EcoHealth 19, 402–416, 2022 https://doi.org/10.1007/s10393-022-01612-9  2022 The Author(s). This article is an open access publication Original Contribution Haemosporidians from a Neglected Group of Terrestrial Wild Birds in the Peruvian Amazonia Merit González-Olvera,1 Arturo Hernandez-Colina,1,8 Jocelyn G. Pérez,1 Gabriela M. Ulloa,2,3 Stephanie Montero,4 Jorge L. Maguiña,4 Andrés G. Lescano,4 Meddly L. Santolalla,4 Matthew Baylis,1,5 and Pedro Mayor2,6,7 1 Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, IC2 Liverpool Science Park, 146 Brownlow Hill, Liverpool L3 5RF, UK 2 Programa de Pós-Graduação em Saúde e Produção Animal na Amazônia, Universidade Federal Rural da Amazônia (UFRA), Belém, Pará, Brazil 3 Grupo Enfermedades Emergentes, Universidad Cientı́fica del Sur, Lima, Peru 4 Emerge, Emerging Diseases and Climate Change Research Unit, School of Public Health and Administration, Universidad Peruana Cayetano Heredia, Lima, Peru 5 Health Protection Research Unit in Emerging and Zoonotic Infections, University of Liverpool, Liverpool, UK 6 Departamento de Sanitat i Anatomia Animals, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain 7 ComFauna, Comunidad de Manejo de Fauna Silvestre en la Amazonı́a y en Latinoamérica, 332 Malecón Tarapacá, Iquitos, Peru 8 Coventry, UK Abstract: Haemosporidians are a widespread group of blood parasites transmitted by vectors. Despite their relevance for bird conservation, few studies have been conducted in the Amazonia and even less in terrestrial wild birds. We analysed blood samples from 168 game birds, collected from 2008 to 2015 by subsistence hunters of an indigenous rural community in the Peruvian Amazonia. DNA was tested for Haemoproteus spp., Plasmodium spp. and Leucocytozoon spp. and positive amplicons were sequenced and curated for phylogenetic analysis. Haemosporidian prevalence was 72% overall, 66.7% for Haemoproteus spp. and 5.4% for Plasmodium spp. and respectively by bird species: Spix’s Guan (Penelope jacquacu, n = 72) 87.5% and 0%, Razor-billed Curassow (Mitu tuberosum, n = 45) 77.8% and 6.7%, White-winged Trumpeter (Psophia leucoptera, n = 20) 6.3% and 12.5%, Blue-throated Piping-guan (Pipile cumanensis, n = 16) 73.3% and 6.7%, and Great Tinamou (Tinamus major, n = 15) 10% and 15%. Leucocytozoon spp. was not found. P. leucoptera and T. major were less likely to be infected with Haemoproteus spp. Fruit abundance had a negative association with Haemoproteus spp. prevalence and precipitation was negatively associated with Plasmodium spp. prevalence. The 106 se- quences examined represented 29 lineages, 82.8% of them were new lineages (Plasmodium n = 3, Haemo- proteus n = 21). Novel host-parasite associations and lineages were unveiled, including probably new species of Plasmodium spp. Our results highlight the scientific value of alternative sampling methods and the collabo- ration with local communities. Keywords: Amazonia, Haemosporidia, Haemoproteus, Plasmodium, Terrestrial birds, Subsistence hunting Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1007/s10393-022-01612-9. Published online: August 27, 2022 Correspondence to: Arturo Hernandez-Colina, e-mail: arturo.hernandez-colina@liver pool.ac.uk Haemosporidians from a Neglected Group of Terrestrial Wild Birds 403 INTRODUCTION AND PURPOSE Rainforest game birds are vital links in the complex dynamics of Neotropical rainforest systems and provide key The Amazon rainforest is renowned for its high biodiver- ecosystem services; additionally, they represent a traditional sity, which has not been fully recorded yet, since new resource for many indigenous cultures for various purposes species are being described constantly (Metcalf et al. 2020). including a dietary source (Whitworth et al. 2018). Despite The Peruvian Amazonia has the second largest portion of their key role, these birds are highly threatened, in partic- the Amazon rainforest (Marzal et al. 2015) and it is one of ular, large terrestrial birds such as trumpeters (Psophiidae), the most biodiverse regions in the world (Metcalf et al. various cracids (Cracidae) and tinamous (Tinamidae) are 2020), its ornithological fauna represents 20% of bird the first birds to be hunted in the proximities of human diversity worldwide and, due to their high degree of en- settlements (Lloyd 2004). demism and risk of extinction, many of these bird species In the Peruvian Amazonia, cracids are threatened due are a conservation priority (Marzal et al. 2015). to unsustainable hunting practices, and populations of Haemosporidian parasites have important effects on Mitu tuberosum, Penelope jacquacu and Pipile cumanensis their hosts like reducing survival, body condition and have been reduced by over 90% in heavily hunted areas reproductive success (Garcı́a-Longoria et al. 2014; Bush (Begazo and Bodmer 1998). As these birds have intrinsi- and Clayton 2018). These parasites may constitute another cally low rates of reproduction and are dependent on conservation threat for wild birds (Sijbrandra et al. 2017; undisturbed forest, their populations cannot be maintained Chaisi et al. 2019), particularly the lineage SGS-1 of Plas- under high hunting pressure (Barrio 2011). According to modium relictum, which was recorded for the first time in the Red List of the International Union for Conservation of Peru bordering the Amazonia as an invasive species Nature (IUCN 2021), globally, Tinamus major and Psophia (Marzal et al. 2015). Plasmodium spp., Haemoproteus spp. leucoptera are classified as nearly threatened and P. jac- and Leucocytozoon spp. are the most common genera of quacu, M. tuberosum and P. cumanensis remain at least avian haemosporidian parasites on a global scale; however, concern. their prevalence varies according to environmental factors Most of the research on avian haemosporidians has (i.e., altitude, wetland cover, forest cover, vegetation den- been carried out employing mist nets; hence, it describes sity) and