Research Article |
Corresponding author: Paulo Vitor Alves Ribeiro ( paulovitorbio@gmail.com ) Academic editor: Luis Fabio Silveira
© 2020 Paulo Vitor Alves Ribeiro, Camilla Queiroz Baesse, Márcia Cristina Cury, Celine de Melo.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Ribeiro PVA, Baesse CQ, Cury MC, de Melo C (2020) Leukocyte profile of the helmeted manakin, Antilophia galeata (Passeriformes: Pipridae) in a Cerrado forest fragment. Zoologia 37: 1-9. https://doi.org/10.3897/zoologia.37.e46441
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Changes in the amounts and proportions of leukocytes, known as leucocyte profiles, have been documented for several bird species and have been used to measure stress levels in these animals. The present work ascertained the biological and ecological attributes that influence the leukocyte profile of Antilophia galeata (Lichtenstein, 1823), the helmeted manakin. This species has been deemed useful in ecological studies because it responds to environmental changes. Blood samples drawn from 89 individuals of A. galeata captured in a Cerrado forest fragment were subjected to analysis under optical microscopy to identify and quantify leukocytes and micronuclei. The number of lymphocytes was greater for males, non-reproductive individuals and individuals infected with ticks. None of the leukocyte components differed in relation to age, molting or body condition index. The amount of micronuclei was correlated with values for total leukocytes, H/L ratio, heterophils, basophils and monocytes. The results suggest that reproduction may be an immunosuppressive factor for the species, producing sexual differences in lymphocyte availability. In addition, biomarkers of genotoxic damage (micronuclei) were related to the amount of leukocytes, indicating that individuals may be sensitive to environmental disturbances. Leukocyte profiles can be considered a useful tool for addressing ecological questions that are relevant to the conservation of species in degraded environments.
Avian hematology, Brazilian birds, micronuclei, stress, white blood cells
Various biological and physiological parameters can be used to assess stress in birds, including hematological exams such as the evaluation of leukocyte profiles (
Avian leukocytes can be classified into granulocytes and agranulocytes or mononuclear cells. The former include basophils, eosinophils and heterophils, while the latter include lymphocytes and monocytes (
Heterophils and lymphocytes together make up approximately 80% of the leukocytes of birds and their amounts and proportions can change according to variation in corticosteroid concentrations (
Evidence has shown that the leukocyte profiles of birds differ between males and females, since sex hormones act as important modulators of the immune response: testosterone is immunosuppressive and estrogen is immune-enhancing (
Birds undergo other stressors that may influence their leukocyte profile, such as parasitism, food shortage and molting, among others.
Another alteration evidenced in hematological examinations is the presence of erythrocyte micronuclei, which has been associated with anthropic influences (
Few studies have documented the leukocyte components in Brazilian bird species, and the influence of stressful situations on them (
The helmeted manakin has been considered useful in ecology studies because it is territorial, dependent on the forest environment and is sensitive to the effects of disturbances, being a species that can show responses to environment changes (
The study was carried out in a Cerrado forest fragment at the Fazenda Experimental do Glória (18°57’03”S; 48°12’22”W), in the municipality of Uberlândia, Minas Gerais, Brazil. The fragment encompasses 30 ha composed of seasonal semideciduous forest with gallery forest at is lower limit and abrupt transitions at artificial borders with pastures at the upper and lateral limits (
Seven field campaigns of five days each were undertaken from June 2013 to December 2015. Birds were captured using 20 to 25 mist nets (12 m long x 3 m high) exposed on trails between 6:30 am and 5:00 pm. The nets were checked at intervals of approximately 30 minutes. When birds were found they were removed and placed in clothe bags for subsequent screening. Individuals were weighed in the bags using a hand dynamometer (Pesola®), with the weight of the bag then being subtracted. The tarsal length of each bird was measured using a digital caliper (Lotus®). Individuals were identified and banded with metal bands provided by Center for Research and Conservation of Wild Birds (CEMAVE/ICMBio – Authorization: 3730, Registry: 359076).
