Bioscience Journal | 2023 | vol. 39, e39071 | ISSN 1981-3163 1 Paulo Vitor Alves RIBEIRO1 , Luís Paulo PIRES1 , Márcia Cristina CURY2 , Celine de MELO1 1 Laboratory of Ornithology and Bioacoustic, Institute of Biology, Universidade Federal de Uberlândia, Uberlândia, Minas Gerais , Brazil. 2 Laboratory of Serology and Molecular Biology of Parasites, Institute of Biomedical Sciences, Universidade Federal de Uberlândia, Uberlândia, Minas Gerais, Brazil. Corresponding author: Paulo Vitor Alves Ribeiro paulovitorbio@gmail.com How to cite: RIBEIRO, P.V.A., et al. Haemosporidian parasites in Antilophia galeata (Aves: Pipridae) in a Cerrado forest fragment. Bioscience Journal. 2023, 39, e39071. https://doi.org/10.14393/BJ-v39n0a2023-53589 Abstract Haemosporidian parasites can cause pathogenic infections, leading to death or a reduction in the physical and reproductive abilities of the host. Several studies have identified haemosporidian infections in neotropical bird communities, but few have been conducted in populations, relating the infection to the biological attributes of the species. To determine haemosporidian prevalence in a population of Antilophia galeata and to assess factors that may be associated with parasitaemia, we analysed blood smears of 62 individuals from a Cerrado forest fragment. For each individual, the body mass, length of tarsus, sex, presence/absence of brood patch and feather moult were recorded. In total, 33 (53.2%) individuals were infected with haemosporidian parasites, 32 (51.6%) were infected with Plasmodium spp. and one (1.61%) was infected with Haemoproteus sp. Parasitaemia was not related to seasons, sex, reproduction, moulting or body condition but correlated positively with total leucocyte count, suggesting that individuals may b e effective in infection control. This population may be tolerant to haemosporidian parasites because, despite the high prevalence, parasitaemia was low and constant; this is a potentially chronic infection that showed no adverse effects on the parameters analysed in this population. Keywords: Avian Malaria. Leukocytes. Parasite-host Relationship. Wild Birds. 1. Introduction Birds can be affected by a diverse parasitic fauna, including protozoa, helminths and arthropods (Atkinson et al. 2009). A group of well-studied bird parasites are the haemosporidian protozoa that cause malaria (genus Haemoproteus and Plasmodium); they have a wide geographical distribution, occurring in birds throughout the world (Clark et al. 2014). These parasites are responsible for acute and/or chronic infections and may compromise host reproductive success and survival survival (Knowles et al. 2010; Vanstreels et al. 2014; Dinhopl et al. 2015). This occurs mainly when the parasites are introduced in non - adapted populations, which can generate high pathogenicity, mortality and even extinction (Atkinson and Van Ripper III 1991; Atkinson and La Point 2009). Haemosporidian parasites are transmitted by hematophagous insects (Diptera: Culicidae, Ceratopogonidae, Hippoboscidae and Simuliidae), which play a fundamental role in maintaining infection in bird populations (Valkiunas 2005). Factors related to vector biology (such as greater activity in hot months) have been attributed as one of the causes of seasonal variation in haemosporidian prevalence in temperate climate environments (Bensch et al. 2007; Cosgrove et al. 2008). However, few studies have HAEMOSPORIDIAN PARASITES IN Antilophia galeata (AVES: PIPRIDAE) IN A CERRADO FOREST FRAGMENT https://orcid.org/0000-0002-8232-6447 https://orcid.org/0000-0003-1255-8880 https://orcid.org/0000-0003-2939-9046 https://orcid.org/0000-0002-6603-8314 Bioscience Journal | 2023 | vol. 39, e39071 | https://doi.org/10.14393/BJ-v39n0a2023-53589 2 Haemosporidian parasites in Antilophia galeata (Aves: Pipridae) in a Cerrado forest fragment found seasonality in haemosporidian prevalence among birds in the Neotropics (Ferreira-Junior et al. 2017; Hernández-Lara et al. 2017). A host’s biological traits can also influence haemosporidian prevalence and parasitaemia (Zuk and Stoehr 2010; Calero-Riestra and García 2016). For example, sex- biased parasitism may be due to physiological or ecological factors (Zuk and Mckean 1996). Physiological factors are related to the effects of sex hormones, especially testosterone, which is considered immunosuppressive, leading to higher susceptibility to infection among adult males (Foo et al. 2016; Roved et al. 2016). Ecological factors include differential exposure to vectors due to sex-specific behaviours, such as incubation (Zuk and Mckean 1996; Fecchio et al. 2015). Reproduction is energetically costly for birds due to nest building and parental care (Saino