host traits (i.e., body mass, foraging habits, bird the situation exclusively in aerial birds and information species richness, migration distance), across different re- about terrestrial birds is minimal. The study of haemo- gions (Fecchio et al. 2021). The latest synthesis on sporidian parasites in the Amazonia is beset by logistical haemosporidian prevalence shows prevalence variations for and financial restrictions, and information regarding the Haemoproteus spp. (7.1–38.4%), Plasmodium spp. (5.8– diversity of their vertebrate hosts and the environmental 28.6%), and Leucocytozoon spp. (0–30.1%) across zoogeo- influence on their prevalence and distribution is incom- graphical regions (Fecchio et al. 2021). In the Amazonia, plete. Therefore, this study aims to characterize haemo- prevalence and diversity of these parasites have marked sporidian infections in terrestrial game birds in the differences due to climatic conditions (Fecchio et al. 2017, Peruvian Amazonia by exploring parasite diversity, host- 2018c). For instance, Plasmodium spp. and Haemoproteus parasite associations, prevalence, environmental influences, spp. species and lineages were highly diverse and dispersed and parasite phylogeny. In the Amazonia, local commu- (Fecchio et al. 2017, 2018a); whereas a single lineage of nities rely on subsistence hunting for food and could be- Leucocytozoon spp. has been recorded at low prevalence in come active samplers of valuable biological material that is one bird species (Fecchio et al. 2018c). Haemosporidian usually discarded. To overcome accessibility and conven- lineages in the Amazonia coexist in the same bird com- tional fieldwork constraints, this study used blood samples munities presenting different levels of host prevalence and from game birds sustainably hunted over eight years by the specialization, ranging from generalists to specialists indigenous Yagua community of Nueva Esperanza, Peru. (Svensson-Coelho et al. 2013; Fecchio et al. 2017, 2018a, 2019). Therefore, habitat choice, migratory behaviour and coloniality could affect the prevalence of blood parasites in birds (Garcı́a-Longoria et al. 2014). 404 M. González-Olvera et al. METHODS Local hunters were asked to impregnate blood from the cranial or caudal cava vein of the hunted birds on either Study Area Whatman filter paper No. 3, FTA cards or protein saver cards. Samples were collected in all seasons, and hunters The sampling area is located around the indigenous Yagua recorded the species, date, location, and sex in the case of community of Nueva Esperanza (04 190 5300 S; 71 570 3300 sexual dimorphism. Cards were placed individually in W; UT5:00), comprised of 281 people (2015) and estab- tagged paper envelopes and kept at environmental tem- lished in the Yavarı́-Mirı́n River, within the Loreto state in perature protected from light and humidity until sent for Peru (Fig. 1). The area is composed of 322,500 ha of storage (- 20C) and processing at the EmergeLab, LID continuous predominantly non-flooding terra firme forest; 412 of Universidad Peruana Cayetano Heredia. in 2010, the total disturbed area was 0.8%, mostly caused by logging camps and agriculture (Mayor et al. 2015). Main occupations of local people are small-scale agriculture, Molecular Methods fishing, logging, and subsistence hunting within a hunting Whatman filter paper No. 3, FTA cards or protein saver area of approximately 42,200 ha (Mayor et al. 2015). The cards were placed into a petri dish and a 6 mm2 piece was climate is typically equatorial with an annual temperature cut into smaller pieces with a scalpel. Prior to DNA of 22–36C, a relative humidity of 80–100%, and an annual extraction, sample preservation condition was recorded rainfall of 1500–3000 mm (El Bizri et al. 2018). (good, medium, bad) as well as fungal presence (abundant, present, scarce, none). The extraction was done using a Sample Collection QIAamp DNA Mini Kit following the manufacturers instructions, except for the incubation, which elapsed From 2008 to 2015, blood samples from 168 terrestrial wild overnight to increase the DNA yield. Extracted DNA was birds were collected by subsistence hunters as part of a tested for the most common haemosporidians (Haemo- wildlife conservation program. A total of 168 game birds proteus spp., Plasmodium spp. and Leucocytozoon spp.) belonging to three orders (Galliformes, Gruiformes, following the protocol by Hellgren et al., (2004), which Struthioniformes), three families (Cracidae, Psophiidae, consists of a nested PCR that amplifies a 479-bp fragment Tinamidae) and five species were collected by the Yagua of the cytochrome b (cytb) gene. In the first part of the hunters. The catches per bird were: Spix’s Guan (P. jac- reaction 1 ll of DNA template, 1 ll of forward primer quacu) (n = 72), Razor-billed Curassow (M. tuberosum) HaemNF1, 1 ll of reverse primer HaemNR3, 10 ll of (n = 45), Great Tinamou (T. major) (n = 20), White- Bioline mix, 1 ll of BSA (bovine serum albumin), and 6 ll winged Trumpeter (P. leucoptera) (n = 16), and Blue- of nuclease-free water were mixed to reach a final volume throated Piping-guan (P. cumanensis) (n = 15) (Table S1). of 20 ll. The PCR profile was 22 cycles at 94C for 3 min, 94C for 30 s, 50C for 30 s, 72C for 45 s, followed by a final extension at 72C for 10 min. For the second part, 2 ll of PCR product from the previous reaction were used as template, 10 ll of MyTaqTM Red MixPCR master mix, 1 ll of BSA and 5 ll of nuclease-free water were employed. The samples had a DNA concentration range of 50–700 ng/ ll (median 200 ng/ll), all primers were used at a 10 lM concentration, and BSA was used at a 0.5% concentration. For detection of Plasmodium spp. and Haemoproteus spp., 1 ll of forward primer HaemF and 1 ll of reverse primer HaemR2 were added, whereas for the detection of Leuco- cytozoon spp. 1 ll of forward primer HaemFL and 1 ll of reverse primer HaemR2L were used. The PCR profile for Figure 1. Map of the study area in the territory of the local the second part was 36 cycles at 94C for 3 min, 94C for community of Nueva Esperanza in the Yavarı́-Mirı́n River in the 30 s, 50C for 30 s, 72C for 45 s, followed by extension at Peruvian Amazonia. 