Helminth and ectoparasitic faunas of the Harris’s hawk, Parabuteo unicinctus (Accipitriformes: Accipitridae), in Chile: new data on host-parasite associations for Neotropical raptors

Birds of prey harbor a wide spectrum of various parasites, mostly with a heteroxenous life cycle. However, most reports on their parasites come from Europe. Although the Harris’s hawk ( Parabuteo unicinctus ) is a widespread species in America, parasitological surveys on this hawk are mostly focused on coprological findings and ectoparasites, with poor attention paid to helminths. The aim of this study was to gather new and additional data on host-parasite associations for the Harris’s hawk. Twenty-nine birds from central and southern Chile were necropsied. Further, nine birds from a rehabilitation center and 22 museum specimens were inspected for ectoparasites. Sixty-eight percent of birds hosted at least one parasite species. Four lice species, one mite species and eight helminth species (five nematodes, two platyhelminthes and one acanthocephalan) were recorded. Parasitic lice Colpocephalum nanum and Nosopon chanabense , and a nematode Cyathostoma ( Hovorkonema ) americana were recorded for the first time in raptors from the Neotropics. A feather mite, Pseudalloptinus sp., nematodes, Physaloptera alata and Microtetrameres sp., and a trematode Neodiplostomim travassosi , were recorded for the first time in Chile. The presence of diverse heteroxenous helminths reported here in the Harris’s hawk could be explained by the generalist diet of this raptor.


Introduction
The parasitic fauna of birds of prey is rich in species with direct and indirect life cycles. In most cases, birds of prey are the definitive hosts of helminths, acquiring them through the ingestion of intermediate and paratenic hosts, such as birds, micromammals, earthworms, and arthropods (Krone, 2000;Krone & Cooper, 2002;Atkinson et al., 2008). Despite the wide geographical distribution of birds of prey, most studies of their host-parasite associations have been carried out in European countries (Krone, 2000). Additionally, parasitism can be costly to the host, as pathological changes in tissues, immune response, and loss of nutrients may occur (Krone & Cooper, 2002;Atkinson et al., 2008). However, the possible effects of parasitism on the health of birds of prey is still largely unknown, as particular records have been identified in certain species (Lavoie et al., 1999;Krone & Cooper, 2002;Atkinson et al., 2008;Santoro et al., 2010;Vaughan-Higgins et al., 2013).
The Harris's hawk Parabuteo unicinctus (Temminck, 1824) (Accipitriformes, Accipitridae) is a raptor that is widely distributed in the Americas with two recognized subspecies: P. u. harrisi (Audubon, 1837) in North America and part of South America; and P. u. unicinctus restricted to the Neotropics (Ferguson-Lees & Christie, 2005). This raptor is widely distributed in various environments occurring in forests, agricultural lands, and also in urbanized areas with patches of arboreal cover (Pavez, 2019).
There are 35 species of birds of prey in Chile (Pavez, 2019), of which 23 have been recorded as hosts of some parasitic species. However, most of these studies focused on ectoparasites, with only 4 species reported as hosts of helminths (Moreno & González-Acuña, 2015;Oyarzún-Ruiz et al., 2016;Grandón-Ojeda et al., 2018, 2019. Additionally, parasite surveys of the Harris's hawk in Chile previously documented only one record for lice (González-Acuña et al., 2008), with no data on mites or helminths. Thus, the aim of the present study was to detail the ectoparasitic and helminth fauna of the Harris's hawk in Central and Southern Chile, establishing additional and new records for Neotropical raptors.