The reproductive stage of the captured birds was determined by the presence of an incubation patch; when the abdominal region lacks feathers and has thin, wrinkled skin with a darker coloration due to increased vascularization (Cemave 1994). The feathers covering the abdominal region were blown to allow visualization of the incubation patch and determine its presence. In addition, feathers from other regions of the body (head, neck, back) were blown to detect the presence of ticks and molting (appearance of new feathers).
When possible, the age group (young or adult) of each individual was determined, since young birds usually have a yellowish labial commissure and the absence of, or minimal, cranial ossification. To determine sex, blood samples were colleted from all individuals of green color (05 µL of blood from the tarsal vein) with the aid of sterile disposable needles (8 × 0.3 mm) (SISBIO/ICMBio – Authorization: 44901). The collected blood samples were stored in specific kits provided by the sexing laboratory. The samples were sent to a private laboratory for molecular sexing (©Unigen Tecnologia do DNA – from São Paulo, SP, Brazil).
A drop of blood (05 µL) was collected from the tarsal vein and placed on a clean, sterile microscope slide (SISBIO/ICMBio – Authorization: 44901). With the aid of a second slide inclined at 45°, the blood was drawn across the first slide. After drying, the slides were fixed with absolute methanol while still in the field. In the laboratory the slides were stained with a solution of Giemsa (5%) and phosphate buffer (pH 5.8) for 30 minutes. The slides were then washed in distilled water, dried at room temperature and identified with the individual’s band number.
Slides were analyzed under an optical microscope with a 100x objective using immersion oil, with 200 fields per individual (100 per slide). The components of the leukocyte profile were identified according to the descriptions of
Micronuclei analysis involved counting the number of micronuclei in each of the 5,000 erythrocytes on each slide for a total of 10,000 erythrocytes per individual (
The capacity of individuals to store resources and survive adverse situations can be evaluated by analyzing the body condition (Schulte-Hostedde et al. 2005). Body condition can be estimated by the Relative Mass Index (RMI), which was calculated by simple linear regression between the log-transformed values of right tarsus length and individual biomass. The residual values of the regression were used as RMI, with negative values representing poorer body conditions than positive values (Schulte-Hostedde et al. 2005).
Parametric tests (Student’s t test and Pearson correlation) were performed to determine whether the components of the leukocyte profile differed among the analyzed variables (sex, age, reproductive period, molting, ectoparasites, body condition and micronuclei). To meet the assumptions of the tests, percentage data (heterophils, lymphocytes, eosinophils, basophils, monocytes and H/L ratio) were transformed (arcsine of the square root), while the continuous variables (TLC and micronuclei) were log-transformed. The analyses were conducted using Systat 10.2 software with a level of significance of p < 0.05.
The following numbers of individuals were captured at each year of the research: 2013, 15 individuals; 2014, 14 individuals (eight in February and six in July); 2015, 60 individuals (25 in June, 18 in August, 12 in October and five in December). In total, 89 individuals were captured, of which 40 (44.9%) were females, 43 (48.3%) were males. It was not possible to determine the sex of six (6.74%) individuals. The sample included 58 (65.1%) adults, 14 (15.7%) juveniles and 17 (19.1%) individuals for which the development phase could not be determined. The birds for which sex and development could not be estimated were disregarded from analyses involving sexes and age groups. A total of 26 (29.2%) individuals possessed an incubation patch while 63 (70.7%) did not. Indications of molting were present in 23 (25.8%) individuals and absent in 66 (74.1%). Ticks were found infesting 17 (19.1%) individuals, while 72 (80.8%) were tick free. Micronuclei were present in 55 (61.7%) individuals and absent in 34 (38.2%).
The number of lymphocytes was greater for males (t = -2.001, df = 81, 0.049, Table
Mean ± standard deviation for the components of the leukocyte profile of Antilophia galeata in relation to sex, reproductive period, and ticks. Statistically significant differences are shown in bold (t = Student’s t test, df = degrees of freedom, p = significance probability < 0.05).