72C for 10 min. Molecular grade water was used as a Haemosporidians from a Neglected Group of Terrestrial Wild Birds 405 negative control and genomic DNA from P. bergei ANKA lineages from a single host species. New lineages were de- or genomic DNA from Leucocytozoon spp. was used as a posited in GenBank under the accession numbers positive control. The amplicons were visualized on a 1.5% MZ614915–MZ614938 and ON246344. agarose gel with SYBRTM Safe DNA gel satin (Thermo Fisher Scientific). Positive PCR products were sent for Phylogenetic Analysis sequencing in forward and reverse direction with the San- ger method employing the primers HAEMF and HAEMR2. To estimate a phylogeny of our sequences, representative Resulting sequences were visualized, assembled, and man- reference sequences from the clusters identified in the CD- ually corrected using BioEdit. Sequence reads were com- HIT analysis, and representative sequences of each new pared to previously published avian Haemosporidia in the lineage from our collection were employed. The sequences GenBank nucleotide database using BLASTn to identify the were aligned using ClustalW [33] and Leucocytozoon was genus of the parasite. Mixed sequences (n = 6), indicated used as outgroup to root the tree. A Maximum Likelihood by electropherograms with consistent double peaks in the (ML) phylogeny was estimated using MEGA X (Kumar, same base position, were considered as mixed infections et al. 2018) with a GTR + G + I nucleotide substitution (Lutz et al. 2015) and were excluded from further analysis. model (estimated by Smart Model Selection using the Akaike Information Criterion). The data set was parti- tioned to allow independent modelling of base substitution Sequence Clustering rates at each codon position, a measure that will reduce the Sequence clustering and phylogenetic analysis were used to effect of homoplasy at the third position. Node robustness relate each observed sequence to all previously published was evaluated with an SH-like log-Likelihood ratio test. sequences. To guarantee the most thorough comparison of The software FigTree v1.3.1 was used to visualize the new sequences with those recorded previously, a randomly phylogenetic tree. Sequences in clades containing sequences chosen Haemoproteus cytb sequence, was used for the derived from a named morphospecies were assigned to that GenBank search. The query showed 10,000 matches ranked morphospecies. in descending sequence identity, which included non-avian Plasmodium, Haemoproteus, and Leucocytozoon sequences Data Analysis at the lower end, ensuring that all available avian Plas- modium and Haemoproteus sequences were comprised. All We analysed (1) the biological susceptibility, including bird avian Plasmodium and Haemoproteus sequences from the species, family, and sex, and (2) the environmental drivers query were selected and joined with an additional 224 of infection, with the year, precipitation, season, tempera- voucher sequences from MalAvi. After removing dupli- ture, river level and ripe fruit availability as explanatory cates, partial sequences, and sequences containing variables. Precipitation and river level values were obtained ambiguous bases, the reference sequences were aligned in from the HidroWeb portal of the National System of Water Bioedit (Hall 1999) using ClustalW (Chenna et al. 2003) Resources Information (SNIRH) of Brazil. The data from with all new sequences identified in this study (n = 106) to the four closest stations to the study area in the Yavari produce a 475 bp multiple sequence alignment (n = 5810). River with complete records of the day the birds were To simplify the phylogeny, CD-HIT was used to cluster hunted were used. We classified the months according to sequences into common lineages, defined by a 1% sequence their average precipitation over the years of our sampling. divergence threshold, as this value retained the best mor- The months that received more rain than the overall phospecies of the reference lineages used. Sequences found average + 1 standard deviation (SD) were classified as rainy in the same cluster were considered to be the same lineage. months, the ones that received less than the average—1 SD Hence, Peruvian sequences found in clusters with previ- as dry months and those between ± 1 SD as intermediate ously described lineages were identified as such, and se- months (normality was confirmed with a Shapiro–Wilk quences in clusters without reference lineages were normality test, P = 0.452). In this way, the rainy months established as new lineages and were named following the were January and March (294–329 mm), the intermediate standard protocol of Bensch et al. (2009), creating a six- months were February, May–June, October–December letter code formed by the first three letters of both, the host (165–294 mm) and the dry months were July–September genus and species, followed by a number to denote multiple (142–165 mm). The temperature data was obtained from 406 M. González-Olvera et al. Figure 2. Distribution of terrestrial bird species studied (n = 168) Figure 3. Overall Haemosporidian prevalence in terrestrial birds according to infection by Haemoproteus spp. and Plasmodium spp. in (n = 168) in the territory of the local community of Nueva the territory of the local community of Nueva Esperanza in the Esperanza in the Yavarı́-Mirı́n River by monthly rain seasonality. Yavarı́-Mirı́n River. P. leucoptera and T. major were less likely to be Rainy: January and March (294–329 mm); Intermediate: February, infected by Haemproteus spp. (P < 0.001). May–June, October–December (165–294 mm); Dry: July–September (142–165 mm). the NCEI Climate Data Online service requesting