at large geographic scales.

Landscape scale thermoregulatory costs from sublethal exposure to Deep Water Horizon oil in the double-crested cormorant.

Toxic effects of heavy oiling to wildlife are well known from oil spills, although sublethal oil exposure effects are poorly understood. We used Niche Mapper™, to compute spatially and temporally specific energetic and behavioral impacts of repeated sublethal oil exposure to double-crested cormorants (Phalacrocorax auritus). During winter (October-March) cormorants exposed to 13 g, 39 g, and 65-78 g of oil, had on average a 31%, 59%, and 76% predicted increase in total resting energetic requirements (RMR) compared to unoiled birds, respectively. Increased RMR resulted in a mean (±SD) predicted increase in time spent foraging of 36 (±13) min·d-1. During the breeding season (April-September), cormorants had on average a 29%, 57% and 73% increase in total RMR and the mean predicted increase in time spent foraging was 131 (±49) min·d-1. Thermoregulatory effects of sublethal oil exposure may cause greater impacts to bird populations than is currently understood.

Fine scale characteristics of catfish aquaculture ponds influencing piscivorous avian species foraging use in the Mississippi Delta.

Piscivorous avian species are the main source of catfish depredation at aquaculture facilities in Mississippi, resulting in the economic loss of millions of dollars every year. Most notable of these avian species are the double-crested cormorant (Phalacrocorax auritus), great blue heron (Ardea herodias), and great egret (A. alba). Understanding why these species select specific ponds can increase management efficiency directed at avian dispersal and provide insight into their decision making with respect to foraging behavior. We collected species presence data on catfish ponds by flying 35 surveys from October through April of 2015-2017, during which an average of 973 catfish ponds were observed each year. We collected data associated with each pond's physical surroundings and contents and used occupancy modeling to determine their influence on avian occupancy probability. We also collected data associated with stocking practices and catfish health on a subset of ponds, and constructed resource selection functions to model their influence on avian presence. Pond area was positively related to occupancy probability of each species. Cormorant occupancy increased as pond distance from forest cover and activity centers, such as workshops and offices, increased. Distance to nearest activity center was positively related to egret occupancy, while distance to nearest forested area was negative. Ponds containing diseased catfish had an increased probability of use by both herons and egrets. In general, cormorants and egrets showed greater probability of use on the periphery of pond clusters. The abundance of catfish was positively related to cormorant and heron presence. Specific pond contents and characteristics influenced presence of each avian species in different ways, including fish species cultured, production methods, pond systems, and fish types. Many pond selection relationships were species-specific, illustrating inherent differences in their foraging ecology. Consequently, specific management actions aimed to reduce avian presence will depend on the targeted species.

Data of soil, vegetation and bird species found on double-crested cormorant colonies in the southeastern United States.

This data article provides the methods and procedures followed to collect and analyse soil, vegetation and bird data on three different treatment islands in Guntersville Reservoir, Alabama. Samples were collected from randomly selected plot points from islands that were placed into three different treatment types: Colony (currently occupied by Double-crested Cormorants) (Phalacrocorax auritus; n = 5), Historic (historically occupied by cormorants and currently abandoned; n = 3) and Reference (never occupied by cormorants; n = 4). We compared vegetation and tree metrics such as structure and diversity, as well as soil chemistry and bird diversity and communities among islands within Guntersville Reservoir. These data document for the first time that we are aware of the long-term effects of soil chemistry changes, vegetation changes, and impacts to avian diversity, in temperate forest ecosystems, by cormorant colonies. All data is associated with the recent article by Veum et al. [1] and provided here as raw data.

Effects of Repeated Sublethal External Exposure to Deep Water Horizon Oil on the Avian Metabolome.

We assessed adverse effects of external sublethal exposure of Deepwater Horizon, Mississippi Canyon 252 oil on plasma and liver metabolome profiles of the double-crested cormorant (Phalacrocorax auritus), a large (1.5 to 3.0 kg) diving waterbird common in the Gulf of Mexico. Metabolomics analysis of avian plasma showed significant negative effects on avian metabolic profiles, in some cases after only two external exposures (26 g cumulative) to oil. We observed significant (p < 0.05) changes in intermediate metabolites of energy metabolism and fatty acid and amino acid metabolic pathways in cormorants after repeated exposure to oil. Exposure to oil increased several metabolites (glycine, betaine, serine and methionine) that are essential to the one-carbon metabolism pathway. Lipid metabolism was affected, causing an increase in production of ketone bodies, suggesting lipids were used as an alternative energy source for energy production in oil exposed birds. In addition, metabolites associated with hepatic bile acid metabolism were affected by oil exposure which was correlated with changes observed in bile acids in exposed birds. These changes at the most basic level of phenotypic expression caused by sublethal exposure to oil can have effects that would be detrimental to reproduction, migration, and survival in avian species.

Experimental and modeled thermoregulatory costs of repeated sublethal oil exposure in the Double-crested Cormorant, Phalacrocorax auritus.

To fully understand the impact of oil exposure, it is important to understand sublethal effects like how increased thermoregulatory costs may affect survival and reproduction. However, it is difficult and time-consuming to measure these effects in wild animals. We present a novel use of a bioenergetics model, Niche Mapper™, to estimate thermoregulatory impacts of oiling, using data from captive Double-crested Cormorants (Phalacrocorax auritus) experimentally exposed to oil. Oiled cormorants had significant increases in surface body temperatures following exposure. Niche Mapper accurately predicted surface temperatures and metabolic rates for unoiled and oiled cormorants and predicted 13-18% increased daily energetic demands due to increased thermoregulatory costs of oiling, consistent with increased food consumption observed in experimentally oiled cormorants. We show that Niche Mapper can provide valuable insight into sublethal oiling effects by quantifying the extent to which thermoregulatory costs divert energy resources away from important life processes like maintenance, reproduction and migration.