et al. 2002; Mainwaring and Hartley 2013), egg production for females (Williams 2005), territorial defence and courtship for males (Edler 2011). These high energetic demands can increase stress in both sexes, compromise health and result in immunosuppression (Norris and Evans 2000; Frigerio et al. 2017; Ribeiro et al. 2020a). Moulting has also been linked to parasitic infections. Studies have shown that infected in dividuals delayed moulting or had lower daily feather growth than healthy individuals (Langston and Hillgarth 1995; Tarello 2007; Marzal et al. 2013a). In addition, parasitic infections can negatively affect body condition (Marzal et al. 2013b; Gethings et al. 2016), which estimates the ability of individuals to store energy resources and to survive in adverse situations (Schulte-Hostedde et al. 2005). Parasitic infections have also been associated with other indicators of bird health status, such as leukocyte counts and heterophil/lymphocyte (H/L) ratios (Norte et al. 2009; Lüdtke et al. 2013). The total leukocyte count is a good indicator of immune system status; high leukocyte counts can indicate inflammation or infection, while low counts can indicate immunosuppression (Campbell 2015). The H/L ratio is considered an efficient indicator of stress since high levels of stress hormones (glucocorticoids) trigger a greater release of heterophils in relation to lymphocytes, and this increases the H/L ratio values, which are usually associated with chronic stressors (Davis et al. 2008; Davis and Maney 2018; Ribeiro et al. 2022). Several studies have addressed haemosporidian infections in Cerrado birds (Fecchio et al. 2007, 2011, 2013; Belo et al. 2011; Leite et al. 2013; Lacorte et al. 2013; Ribeiro et al. 2020b). However, few have investigated the role of host attributes and seasonality on the prevalence of blood-parasites within populations of a single species (Lobato et al. 2011; Fecchio et al. 2015). The present study examined a population of Antilophia galeata, a passerine bird endemic to the Cerrado that inhabits the understory of riparian forests (Marini 1992; Sick 2001). Antilophia galeata is predominantly territorial and frugivorous, with sexual dimorphism in adults (Silva and Melo 2011). Adult males possess black plumage with red feathers on the top of the head, while adult females and juveniles of both sexes have a discrete greenish plumage throughout the body (Marini 1992; Sick 2001). The objectives of the study were to investigate the haemosporidian prevalence in the A. galeata population and to evaluate factors possibly associated with parasitaemia. Specifically, we evaluated three hypotheses: i) parasitaemia does not vary between seasons, considering that vectors can be active throughout the year in tropical regions; ii) parasitaemia is higher among males and in reproducing, moulting and reduced body condition individuals, because these traits are related to immunosuppression; iii) parasitaemia increases the count of leukocytes and the H/L ratio, as parasitic infections can influence leukocyte profiles. 2. Material and Methods Study site This study was carried out in a Cerrado remnant (18º57'03’’S and 48º12’22’’W) at the Fazenda Experimental do Glória (Federal University of Uberlândia) in Uberlândia, Minas Gerais, Brazil. The phytophysiognomies that constitute the study site are seasonal semideciduous forest and gallery forest. The climate is Aw type according to the Köppen climate classification, with a dry season (April to September) and a rainy season (October to March). The annual rainfall is 1,500 mm, and the average temperature is 22°C (Rosa et al. 1991). Bioscience Journal | 2023 | vol. 39, e39071 | https://doi.org/10.14393/BJ-v39n0a2023-53589 3 RIBEIRO, P.V.A., et al. Capture of individuals Seven field campaigns were carried out in 2016 and 2017, each lasting five days. Four of these campaigns took place in the dry season and three in the rainy season. To capture the birds, 20 mist nets (12 m long/3 m high) were used and exposed on trails between 6:00-17:00. Mist nets were checked every 30 minutes. The captured birds were removed and placed in cotton bags for weighing (Pesola®) and for subsequent screenings, such as tarsal measurements (using a digital calliper - Lotus®), to determine the reproductive stage (presence/absence of brood patch) and presence of moulting. The individuals were identified, marked with metal rings provided by CEMAVE/ICMBio (Projects: 3238/3740 - Registration: 359076) and released. Sexing of individuals All green individuals had blood samples (5 µL) collected from the tarsal vein with the aid of sterile disposable needles (8 mm x 0.3 mm) (SISBIO/ICMBio - Authorization: 44901). The samples were stored