the daily summaries for Iquitos station (Station ID: the models simultaneously (non-independent variables: GHCND:PE000084377) given that it is the closest to the family and species; colinear variables: river level, precipi- sampling area (approximately 195 km). As the minimum tation, season, and fruit abundance in upland forest and in and maximum temperature records were incomplete, only swamp forest; variance inflation factors > 3). As the the daily average temperature was extracted and then number of infected birds was correlated with the number of averaged by month for analysis. In all cases, the search lineages found by bird species (Pearson’s correlation, period was from the 1st of January 2008 to the 31st of r2 = 0.999, P < 0.001), the number of lineages in relation December 2015. to the biological susceptibility was not analysed as the re- Body condition and fitness of individuals have been sults were very similar to the ones for the parasite preva- shown to influence their immune response against para- lence. However, the number of lineages of Haemoproteus sitic, viral, and bacterial infections (van Hoesel et al. 2020). spp. in relation to environmental variables was aggregated Thus, we used seasonal fruit availability to relate the by month and analysed using a negative binomial regres- environmental stress with infection rates in these frugivo- sion model. The number of Plasmodium spp. lineages was rous bird species. The percentage of ripe fruit in the study insufficient for analysis. area was obtained from El Bizri et al. (2018), were the The influence of the card types for blood storage (Filter authors observed the canopy of trees and vines monthly paper W3, FTA cards and Protein saver cards), sample along two transects in upland forest and one in upland preservation (good, medium, bad) and fungal presence swamp forest dominated by palms; the length and area of (abundant, present, scarce, none) on the test result was each transect were determined by the discovery rate of new assessed using logistic regression; all variable combinations species and as it plateaued, it was considered representative were explored. of fruit abundance in the area. For all models, observations without complete data for Generalized linear models (GLM) were used to analyse all variables were excluded. Candidate models were selected the prevalence and number of lineages of Haemoproteus using the Akaike’s Information Criterion (AIC) theoretic and Plasmodium, separately. We used logistic regression to approach and the ones within two AIC units (i.e., estimate parasite prevalence. All combinations of variables DAIC < 2) were considered as the best-supported by the were explored, but variables that are not independent of evidence in the data. The second order AIC (AICc) was each other or that showed collinearity were not included in used instead for the models assessing the number of lin- Haemosporidians from a Neglected Group of Terrestrial Wild Birds 407 408 M. González-Olvera et al. b Figure 4. Association between environmental variables and Hae- modium spp. Regarding sample storage, the best models mosporidian prevalence in terrestrial birds (n = 168) in the territory showed that FTA cards had higher positivity (P = 0.002), of the local community of Nueva Esperanza in the Yavarı́-Mirı́n whereas sample preservation and fungal contamination River. Regression curves were constructed for each variable were not associated with positivity differences. independently. Upper histogram shows positive individuals and Haemoproteus spp. infections were observed in all years lower histogram shows negative individuals. For Haemoproteus spp., with the highest prevalence in 2013 (88.5%, 23/26) and the the percentages of fruit in upland forest was significant (P = 0.002), lowest in 2008 (40.9%, 9/22). Haemoproteus spp. preva- and for Plasmodium spp. precipitation was significant (P = 0.032). lence was significantly higher in 2013 (P = 0.017) and 2015 (P = 0.04). Plasmodium spp. infections were not observed between 2008 and 2014; the highest prevalence was ob- eages to account for the small sample size (n = 37). The served in 2011 (12.5%, 1/8) and the lowest in 2009 (3.7%, variables used in each model, AIC, DAIC and AIC weights 1/27) (Table S1). For the environmental influence on are shown in Supplementary Tables S3–S6. Given P values Haemoproteus spp. infection, the three best-supported are for the best-supported models. All analyses were per- models indicated a negative association with the abundance formed in R (version 4.1.1). of fruits in upland forest habitats (P = 0.002). In the case of Plasmodium spp. infection, only the precipitation had a Ethics negative relation in the selected models (P = 0.032). No All applicable institutional and national guidelines for the significant environmental association was found for the care and use of animals were followed. Birds were killed number of Haemoproteus spp. lineages and only one model exclusively as part of the usual activities of local hunters. including precipitation was supported. Figures 3 and 4 The research protocol and export permits were approved show parasite prevalence by monthly seasonality and by Servicio Nacional Forestal y de Fauna Silvestre of Peru environmental variables, respectively. (Peruvian Forestry and Wildlife Agency) (Research Ethics Committee for Experimentation in Wildlife Protocols res- Haemosporidian Genetic Analysis olutions 041-2007- DGGFS-DGEFFS, 0350-2012-DGFFS- From the 121 cytb sequences obtained in this study, 14 were DGEFFS, 258-2019-MINAGRI-SERFOR-DGGSPFFS, and either mixed, partial, or poor-quality sequences; hence, export permit 003457-SERFOR) and the Institutional they could not be further analysed. The remaining se- Animal Use Ethics Committee from Universidad Peruana quences were clustered at 1% divergence thresholds since it Cayetano Heredia (resolution 029-03-19, protocol 102142). was observed that it