1 Molecular Insights Into Neotropical Bird

Molecular insights into Neotropical bird – tick ecological associations and the role of birds in tick-borne disease ecology Matthew J. Millera, Helen J. Essera,b, Jose R. Loaizaa,c,d, Edward Allen Herrea, Celestino Aguilara,c, Diomedes Quinteroe, Erick Alvareza,d, Eldredge Berminghama,f a Smithsonian Tropical Research Institute, Apartado Postal 0843-03092, Panamá, Republic of Panama b Department of Environmental Sciences, Wageningen University, PO Box 47, 6700 AA Wageningen, the Netherlands c Instituto de Investigaciones Científicas y Servicios de Alta Tecnología, Clayton, Panamá, República de Panamá d Programa Centroamericano de Maestría en Entomología, Vicerrectoría de Investigación y Postgrado, Universidad de Panamá, República de Panamá e Museo de Invertebrados G. B. Fairchild, Estafeta Universitaria 0824, Universidad de Panamá, República de Panamá f Patricia and Phillip Frost Museum of Science, Miami, FL 33129, USA Corresponding Author: Matthew J. Miller, [email protected] Key words: avian ectoparasites, vector ecology, host-parasite interactions, host specificity, interaction network analysis 1 ABSTRACT Ticks are important vectors of emerging zoonotic diseases. While adults of many tick species parasitize mammals, immature ticks are often found on wild birds. In the tropics, difficulties in species-level identification of immature ticks hinder studies of tick ecology and tick-borne disease transmission, including any potential role for birds. In Panama, we found immature ticks on 227 out of 3,498 birds representing 93 host species, about 1/8th of the entire Panamanian terrestrial avifauna. Tick parasitism rates did not vary with temperature or rainfall, but parasitism rates did vary with host ecological traits: non-migratory residents, forest dwelling birds, bark insectivores, terrestrial foragers and lowland species were most likely to be infested with ticks. Using a molecular library developed from adult ticks specifically for this study, we identified 130 immature ticks obtained from wild birds, corresponding to eleven tick species, indicating that a substantial portion of the Panamanian avifauna is parasitized by a variety of tick species. Furthermore, we found evidence that immature ticks show taxonomic or ecological specificity to avian hosts. Finally, our data indicate that Panamanian birds are not parasitized regularly by the tick species responsible for most known tick-borne diseases. However, they are frequent hosts of other tick species known to carry a variety of rickettsial parasites of unknown pathogenicity. Given the broad interaction between tick and avian biodiversity in the Neotropics, future work on emerging tropical tick-borne disease should explicitly consider wild birds as vertebrate hosts. 2 INTRODUCTION Wild birds are increasingly recognized as playing an important role in human and animal health. Emerging zoonotic diseases such as avian influenza, West Nile Virus, and Lyme disease (which is transmitted by ticks) often have wild birds in their transmission cycle (1-3). Wild birds can act as reservoir hosts in endemic areas (4). They also have the potential to introduce diseases into previously naïve populations by spreading pathogens and/or their vectors over large distances via migratory flyways (5,6). Ticks (Ixodidae) are haematophagous ectoparasites that are regularly found on wild birds, providing them habitat and blood-meal resources, and are known to transmit to humans a larger variety of pathogens than any other arthropod vector group (7). Migratory birds captured in temperate regions upon their return from wintering grounds have been observed infected with larval and nymphal stages of tropical tick species (5). The ability of migratory birds to move tropical ticks over long distances, in concert with global climate change, potentially exposes extra-tropical regions to novel tropical tick-borne pathogens and vice versa (7, 21, 22). Therefore, empirical studies are needed that evaluate which tick species are frequently involved in bird parasitism and which ecological characteristics of bird species are related to increased tick infestation levels. In the New World tropics, the greatest risk of tick-borne disease comes from Rickettsia rickettsii, the etiological agent of Rocky Mountain Spotted Fever. Here, its primary Neotropical vector is Amblyomma cajennense sensu lato (8), although R. rickettsii has also recently been discovered in A. aureolatum in Brazil (9). Recent improvements in molecular detection of tick endosymbionts have uncovered many novel Rickettsia species of unknown pathogenicity in Neotropical Amblyomma (10-12). Although adults of the genus Amblyomma and several other Neotropical tick species typically exploit mammals, or reptiles and amphibians to a lesser degree (13), immature forms are routinely found on birds (7, 12-19) including, rarely, nymphs of the A. cajennense species complex (20). Our understanding of Neotropical bird-tick associations and hence their role in disease 3 transmission has been hampered by species identification problems; most immature Neotropical ticks, especially those of the genus Amblyomma, are not readily identifiable to species by morphology alone. Amblyomma species diversity peaks in the Neotropics, where taxonomic keys serve to identify the nymphal stage of few Amblyomma species (23). For example, in a recent survey of ticks found parasitizing humans in Panama, only 38% of the recovered specimens of Amblyomma could be identified to species (24). Similarly, in a study documenting tick infestation patterns in wild birds from southeastern Brazil, nearly 48% of the specimens of Amblyomma could not be identified to species (20). This study and others (12, 14) demonstrate that new tools and approaches are needed to properly assess the role of wild birds in tick ecology and tick-borne disease transmission in the Neotropics. Here, we: 1) identify the ecological characteristics of Panamanian bird species frequently parasitized by ticks, and determine if climate (e.g., rainfall and temperature) affects tick parasitism rates; 2) produce a robust DNA barcode library capable of identifying most of the commonly encountered ticks in Panama; 3) use the DNA barcode library to identify to species immature ticks collected off of Panamanian wild birds and 4) provide the first analysis of bird- tick ecological associations at the species level in the northern Neotropics, including an assessment of the potential role that birds may have in the transmission of tick-borne pathogens. RESULTS Ecological traits of Panamanian wild birds parasitized by ticks We evaluated patterns of tick parasitism in 3,498 bird specimens from the STRI bird collection, representing 384 species, i.e. nearly half of the roughly 800 non-aquatic bird species recorded from Panama. Ticks parasitized a total of 227 specimens of Panamanian birds (6.5%; Supplementary Table S1) representing 93 avian species and 24 families. Among avian families with at least 25 specimens evaluated, the families with the greatest proportion of specimens parasitized by ticks (%) were: Thamnophilidae (antbirds: 18%; 12 of 20 species); Furnariidae (ovenbirds and woodcreepers: 15%; 11 of 20 species); Polioptilidae (gnatcatchers: 15%; 1 of 3 species); Turdidae (thrushes: 15%; 7 of 15 species); and Troglodytidae (wrens: 14%; 9 of 16 species). Resident birds (i.e. species that breed in Panama) were 3.8 times as likely to be 4 parasitized by ticks compared to non-breeding long-distance migrant species (Fisher’s exact test, P = 0.001). Although females had a slightly higher prevalence of tick parasitism than males, the difference was not significant (Fisher’s exact test, P = 0.23). Among resident birds, a logistic model indicated that only forest habitation, terrestrial foraging, bark insectivory, and lowland residency were significantly associated with tick parasitism (Table 1). Among 26 sites with at least 20 sampled birds, we found no relationship between the frequency of tick parasitism and annual mean temperature, temperature seasonality, annual precipitation, or precipitation seasonality, however we did recover an affect of taxonomic composition, specifically, the proportion of the sampled avifauna belonging to the five families most frequently parasitized by ticks (Supplementary Table S2). DNA identifications of adult ticks agree with morphological taxonomy The 96 individuals in the adult reference library of morphologically identified ticks from central Panama formed 20 clusters with pairwise Kimura-2 parameter (K2P) genetic distances greater than 5% (Supplementary Figure 1). All 96 could be placed in clusters in agreement with the original morphological identification of the voucher with bootstrap support values of at least 99%. Among the 20 species -clusters, average nearest-neighbor K2P distance to another cluster was 15.6% and the minimum nearest-neighbor K2P distance was 12.5% (range: 12.5% – 20.5%). Two species contained DNA barcode sub-clusters with between-cluster sequence divergence greater than 3.0%. Haemaphysalis juxtakochi comprised two clusters that differed by 3.0% pairwise K2P distance, with each cluster supported by 99% bootstrap support, while Amblyomma ovale contained one cluster of four individuals supported by 97% bootstrap and a fifth individual that varied by an average K2P distance of 3.2%. In all both cases, individuals from both clusters were collected at a shared location, suggesting that these might represent cryptic biological species. Thus, the BIN (barcode identification number) numerical

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