in specific kits provided by a private molecular sexing laboratory (Unigen Tecnologia do DNA - São Paulo, SP, Brazil) and sent to the laboratory for the exams. Preparation and analysis of blood smears Two blood smears were made for each individual. For each smear, 5 µL of blood was collected from the tarsal vein, placed on a microscope slide and distributed with a second slide tilted at 45°. The slides were fixed with absolute methanol and stained with a solution of Giemsa (Braga et al. 2010). The slides were analysed under an optical microscope (Nikon Eclipse E200) with a 100x magnification while using immersion oil. Haemosporidian parasites were identified according to genera (Haemoproteus/Plasmodium) as described by Valkiunas (2005) and quantified according to the number observed in 200 microscopic fields per individual (Godfrey et al. 1987). Leukocytes were identified, classified and quantified according to the descriptions by Campbell (2015). The H/L ratio was calculated from the division between the numbers of heterophils per lymphocyte (Ribeiro et al. 2022). Body condition Body condition was estimated for each individual of Antilophia galeata by the Scaled Mass Index (SMI) proposed by Peig and Green (2009). This index standardises body mass to a specific fixed linear measurement of the organism using the following equation: SMI = Mi(L0/Li)bSMA Where Mi and Li are the body mass and the linear body measurement of individual i, respectively; bSMA is the scaling exponent estimated by the SMA regression of M on L; L0 is an arbitrary value of L (e.g. the arithmetic mean value for the study population); and SMI is the predicted body mass for individual i when the linear body measure is standardised to L0 (Peig and Green 2009). We used the right tarsus length as Li, and the SMI was calculated in the R software (R Core Team 2022). Statistical analyses We used general linear models (GLM) to analyse the effect of seasons, sex, reproduction, moulting, body condition and leukocyte counts on the parasitaemia, using the function GLM in the lme4 package (Bates et al. 2011). We generated models with all possible combinations of predictor variables, including a null model (a model containing only the intercept) using the dredge function in the package MuMIn (Barton and Barton 2015). We only considered the models with ΔAICc ≤ 2 as top-ranked candidate models. When multiple models were equally top-ranked candidates, we averaged them to produce the conditional Bioscience Journal | 2023 | vol. 39, e39071 | https://doi.org/10.14393/BJ-v39n0a2023-53589 4 Haemosporidian parasites in Antilophia galeata (Aves: Pipridae) in a Cerrado forest fragment estimates and the relative importance (i.e. the sum of the model weights) of each parameter (Grueber et al. 2011). We considered a predictor to be significant in the averaged model when relative importance > 0.8 (Barton and Barton 2015). The analyses were performed in the R software (R Core Team 2022). 3. Results In total, 62 individuals were captured, 52 (83.3%) in the dry season and 10 (16.1%) in the rainy season; 23 (37%) females and 39 (63%) males. Brood patches were present in 12 (19.3%) individuals and absent in 50 (80.6%). Moulting was present in 17 (27.4%) individuals and absent in 45 (72.5%). There were 33 (53.2%) individuals infected by haemosporidian parasites; 32 (51.6%) were infected by Plasmodium spp.; and one (1.61%) was infected by Haemoproteus sp. The number of infected individuals, prevalence and parasitaemia according to the seasons, sexes, reproduction (presence/absence of brood patch) and moulting are described in Table 1. We found that the number of leukocytes appeared in all the top-ranked models explaining parasitaemia in birds (Table 2), and it was also the only significant predictor in the averaged model (Table 3, Figure 1). Table 1. Haemosporidian prevalence (%) and parasitaemia (mean ± standard deviation) in Antilophia galeata in relation to seasons, sex of individuals, presence/absence of brood patch and moulting. Factors n Infected individuals (%) Parasitaemia (X ± SD) Season Dry 52 26 (50%) 3.48 ± 5.42 Rainy 10 06 (60%) 2.50 ± 2.32 Sex Female 23 11 (47.8%) 2.39 ± 2.80 Male 39 21 (53.8%) 3.87 ± 5.96 Brood Patch Present 12 07 (58.3%) 3.50 ± 4.03 Absent 50 26 (52.0%) 3.28 ± 5.30 Moulting Present 17 10 (58.8%) 2.76 ± 2.77 Absent 45 23 (51.1%) 3.53 ± 5.69 Table 2. The top-ranked models used to explain parasitaemia in Antilophia galeata. Model df logLik AICc delta weight Intercept + Leukocytes 3 -177.53 361.48 0.00 0.35 Intercept + Leukocytes + Season 4 -176.48 361.66 0.18 0.32 Intercept + Leukocytes + Sex 4 -176.95 362.61 1.13 0.20 Intercept + Leukocytes + Moulting 4 -177.31 363.32 1.84 0.14 Table 