Hematologic parameters | Sex | Incubation patch | Ticks | ||||||||||||||
Females (n = 40) |
Males (n = 43) |
Statistics | Present (n = 25) |
Absent (n = 61) |
Statistics | Present (n=17) |
Absent (n=72) |
Statistics | |||||||||
t | df | p | t | df | p | t | df | p | |||||||||
TLC | 7.80 ± 9.88 | 8.74 ± 12.2 | 0.387 | 62 | 0.700 | 12.9 ± 11.9 | 6.06 ± 8.20 | -0.229 | 66 | 0.766 | 6.23 ± 8.07 | 8.25 ± 10.1 | 0.122 | 66 | 0.904 | ||
H/L ratio | 0.49 ± 1.50 | 0.71 ± 1.68 | -1.929 | 81 | 0.057 | 1.55 ± 2.36 | 1.14 ± 4.66 | -0.227 | 86 | 0.821 | 2.66 ± 8.44 | 0.88 ± 1.92 | -1.012 | 86 | 0.314 | ||
Heterophils (%) | 32.3 ± 37.6 | 24.7 ± 31.5 | 0.750 | 81 | 0.456 | 43.5 ± 28.8 | 26.9 ± 35.6 | -1.482 | 86 | 0.142 | 22.8 ± 33.5 | 32.5 ± 34.4 | 1.323 | 86 | 0.188 | ||
Lymphocytes (%) | 25.8 ± 36.0 | 37.8 ± 39.4 | -2.001 | 81 | 0.049 | 32.3 ± 31.8 | 34.1± 39.9 | 2.565 | 86 | 0.012 | 27.6 ± 38.3 | 35.0 ± 37.8 | -3.064 | 86 | 0.003 | ||
Eosinophils (%) | 0.49 ± 1.50 | 0.77 ± 2.20 | 0.979 | 81 | 0.094 | 5.75 ± 10.8 | 0.80 ± 2.47 | -0.992 | 86 | 0.324 | 0.49 ± 2.02 | 2.25 ± 7.03 | * | * | * | ||
Basophils (%) | 0.90 ± 3.56 | 0.99 ± 2.60 | -0.216 | 81 | 0.829 | 1.74 ± 5.65 | 0.72 ± 2.25 | -0.884 | 86 | 0.379 | 1.07 ± 3.05 | 0.96 ± 3.13 | * | * | * | ||
Monocytes (%) | 4.56 ± 13.8 | 2.67 ± 5.94 | 0.643 | 81 | 0.522 | 6.07 ± 16.6 | 2.08 ± 5.34 | -0.057 | 86 | 0.178 | 2.04 ± 4.15 | 3.35 ± 10.7 | * | * | * |
Mean ± standard deviation for the components of the leukocyte profile of Antilophia galeata in relation to age groups and molting (t = Student’s t test, df = degrees of freedom, p = significance probability < 0.05, * = insufficient data for the test).
Hematologic parameters | Age group | Molting | |||||||||
Adult (n = 58) | Young (n = 14) | Statistics | Present (n = 23) | Absent (n = 66) | Statistics | ||||||
t | df | p | t | df | p | ||||||
TLC | 14.4 ± 23.78 | 5.33 ± 6.17 | 0.543 | 55 | 0.589 | 7.60 ± 8.14 | 7.29 ± 10.3 | -1.450 | 66 | 0.155 | |
H/L ratio | 1.54 ± 4.93 | 0.85 ± 1.52 | 0.452 | 70 | 0.652 | 0.49 ± 1.16 | 1.47 ± 4.64 | 0.427 | 86 | 0.671 | |
Heterophils (%) | 37.2 ± 65.1 | 29.5 ± 34.5 | 1.655 | 70 | 0.102 | 28.2 ± 30.8 | 31.5 ± 35.6 | 0.965 | 86 | 0.337 | |
Lymphocytes (%) | 30.8 ± 36.0 | 39.2 ± 40.5 | -1.236 | 70 | 0.221 | 34.7 ± 38.1 | 33.2 ± 37.9 | -1.335 | 86 | 0.185 | |
Eosinophils (%) | 2.66 ± 7.61 | 0.42 ± 1.52 | 1.099 | 70 | 0.276 | 2.35 ± 5.26 | 2.10 ± 6.82 | -0.050 | 86 | 0.960 | |
Basophils (%) | 1.11 ± 3.45 | – | * | * | * | 2.10 ± 4.79 | 0.59 ± 2.16 | -1.286 | 86 | 0.202 | |
Monocytes (%) | 4.05 ± 11.7 | 0.8 ± 2.91 | * | * | * | 7.92 ± 17.9 | 1.49 ± 3.75 | -0.507 | 86 | 0.614 |
Pearson correlations (r) between components of the leukocyte profile of Antilophia galeta and the Relative Mass Index (RMI) and the amount of micronuclei. Statistically significant correlations are shown in bold (df = degrees of freedom; p = probability of significance < 0.05).