adequately retained morphospecies. A total of 106 cytb sequences were identified in this study, 100 RESULTS within the genus Haemoproteus and six within the genus Plasmodium. In total, 29 different lineages were identified, The haemosporidian prevalence was 72% overall, for 24 (82.8%) were recorded for the first time, three belonged Haemoproteus spp. it was 66.7% (112/168) and for Plas- to Plasmodium (10.4%) and 21 to Haemoproteus (72.4%). modium spp. it was 5.4% (9/168). Leucocytozoon spp. was The majority of novel lineages were isolated from M. not found. Haemoproteus spp. prevalence was higher in P. tuberosum. Most lineages for both Haemoproteus and jacquacu (87.5%, 63/72), M. tuberosum (77.8%, 35/45) and Plasmodium were observed once. The most prevalent lin- P. cumanensis (73.3%, 11/15); whereas the highest Plas- eage (TOFLA03) was recorded on 49 birds from four spe- modium spp. prevalence was found in T. major (15%, 3/ cies with most of its observations coming from P. jacquacu 20), and P. leucoptera (12.5%, 2/16) (Fig. 2). For the (Table 1). assessment of the biological susceptibility, the highest For Plasmodium, five different lineages were observed, supported models included family or species; nevertheless, and three were reported for the first time (Table 1). Pre- the model with the lowest AIC was the one for species viously reported parasite lineages that matched sequences alone, which showed a significant negative association be- from this study were: TOFLA03 (Accession number tween Haemoproteus spp. prevalence and P. leucoptera JX029916) (n = 50), PENOBS01 (Accession number (P < 0.001) and T. major (P < 0.001). No significant KX171627) (n = 6), PENJAC01 (Accession number effects were observed in the supported models for Plas- KF482345) (n = 6), and PSOOCH01 (Accession number Haemosporidians from a Neglected Group of Terrestrial Wild Birds 409 Table 1. Plasmodium and Haemoproteus lineages by bird species from the Nueva Esperanza settlement of the Peruvian Amazonia. Lineages Spix’s guan Razor-billed White-winged Blue-throated Great Tinamou Total (Penelope jac- Curassow Trumpeter Piping-guan (Tinamus quacu) (Mitu tubero- (Psophia leu- (Pipile cuma- major) sum) coptera) nensis) DENVID01P 1 1 *MITTUB02P 1 1 *MITTUB03 1 1 *MITTUB04 1 1 *MITTUB05 1 1 *MITTUB06 1 1 *MITTUB07 1 1 *MITTUB08 1 1 *MITTUB09 1 1 *MITTUB10 1 1 *MITTUB11 2 2 *MITTUB12 4 4 *MITTUB13 5 5 *MITTUB14 8 8 *MITTUB15 1 1 PENJAC01 6 6 *PENJAC02 1 1 *PENJAC03 1 1 *PENJAC04 1 1 *PENJAC05 3 3 PENOBS01b 6 6 *PIPCUM02 1 1 *PIPCUM03 1 1 *PIPCUM04 1 1 *PSOLEU02P 1 1 *PSOLEU03 1 1 PSOOCH01P 1 1 *TINMAJ01P 3 2 TOFLA03a 38 4 6 1 49 Sequences analysed 56 33 3 10 4 106 Found lineages• 7 15 3 5 2 29¤ a Haemoproteus paraortalidum. b Haemoproteus ortalidum. P Plasmodium spp. lineages. Other lineages without superscripts belong to Haemoproteus spp. *New lineages found in this study. •Number of different lineages observed in each bird species. ¤Number of different lineages observed in the birds. KU562606) (n = 1) and DENVID01 (Accession number and TOFLA03 to Haemoproteus paraortalidum. No Plas- KU057966). Three lineages belong to unidentified mor- modium morphospecies could be identified in this study. phospecies, PENJAC01 (Haemoproteus) and PSOOCH01 For the new lineages, the closest related sequences had an and DENVID01 (Plasmodium), whereas PENOBS01 cor- identity of 95–99% and a nucleotide correspondence of responds to the morphospecies Haemoproteus ortalidum 444–467 bp out of 469 bp (Table S2). 410 M. González-Olvera et al. Figure 5. Maximum likelihood phylogeny of the cytb sequences obtained in this study and reference sequences. The phylogeny was estimated from a 49 bp multiple sequence alignment using a GTR + C + I model (a = 0.488; proportion of invariant sites = 0.248). The tree is rooted with an outgroup of Leucocytozoon sequences (boxed in black). Node accuracy is indicated by an SH-like log-Likelihood ratio metric; lower bootstrap values (< 50) are omitted. All sequences with different lineage names were obtained with a 1% sequence divergence threshold. Reference sequences obtained from MalAvi and GenBank are shaded in black, the rest of the sequences were obtained from this study and are shaded according to the bird they were obtained from Penelope jacquacu (Green), Pipile cumanensis (Pink), Mitu tuberosum (Blue), Psophia leucoptera (Brown) Tinamous major (Red). Sequences are named by their lineage, and their accession numbers are indicated in parenthesis. Known parasite species for the lineages are listed to the right. Different parasite genus and subgenus are indicated by boxes: Haemoproteus (Parahaemoproteus) (Blue), Plasmodium (Yellow), Haemoproteus (Haemoproteus) (Red). Branches lengths are drawn proportionally to evolutionary distance. Lineages AUTOCH3, PHLNIG04 and COLL12 are listed as Haemoproteus spp. (MalAvi 2021) (Color figure online). Plasmodium sequences formed a single clade in the ML clades with one of them containing all the new tree and newly described lineages, TINMAJ01, MITTUB02 Haemoproteus lineages found in this study as well as ref- and PSOLEU02 identified as Plasmodium by NCBI blast, erence sequences described in the Amazonia. Interestingly, were placed in that clade. In addition, the lineages in the Plasmodium clade, there were two subclades that PSOOCH01 and DENVID01, which matched a sequence contained Haemoproteus sequences, one containing only isolate in this study, were also positioned in the Plasmod- Haemoproteus lineages (AUTOCH03 and PHLNIG04) and ium clade (Fig. 5). Another two well-supported clades for the other one grouped Haemoproteus (CATAUR01 and both Haemoproteus subgenus were produced. The Para- COLL12) and Plasmodium (PSOOCH01) lineages. The haemoproteus subgenus clade formed two distinctive sub- lineages PSOLEU03 and MITTUB15, identified as Hae- Haemosporidians from a Neglected Group of Terrestrial Wild Birds 411 moproteus spp., formed a single clade isolated from the