Materials and Methods
A total of 29 Harris's hawk carcasses from different localities ( Figure 1) were necropsied from 2017 to 2020. Eight were obtained from the Metropolitan region (San Bernardo and Providencia commune, n=1 each; and with no locality recorded, n=6), three -from O'Higgins region (San Ignacio commune, n=1; and with no locality recorded, n=2), 17 -from Ñuble region (Chillán,n=9;Cato,n=1;San Ignacio,n=3;San Carlos,n=1;Pinto,n=1;Bulnes commune,n=1;and Vegas de Itata,n=1), and one -from Los Ríos region (Valdivia) (Figure 1). All of these birds were found dead or were euthanized in rehabilitation centers for humanitarian reasons. All birds were transported for parasitological examination to the Laboratorio de Parásitos y Enfermedades de Fauna Silvestre, Facultad de Ciencias Veterinarias, Universidad de Concepción, Chillán, Chile, where they were immediately necropsied or frozen until analyses were performed. No ethical committee's approval was necessary for the use of these carcasses. The age of each bird was determined according to the chromatism of feathers (Pavez, 2019), and then confirmed through the presence/absence of the bursa of Fabricius during necropsy; the sex of each bird was determined via inspection of its gonads. Of these, 10 birds were males (2 immatures, 6 juveniles, 1 adult, and 1 with no age data), 12 were females (4 immatures, 7 juveniles, and 1 with no age data), and 7 had no age or sex data.
Additionally, between 2013 and 2017, nine Harris's hawks were screened for ectoparasites and rehabilitated at the facilities of the Centro de Rehabilitación de Fauna Silvestre (CR), Universidad de Concepción, Chillán, Chile. The skins from 22 Harris's hawks that had been deposited in the Museo Nacional de Historia Natural (MNHN), Santiago, Chile were included in the present study and inspected for ectoparasites.
The presence of ectoparasites was determined by inspecting feathers of the head, body, wings, and rectrices under a stereomicroscope. Then, using an insecticide based on pyrethrin (Cyperkill 25 EC), the "dust-ruffling" technique described by Walther & Clayton (1997) was performed to collect additional ectoparasites. To determine the presence of nasal mites, the modified Yunker's method was performed (Wilson, 1964) in the necropsied birds, as achieved through nasal flushing using a water-soap solution and rinsed with tap water. Then, the nasal sinuses, nasal turbinates, orbital cavities, and nares were exposed and inspected under stereomicroscope. All collected ectoparasites were deposited in 70% ethanol.
Parasitic necropsy was performed following the methods of Lutz et al. (2017); thus, the eyes, esophagus, gastrointestinal tract, trachea, lungs, heart, liver, gallbladder, kidneys, bursa of Fabricius, subcutaneous tissue, and articulations were examined under stereomicroscope. Every organ was dissected and/or crushed in citrated saline and then mixed using a bottle, followed by repetitive sedimentations. Once all organs were removed, the coelomic cavity was washed using the former solution. The sediment of every organ and cavity was examined under stereomicroscope. All collected helminths were relaxed in saline and preserved in 80% ethanol according to the methods of Lutz et al. (2017), and Oyarzún-Ruiz & González-Acuña (2020).
All parasites were deposited in the parasitological collection of Laboratorio de Parásitos y Enfermedades de Fauna Silvestre, Universidad de Concepción.

Results
Forty-one of the 60 inspected hawks (68.3%) were parasitized by at least one species of parasite; 17 out 29 necropsied hawks (17/29; 58.6%) were found parasitized by helminth parasites. Also, of the nine hawks screened at the CR, five were found to be parasitized: three birds with chewing lice, and the other two with feather mites. With respect to the skins deposited in the MNHN, 19 out of 22 skins were found with chewing lice and feather mites.