3. Conditional estimates of the parameters in the averaged model predicting parasitaemia in Antilophia galeata. Predictor Estimate Std. Error Importance 95% CI Number of leukocytes 5.45 1.11 1.00 3.23 - 7.68 Season 0.68 1.30 0.32 -0.86 - 5.12 Sex 0.24 0.69 0.20 -1.07 - 3.47 Moulting -0.11 0.54 0.14 -3.27 - 1.66 4. Discussion This study was the first to address haemosporidian infections in Antilophia galeata at the population level, seeking to understand how parasitism occurs in this species. Sebaio et al. (2012) were the first to report Antilophia galeata as a Plasmodium host when examining a single individual of the species within a bird community from the Atlantic Forest. Leite et al. (2013) and Fecchio et al. (2017) examined 9 and 15 individuals of this species, respectively, but neither found haemosporidian parasites. Ribeiro et al. (2020b) found a haemosporidian prevalence of 18% in Antilophia galeata among a bird community from the Cerrado. Most studies that report seasonality in haemosporidian infections were carried out in temperate environments where climatic seasonality is more pronounced, limiting vectors and parasite transmission to the warmer months (Atkinson et al. 1988; Bensch et al. 2007; Cosgrove et al. 2008). In tropical regions, this Bioscience Journal | 2023 | vol. 39, e39071 | https://doi.org/10.14393/BJ-v39n0a2023-53589 5 RIBEIRO, P.V.A., et al. seasonality is uncertain; vectors are abundant and active during most of the year (Atkinson and Van Riper 1991). In addition, previous studies carried out in tropical environments have not found seasonality in haemosporidian infections (Waldenström et al. 2002; Fallon et al. 2004; Chagas et al. 2017), which is in agreement with the results obtained by Fecchio et al. (2015) in a population of white-banded tanagers (Neothraupis fasciata) in the Cerrado and our results. Furthermore, the reduced sample size of birds captured in the rainy season (n=10), compared with the dry season (n=50), may have biased the results. The low capture in the rainy season occurs because individuals of Antilophia galeata increase their stratum breadth and their foraging height during the rainy season, when fruits are available at higher strata (i.e. in the midstory and canopy) (Pires et al. 2022), whereas our mist nets were exposed in the understory. Figure 1. Positive relationship between the number of leukocytes and haemosporidian parasitaemia in individuals of Antilophia galeata from a Cerrado fragment in Southeastern Brazil. No effect of sex on parasitaemia was found, although males were expected to have higher values. Males of Antilophia galeata are highly territorial and display agonistic behaviour towards trespasser males (Marçal and Lopes 2019; Pires and Melo 2020). Such behaviour is related to the increase of testosterone levels (Edler et al. 2011). Once elevated, testosterone can suppress immune function, increasing the susceptibility of infections (Deviche and Parris 2006; Cornelius et al. 2014). Therefore, several studies have reported male-biased parasitism in birds, especially with haemosporidian parasites (Van Oers et al. 2010; Calero-Riestra and Garcia 2016; Rodriguez et al. 2021). However, there are also examples of females with higher haemosporidian parasitaemia (Bichet et al. 2014). In some bird species, females are the main responsible for incubation and spend a long time immobile in their nests, which makes them more exposed to vectors (Norris et al. 1994; Bichet et al. 2014; Ribeiro et al. 2020c). Considering that Antilophia galeata incubation is performed only by females (Marçal and Lopes 2019), and that they can spend about 83.5% of their time in their nests (Bruno et al. 2021), we can assume that the potential immunosuppressive effect of testosterone in males and the greater exposure of females to vectors are likely equivalent factors in the susceptibility to haemosporidian parasites, which could explain the similarity in parasitaemia between sexes. Infections were expected to be greater in individuals with brood patch because, during the reproductive period, birds may be immunosuppressed (Hanssen et al. 2003, 2005; Ribeiro et al. 2020a); however, there were no effects of the reproductive period on parasitaemia. This result suggests that non - Bioscience Journal | 2023 | vol. 39, e39071 | https://doi.org/10.14393/BJ-v39n0a2023-53589 6 Haemosporidian parasites in Antilophia galeata (Aves: Pipridae) in a Cerrado forest fragment breeding birds are also exposed to immunosuppressive conditions. A study with Neothraupis fasciata also found no effects of the reproductive period on infections (Fecchio et al. 2015). This species concentrates their reproduction in the rainy season, like Antilophia