Hematologic parameters | Correlation with RMI | Correlation with micronuclei | |||||
r | df | p | r | df | p | ||
TLC | 0.324 | 66 | 0.569 | 0.343 | 43 | 0.024 | |
H/L ratio | 0.047 | 86 | 0.665 | -0.273 | 53 | 0.044 | |
Heterophils (%) | -0.062 | 86 | 0.568 | -0.335 | 53 | 0.012 | |
Lymphocytes (%) | 0.119 | 86 | 0.270 | 0.164 | 53 | 0.232 | |
Eosinophils (%) | 0.069 | 86 | 0.522 | 0.248 | 53 | 0.068 | |
Basophils (%) | -0.078 | 86 | 0.471 | 0.280 | 53 | 0.039 | |
Monocytes (%) | -0.074 | 86 | 0.495 | 0.277 | 53 | 0.041 |
Although the leukocyte stress profile (H/L ratio) was not correlated to any of the analyzed variables, except for micronuclei, other components of the leukocyte profile varied according to the biological and ecological attributes of the individuals. The amount of lymphocytes was significantly greater in males, contrasting with what was expected: males were expected to have decreased amounts of leucocytes, consistent with studies that have shown that testosterone is an immunosuppressive hormone (
Differences in the number of lymphocytes were also associated with the presence of ectoparasites, with individuals with ticks having higher values. The number of lymphocytes was expected to be lower in infested individuals since, according to the reviews of
Molting is considered an energy demanding process, since it producing new feathers requires energy (
Studies have shown that nutritional status or body condition may influence the leukocyte profile of birds, and that there is a negative relationships between the H/L ratio and body condition indexes; in other words, the higher the stress, the worse the body condition, and vice versa (
Significant positive correlations were found between the quantity of micronuclei and TLC, percentage of basophils and percentage of monocytes. The formation of micronuclei occurs through spontaneous chromosomal changes or environmental disturbances, with findings of increased micronuclei levels due to anthropization (
Although TLC, basophil and monocyte percentages correlated positively with micronuclei in the present study, heterophil percentage and H/L ratio were negatively correlated with micronuclei quantity. Heterophils are the primary cells that proliferate during acute inflammatory responses (
The present study was the first to examine the leukocyte profile of A. galeata, and to determine the aspects of the species’ biology and ecology that influence the leukocytes produced by these birds. Certain components of the leukocyte profile were shown to vary in relation to certain characteristics and life periods of A. galeata. The results suggest that reproduction may be an immunosuppressive factor for the species, thereby producing sexual differences in lymphocyte availability for individuals. Furthermore, biomarkers of genotoxic damage (miocronuclei) were correlated with the H/L ratio, indicating that individuals may be sensitive to environmental disturbances. In this way, it can be concluded that leukocyte profiles are a useful tool for answering ecological questions that can be applied to the conservation of species in degraded environments.
We declare that all proceedings performed with the birds have followed and respected the international rules of animals well-being by means of the parameters required from Animal Ethics Committee. We are grateful to the members of the Laboratório de Ornitologia e Bioacústica (Universidade Federal de Uberlândia – UFU) for their field assistance and to the Programa de Pós-graduação em Ecologia e Conservação de Recursos Naturais (UFU) for their continued support. This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Finance Code 001), the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (APQ-01654-12) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (PELD 403733/2012-0).