Using pentosidine and hydroxyproline to predict age and sex in an avian species.

All living organisms are subject to senescence accompanied by progressive and irreversible physiological changes. The error damage and cross-linking theories suggest that cells and tissues are damaged by an accumulation of cross-linked proteins, slowing down bodily processes and resulting in aging. A major category of these cross-linked proteins are compounds called advanced glycation end products (AGEs). We investigated the relationship between accumulation of the AGE, pentosidine (Ps), and hydroxyproline (HYP) a post-translationally modified amino acid, with age, sex, and breeding status (breeder/nonbreeder) from skin samples of known age (i.e., banded as fledglings), free-ranging Double-crested Cormorants (Phalacrocorax auritus, Lesson 1831). We developed multivariate models and evaluated the predictive capability of our models for determining age and breeding versus nonbreeding birds. We found significant relationships with Ps and HYP concentration and age, and Ps concentration and sex. Based on our two-class model using Ps and HYP as explanatory variables, we were able to accurately determine whether a cormorant was a breeder or nonbreeder in 83.5% of modeled classifications. Our data indicate that Ps and HYP concentrations can be used to determine breeding status of cormorants and potentially age of cormorants although sex-specific models may be necessary. Although the accumulation of Ps explained the greatest amount of variance in breeding status and age, importantly, Ps covaried with HYP and combined improved prediction of these demographics in cormorants. Our data support the error damage and cross-linking theories of aging. Both Ps and HYP increase predictably in cormorants and are predictive of age and breeding status. Given the ubiquity of these biomarkers across taxa, their use in estimating demographic characteristics of animals could provide a powerful tool in animal ecology, conservation, and management.

Using species distribution models to define nesting habitat of the eastern metapopulation of double-crested cormorants.

When organisms with similar phenotypes have conflicting management and conservation initiatives, approaches are needed to differentiate among subpopulations or discrete groups. For example, the eastern metapopulation of the double-crested cormorant (Phalacrocorax auritus) has a migratory phenotype that is culled because they are viewed as a threat to commercial and natural resources, whereas resident birds are targeted for conservation. Understanding the distinct breeding habitats of resident versus migratory cormorants would aid in identification and management decisions. Here, we use species distribution models (SDM: Maxent) of cormorant nesting habitat to examine the eastern P. auritus metapopulation and the predicted breeding sites of its phenotypes. We then estimate the phenotypic identity of breeding colonies of cormorants where management plans are being developed. We transferred SDMs trained on data from resident bird colonies in Florida and migratory bird colonies in Minnesota to South Carolina in an effort to identify the phenotype of breeding cormorants there based on the local landscape characteristics. Nesting habitat characteristics of cormorant colonies in South Carolina more closely resembled those of the Florida phenotype than those of birds of the Minnesota phenotype. The presence of the resident phenotype in summer suggests that migratory and resident cormorants will co-occur in South Carolina in winter. Thus, there is an opportunity for separate management strategies for the two phenotypes in that state. We found differences in nesting habitat characteristics that could be used to refine management strategies and reduce human conflicts with abundant winter migrants and, at the same time, conserve less common colonies of resident cormorants. The models we use here show potential for advancing the study of geographically overlapping phenotypes with differing conservation and management priorities.

Isotopic Discrimination in the Double-Crested Cormorant (Phalacrocorax auritus).

The diet-tissue discrimination factor is the amount by which a consumer's tissue varies isotopically from its diet, and is therefore a key element in models that use stable isotopes to estimate diet composition. In this study we measured discrimination factors in blood (whole blood, red blood cells and plasma), liver, muscle and feathers of Double-crested Cormorants (Phalacrocorax auritus) for stable isotope ratios of carbon, nitrogen and sulfur. Cormorants exhibited discrimination factors that differed significantly among tissue types (for carbon and nitrogen), and differed substantially (in the context of the isotopic variation among relevant prey species) from those observed in congeneric species. The Double-crested Cormorant has undergone rapid population expansion throughout much of its historic range over the past three decades, leading to both real and perceived conflicts with fisheries throughout North America, and this study provides an essential link for the use of stable isotope analysis in researching foraging ecology, diet, and resource use of this widespread and controversial species.

Geographic segregation and evidence of density-dependent changes in sex ratios in an abundant colonial waterbird.

Demographic information, such as geographic segregation of sexes and sex ratio data, is needed to develop, model and evaluate conservation and management strategies for wildlife. A variety of physiological, behavioral and environmental factors can influence segregation of sexes and sex ratios, many of which originate with density-dependent processes. Departure from 50:50 sex ratios of double-crested cormorants (Phalacrocorax auritus) collected during control efforts in breeding and wintering areas across their eastern range of the USA were evaluated using using a Z-test as well as Stouffer's weighted Z-tests. In addition, a specifically-designed randomization test was used to evaluate density-dependent effects on primary sex ratios in cormorants from egg collections and colony nest count data over a 21-year period. Cormorants collected from breeding colonies were strongly male-biased, whereas cormorants collected from feeding flocks were slightly biased toward females. Cormorants were partly segregated by sex on the wintering grounds, with significantly more males found in areas with intensive channel catfish aquaculture. The null hypothesis that females produced a balanced sex ratio independent of number of nesting cormorants was rejected: more male embryos were produced during rapid population growth, whereas at maximum nesting number more female embryos were produced. Once populations stabilized, the sex ratio was more equal. This examination of sex ratios indicates that different management methods and locations result in sex-biased culling of cormorants. Sex-biased culling in cormorants could make population reduction efforts more efficient and reduce overall take. We suggest further research to examine density-dependent effects on primary sex ratios documented here.