galeata (Marçal and Lopes 2019). Fecchio et al. (2015) suggested that the low availability of resources during the dry season in the Cerrado can be a stressful condition that could reduce the immune function. However, this result could also indicate that individuals are equally exposed to parasites and vectors during the breeding and non-breeding seasons, since we did not find seasonality in the infections. The moulting process is highly expensive, as birds need sufficient energy reserves to produce new feathers (Hemborg and Lundberg 1998). Haemosporidian parasites require essential amino acids from the host's plasma to nourish them, while the new feather formation also requires a considerable number of amino acids (Martin and Kirk 2007; Murphy et al. 1996). However, we did not find any effects of moulting on parasitaemia. This may be an indication that parasitaemia was low and did not interfere with the moulting process of Antilophia galeata. Previous studies have found negative relationships between infection and body condition, suggesting that the host's health is compromised (Marzal et al. 2013b; Gethings et al. 2016). However, there are reports of species in which infections did not affect the host’s body condition (Molnár et al. 2013; Maia et al. 2014; Megía-Palma et al. 2016). Certain species may be more sensitive, while others may have a co-evolutionary relationship with the parasites, thus becoming tolerant (Palinauskas et al. 2008). In this study, no significant relationship was found between the infection and SMI, suggesting that body condition was not affected by parasitaemia. In addition, Antilophia galeata is considered a plastic species, capable of adapting well to adverse situations, because its body condition remains stable over different areas, ye ars and seasons, even through variations in the availability of resources (Paniago 2016). This study found that the leukocyte count was the best predictor of parasitaemia in Antilophia galeata, i.e. the greater parasitaemia, the greater the number of available leukocytes. Individuals may be immunologically active and able to control infections, which may explain why parasitaemia did not differ between the other parameters analysed. In addition, no relationship was found between the infection and H/L ratio, which indicates that parasitaemia is not associated with stress. It can be assumed that the parasitaemia in Antilophia galeata remains constant and controlled, characterising it as a chronic infection since it is distributed evenly throughout the population (Norte et al. 2009). 5. Conclusion Haemosporidian parasites (Haemoproteus and Plasmodium) occur in Antilophia galeata in the Brazilian Cerrado. The prevalence was relatively high, but parasitemia remained low, constant and only correlated with the total leukocyte count, which can be an indication of efficiency in controlling the infection. These results show that Antilophia galeata can adapt to adverse situations, such as parasitism. Authors' Contributions: RIBEIRO, P.V.A.: conception and design, acquisition of data, analysis and interpretation of data, and drafting the article; PIRES, L.P.: acquisition of data and analysis and interpretation of data; CURY, M.C.: conception and design and crit ical review of important intellectual content; DE MELO, C.: conception and design and critical review of important intellectual content. All authors have read and approved the final version of the manuscript. Conflicts of Interest: The authors declare no conflicts of interest. Ethics Approval: All bird captures and handling were approved by the Brazilian Biodiversity Information and Authorization System (SISBIO/ICMBio - Authorization: 44901) in compliance with Brazilian laws and regulations. Acknowledgments: We are grateful to the members of Ornithology and Bioacoustic Laboratory (Federal University of Uberlândia) for their field assistance and to the Postgraduate Program in Ecology, Conservation and Biodiversity (Federal University of Uberlândia) for their continued support. This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES - Finance Code 001), the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG - APQ-01654-12) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq - PELD - 403733/2012-0). Bioscience Journal | 2023 | vol. 39, e39071 | https://doi.org/10.14393/BJ-v39n0a2023-53589 7 RIBEIRO, P.V.A., et al. References ATKINSON, C.T., et al. Epizootiology of Haemoproteus meleagridis (Protozoa: Haemosporina) in Florida: seasonal transmission and vector abundance. Journal of Medical Entomology. 1998, 25(1), 45-51. https://doi.org/10.1093/jmedent/25.1.45 ATKINSON, C.T. and LA POINTE, D.A. 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