probability; however, future studies should integrate dif- other two main Haemoproteus clades (Fig. 5). ferent methods to compare haemosporidian prevalence. Prevalence varies greatly among avian species because of differences in immune response (Calero-Riestra 2016), DISCUSSION physiology (Ortego et al. 2008), breeding season (Lachish et al. 2011), and nesting foraging and flocking behaviour To our knowledge, this is the first study of haemosporidian (Gonzalez et al. 2014). Haemosporidian prevalence has infection focused exclusively on wild populations of ter- been evaluated in different avian groups previously (e.g., restrial birds. We present the first record for Haemoproteus Krone et al. 2008; Quillfeldt et al. 2011; Gao et al. 2021; spp. infection in P. leucoptera and in M. tuberosum and the Hernandez-Colina 2021), looking for changes associated first report of haemosporidian parasites infecting T. major with their species, life traits and habitats. Terrestrial lifestyle (Plasmodium spp.) and P. cumanensis (Haemoproteus spp. might change the exposure rate to vectors and may also put and Plasmodium spp.). The only host-parasite association birds in contact with different vector species compared to reported before is Haemoproteus spp. infecting P. jacquacu aerial birds. To our knowledge, there is only one study (MalAvi 2021). reporting haemosporidians in Amazonian terrestrial wild The overall haemosporidian prevalence was 72% and birds, reporting Plasmodium spp. infection in P. leucoptera Plasmodium spp. prevalence (5.4%) was contrasting with (66.7%, 2/3) (Fecchio et. al. 2018b). Chagas et al. (2016) the one for Haemoproteus spp. (66.7%). These parasites reports one out of six individuals of Penelope superciliaris have been recorded previously in the Amazonia with a infected with Plasmodium spp., this terrestrial bird is pre- combined prevalence between 17.4% (Fecchio et al. 2018c) sent in the Amazonia, but in this case, it was sampled in the and 21.7% (Svensson-Coelho et al. 2013), and a similar Atlantic Forest (Chagas et al. 2016). Other studies reported individual prevalence for Plasmodium spp. and Haemo- blood parasites on captive terrestrial birds, Cracidae: proteus spp. of 2.4, 15.3% (Fecchio et al. 2018c) and 6.4, Aburria jacutinga (Haemoproteus spp.; 62.1%, 18/29) 9.6% (Svensson-Coelho et al. 2013), respectively. Preva- (Ferreira-Junior et al. 2018), A. jacitinga (haemosporidians; lence differences may be due to several effects such as the 42.8%, 18/42) (Motta et al. 2013), A. mantelli (Plasmodium sampling area, host fitness and bird species. The ecosystem spp.; 78.0%, 25/32) (Banda et al. 2013), Mitu tormentosum in which this and previous studies were carried out is (Plasmodium spp.; 20.0%, 1/5), Nothocrax urumutum categorised as terra firme forest; however, this is a broad (Plasmodium spp.; 20.0%, 1/5), Penelope obscura (Haemo- definition of a complex ecosystem (Asner et al. 2013). The proteus spp.; 50.0%, 1/2) and Pipile jacutinga (Plasmodium Amazonia consists of a mosaic of different habitats and a spp., 66.7%, 2/3), Phasianidae: Pavo cristatus (Plasmodium habitat classification system that considers all minor habitat spp.; 12.9%, 4/31), Pavo muticus (Plasmodium spp.; 25.0%, types occurring in the region is missing (Harder 2000). 1/4), and Sthrutionidae: Struthio camelus (Plasmodium Habitat type has been described to be associated with spp.; 12.5%, 1/8) (Chagas et al. 2017). parasite host-specialization (Sehgal 2015), and it is known Important differences were observed in parasite to affect vector populations and host-parasite-vector prevalence by bird species, which has also been reported in interactions. In fact, Fecchio et al. (2018c) found significant Western Amazonia (Svensson-Coelho et al. 2013). Here, P. differences in Haemoproteus spp. and Plasmodium spp. leucoptera and T. major were unlikely to be infected by prevalence amongst six areas of endemism. Haemoproteus spp. compared to the other species, but no Previous studies were based on bird netting, whereas in significant influences were found for Plasmodium spp. this study hunted birds were employed. It has been argued infection possibly due to the low number of infected birds. that nets catch mainly healthy birds since this method relies Life traits may influence parasite prevalence, like in the on the active movement of birds, and haemosporidian in- Ecuadorian Amazonia, where differences in prevalence by fected birds have shown a decrease in their locomotion foraging height were found (Svensson-Coelho et al. 2013). (Valkiūnas 2004). Furthermore, Valkiūnas (1991, 1993) Although all the birds studied here share terrestrial habits, demonstrated that birds on the parasitaemia peak are un- their nesting, grouping, and feeding behaviours are differ- der-sampled by mist netting but can be obtained by other ent. For instance, P. jacquacu shows partial arboreous be- collection methods such as shooting. It was not possible to haviour (Mogollón 2016), whereas T. major remains mostly test if haemosporidian infection was related to hunting on ground level (Guerta and Cintra 2014), and P. cuma- 412 M. González-Olvera et al. nensis are capable of gliding for long distances (Mogollón are scarce, birds’ immune response could be suboptimal, 2016) giving them the ability to widen their geographic increasing susceptibility to Haemoproteus infection. range. In the phylogeny, matches to reference sequences from Bird species with high Haemoproteus spp. prevalence our Haemoproteus samples were exclusive with sequences had low Plasmodium spp. prevalence and vice versa. That reported in the Amazonia, suggesting that geographical same pattern has already been recorded in Amazonian birds barriers have impeded the introduction of exotic parasites (Svensson-Coelho et al. 2013) and it could be indicative of to this geographic area (Chagas et al. 2017). Haemoproteus vector host preferences, particularly since each genus is sequences clustered with sequences of the Parahaemopro- vectored by different dipteran insects (Ferreira-Junior et al. teus subgenus and originated from a main branch sug- 2018). Therefore, exposure rates could be also associated gesting that they are highly related. This could be due to with bird behaviours (nesting type and height, social be- their speciation history since these parasites showed low haviour, foraging height, etc.) (Svensson-Coelho et al. rates of speciation events, and parasites from the Para- 2013). Indeed, Fecchio et al. (2018b) found that infection haemoproteus subgenus are known to have high host and diversity of Plasmodium responded mainly to geogra- specificity (Fecchio et al. 2018a). phy, while Haemoproteus was related to host associations. Contrary to the Haemoproteus clustering in the phy- P. jacquacu was the most represented bird in our sampling logeny, our Plasmodium sequences grouped apart from (n = 72), it had the highest haemosporidian prevalence each other, and we observed only two matches, one with a (87.5%) and was infected exclusively with Haemoproteus lineage previously recorded in the Amazonia (PSOOCH01) spp. Hence, either it is refractory to Plasmodium spp. due and one with a lineage obtained in the Brazilian Atlantic to its biology or behaviour, or it is infected at such low Forest (DENVID01). We included other sequences isolated prevalence that further sampling is required to uncover this from the Amazonia in related birds to the ones we studied association. (MYCAME08 and NYCNYC01); however, they did not Climatic variables influence the life cycle of haemo- cluster together or with our sequences. Noteworthy is the sporidian and their vectors, and in consequence could af- clade formed by PSOOCH01, CATAUR01 and COLL12, as fect parasite transmission (Santiago-Alarcon et al. 2012). it is composed of one Plasmodium sequence and two However, no significant positive effects were observed in Haemoproteus sequences and it is placed in the Plasmodium relation to variables typically related to prevalence (i.e., clade. Nevertheless, in this phylogeny, the Plasmodium temperature and precipitation) possibly because in the clade has the lowest nodal support; moreover, it has been Amazonia these variables are maintained within the range suggested that PSOOCH01 and CATAUR01 may corre- that favours vectors’ life cycle and, therefore, parasite spond to a new genus of haemosporidian (Vanstreels et al. transmission (Fecchio et al. 2017). Precipitation can be a 2022), which may explain this odd placing. Haemo- predictor of mosquito abundance and distribution and sporidian phylogenies constantly present inconsistencies therefore, related to the prevalence of avian malaria (Sehgal given the high diversity of these parasites and the lack of 2015). Nonetheless, the negative association of precipita- studies in certain bird species and areas; nonetheless, tion with Plasmodium spp. prevalence observed could be addition of new sequences contributes to the shaping of due to the limiting effect of rain on mosquito flying activity phylogenies and to shed light on the evolutionary origin of and consequently host-seeking behaviour; besides, heavy these parasites. rain can reduce mosquito survival (Karki et al. 2016). It is possible that some of the new lineages observed in We found a negative association between fruit abun- this study, either for Haemoproteus or Plasmodium, repre- dance and Haemoproteus spp. prevalence, and to our sent new species, particularly those with the higher nu- knowledge, the influence of food resources and Haemo- cleotide divergence compared to reported lineages and sporidian prevalence has not been explored before. The diet those whose tree placing is in isolated branches with high of the bird species sampled here is mainly based on fruits nodal support. However, the lack of morphological evi- and complemented with invertebrates, seeds, leaves and dence and the unresolved relationships precludes us from flowers (Mogollón 2016; Schelsky 2004; Sherman 1995). confirming if new species have been found. The availability of resources is related to body condition Some of the sequences obtained in this study clustered and fitness of individuals and consequently, their immune with lineages from GenBank and MalAvi (TOFLA03, PE- response (van Hoesel et al. 2020). Therefore, when fruits NOBS01, PENJAC01 andPSOOCH01). The lineage TO- Haemosporidians from a Neglected Group of Terrestrial Wild Birds 413 FLA03, belonging to H. paraortalidum, was the most fre- Future research should include clinical and quently observed in this study. This lineage was previously histopathological examinations to define health implication observed in Peru and Brazil on Galliformes and Passeri- of haemosporidian and blood smears to complement the formes (MalAvi 2021). The lineage PENOBS01 of the H. classification of these parasites by morphology. Also, ortalidum species has been previously reported in Brazil in studying dipteran vectors present in the area, their com- birds of the Galliformes order (Chagas et al. 2017). The petence, abundance, and host preferences would allow to lineage PENJAC01 was reported in Peru (MalAvi 2021), assess the environmental influence on their life cycles. DENVID01 was isolated from the Brazilian Atlantic Forest Together, this information will help us to understand the (Chagas et al. 2016), and PSOOCH01 (Plasmodium spp.) role of these parasites as an additional pressure on the was recorded in the Brazilian Amazonia (Fecchio et al. conservation of threatened species. 2017). Despite evidence of migrant birds in the area (Harvey et al. 2014) and the recent discover of invasive haemosporidian species in Peru (Marzal et al. 2015), we CONCLUSION found no proof of foreign parasites since the reference lineages have only been reported in the mentioned bird This is one of the first studies to demonstrate high orders and locations. The rest of the sequences obtained haemosporidian infection rates in populations of terrestrial here represented new lineages. Three new lineages were birds from remote areas in the Amazonia. The participation recorded for Plasmodium and 21 for Haemoproteus, cor- of the local community has been key to providing a large responding to 82.8%; a similar proportion of new lineages set of blood samples from species that are usually not has also been reported for the Brazilian Amazonia (91.4%) studied. We also showed that climatic variables and fruit (Fecchio et al. 2018b). Neotropical regions, such as the availability influence the probability of infection. Various Amazonia, are considered as hotspots of avian diversity and lineages and host-parasite associations were recorded for the positive association between host and haemosporidian the first time showing that there is a great diversity in diversity indicates that the diversity of the latter could be Amazonian haemosporidian and that more studies are re- also great and that new species and lineages are likely to be quired to fully describe it. Reporting the distribution and discovered as more studies are performed in the area diversity of avian blood parasites in the Peruvian Amazonia (Chagas et al. 2017). Most of the new lineages were found is an important first step to understand the natural parasite in a single bird species (M. tuberosum) suggesting that they distribution and prevalence in relation to environmental are specialists. Nevertheless, it should be considered that variables. most of the lineages retrieved here had only one observa- tion and they might be found in more bird species over ACKNOWLEDGEMENTS time. Conversely, the lineage TOFLA03 may be a generalist since it was found on four of the five bird species and for We thank the local inhabitants of Nueva Esperanza. We are eight years. Most of the new lineages were isolated from M. especially grateful for the institutional support provided by tuberosum (MITTUB03–MITTUB15), this might suggest the Dirección General Forestal y de Fauna Silvestre from that this bird is exposed to a more diverse array of vectors Peru. We thank Andrew Jackson for his support funding due to its biological or behavioural characteristics. For in- the molecular processing of the samples and for his advice stance, M. tuberosum is reported to be the bird with the on genetic analyses. largest distribution range amongst the ones collected here (Mogollón 2016), and birds with larger ranges may expe- rience a greater risk of infection by more parasite species OPEN ACCESS (Garcı́a-Longoria et al. 2014; Fecchio et al. 2017). Of the three different sampling cards used in this This article is licensed under a Creative Commons study, FTA cards produced a better testing success rate, Attribution 4.0 International License, which permits use, thus their use is recommended for future studies. Although sharing, adaptation, distribution and reproduction in any we did not observe interference by fungal contamination or medium or format, as long as you give appropriate credit to sample preservation, samples should be preserved as best as the original author(s) and the source, provide a link to the possible. Creative Commons licence, and indicate if changes were 414 M. González-Olvera et al. made. The images or other third party material in this hosts based on mitochondrial cytochrome b lineages. Molecular Ecology Resources 9(5):1353–1358 article are included in the article’s Creative Commons Bush SE, Clayton DH (2018) Anti-parasite behaviour of birds. licence, unless indicated otherwise in a credit line to the Philosophical Transactions of the Royal Society b: Biological Sci- material. If material is not included in the article’s Creative ences 373(1751):20170196. https://doi.org/10.1098/ Commons licence and your intended use is not permitted rstb.2017.0196 Calero-Riestra M, Garcı́a JT (2016) Sex-dependent differences in by statutory regulation or exceeds the permitted use, you avian malaria prevalence and consequences of infections on will need to obtain permission directly from the copyright nestling growth and adult condition in the Tawny pipit, Anthus holder. To view a copy of this licence, visit http://creativec Campestris. Malaria Journal 15(1):1–11 ommons.org/licenses/by/4.0/. Chagas CRF, de Oliveira GL, Monteiro EF, Valkiūnas G, Ka- tayama MV, Santos SV, Guida FJZ, Simões RF, Kirchgatter K (2016) Hemosporidian parasites of free-living birds in the São Paulo Zoo. Brazil. Parasitology Research 115(4):1443–1452 FUNDING Chagas CRF, Valkiūnas G, de Oliveira Guimarães L, Monteiro EF, Guida FJ, Simões RF, Rodrigues PT, de Albuquerque Luna EJ, Kirchgatter K (2017) Diversity and distribution of avian malaria MGO and AHC were supported by CONACYT (National and related haemosporidian parasites in captive birds from a Science and Technology Council, Mexico) to perform their Brazilian megalopolis. Malaria Journal 16(1):1–20 PhD studies from which this work derives. This research Chaisi ME, Osinubi ST, Dalton DL, Suleman E (2019) Occurrence was supported by CONCYTEC-FONDECYT within the and diversity of avian haemosporidia in Afrotropical landbirds. International Journal for Parasitology: Parasites and Wildlife framework E035-2017-01, ERANet-LAC (ERANet17/HLH- 8:36–44 0271), research projects (Contract N 136-2018-FONDE- Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins CYT; AC18/00054 Instituto de Salud Carlos III) and NIH/ DG, Thompson JD (2003) Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Research FIC Training Grant D43 TW007393. JGP was supported by 31(13):3497–3500. https://doi.org/10.1093/nar/gkg500 FONDECYT-CONCYTEC (Grant Contract Number 100- El Bizri HR, Fa JE, Bowler M, Valsecchi J, Bodmer R, Mayor P 2016-FONDECYT). (2018) Breeding seasonality in the lowland paca (Cuniculus paca) in Amazonia: interactions with rainfall, fruiting, and sustainable hunting. 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