Discussion
Almost 70% of the inspected hawks were parasitized by at least one taxon of parasite. A total of 13 taxa was collected: four chewing lice, one feather mite, five nematodes, two platyhelminths, and one acanthocephalan, with one helminth and three ectoparasites recorded for the first time in the Neotropics.
Only one unidentified feather mite belonging to the genus Pseudalloptinus Dubinin, 1956 (Pteroichidae: Pterolichinae) was recorded. This genus currently includes six valid species, all restricted to birds of the order Accipitriformes; among them, Pseudalloptinus aquilinus (Trouessart, 1884) infesting Aquila chrysaetos (Linnaeus, 1758) and Haliaeetus leucocephalus (Linnaeus, 1766) was reported from the Holarctic and Paleotropic regions (Gaud & Mouchet, 1959;Gaud, 1988;Galloway et al., 2014;Mironov et al., 2018;Waki et al., 2021). For the Harris's hawk, an unidentified Pseudalloptinus species was previously reported without exact locality, apparently from North America, by Philips (2000). To the best of our knowledge, our finding is the first record of this genus in the Neotropics. The Pseudalloptinus species collected from the Harris's hawk in Chile is supposedly a new species and closest to P. milvulinus (Trouessart, 1884) and P. africanus Gaud, 1988 distributed on various accipitrids in the Old World. This mite differs from the two listed species in having setae ps1 strongly thickened and epimerites IV widened in males and the hysteronotal shield with a pair of deep and narrow incisions in females (Figure 2).
Microtetrameres (Travassos, 1915) inhabits the proventriculus of birds, with females strictly associated with proventricular glands and males with the proventricular mucosa (Anderson, 2000;Díaz et al., 2018). Members of the family Tetrameridae have an indirect life cycle, with arthropods such as grasshoppers and cockroaches serving as intermediate hosts (Anderson, 2000). This genus has been recorded in raptors of orders Accipitriformes, Falconiformes, and Strigiformes worldwide (Illescas Gomez et al., 1993;Krone, 2000;Sanmartín et al., 2004;Honisch & Krone, 2008;Komorová et al., 2017). In South America, there are few records for this genus, with most of them coming from passerine birds and only one record from a bird of prey. Díaz et al. (2018) recently reported a new species for the Neotropics, Microtetrameres urubitinga Díaz, Drago & Núñez, 2018, parasitizing the proventriculus of Buteogallus urubitinga (Gmelin, 1788) in Argentina. The present Microtetrameres sp. is different from M. urubitinga and the other species reported for the Neotropics and North America; thus, it probably corresponds to a new taxon. Additional analysis will be performed to determine its specific taxonomic position. The present finding represents the geographical expansion of the distribution for this genus, considering that this is the first record for Chile (see Oyarzún-Ruiz & González-Acuña, 2021). Additionally, this record represents a new host-parasite association.
Cyathostoma (H.) americana has been reported from the trachea, lungs, and air sacs of birds of prey (Borgsteede & Okulewicz, 2001;Borgsteede et al., 2003;Kanarek et al., 2016). The species of this genus have a direct life cycle, although they could use earthworms as paratenic hosts (Anderson, 2000;Atkinson et al., 2008). These nematodes were found to cause severe pneumonia and pyogranulomatous air sacculitis, which are often associated with high parasitic loads (20-100 worms) (Lavoie et al., 1999;Atkinson et al., 2008;Vaughan-Higgins et al., 2013). However, in the present study, there were no macroscopic pathological changes associated with its presence, probably because of the low parasitic load. The present finding corresponds to the second record for Syngamidae found in a wild bird from Chile (see Oyarzún-Ruiz & González-Acuña, 2021); it also represents the first record of this species in a Neotropical bird of prey. Despite the low prevalence of these respiratory nematodes, as reported in previous studies (Lavoie et al., 1999;Borgsteede & Okulewicz, 2001;Borgsteede et al., 2003;Sanmartín et al., 2004) and as described in the present survey, there is a clear need for the prospection of extraintestinal parasites, considering that most of the studies performed thus far in the country have focused on gastrointestinal parasites (Oyarzún-Ruiz & González-Acuña, 2021). Furthermore, a similar situation occurs in other surveys from the Neotropics.
Neodiplostomum Railliet, 1919 is a cosmopolitan trematode that has been reported in raptors of orders Accipitriformes, Falconiformes, and Strigiformes (Dubois, 1937;Krone, 2000;Gibson et al., 2002;Sanmartín et al., 2004;Papazahariadou et al., 2008;Richardson & Kinsella, 2010). It has an indirect life cycle, with amphibians as intermediate hosts, and reptiles and mammals as paratenic hosts (Gibson et al., 2002). Neodiplostomum travassosi is a Neotropical species recorded from diurnal and nocturnal birds of prey, including toucans and cormorants as hosts (Dubois, 1937;Lunaschi & Drago, 2005 (Lunaschi & Drago, 2005;Drago et al., 2014Drago et al., , 2015 and Brazil (Dubois, 1937). Also, an unidentified Neodiplostomum was recorded for B. magellanicus in Chile by Grandón-Ojeda et al. (2018). The identity of this last taxon is required to establish whether it belongs to the species reported here. For the Harris's hawk, there is a previous record of Neodiplostomum biovatum Dubois, 1937from Brazil (Dubois, 1937. Thus, the present finding represents a new host-parasite association and marks the expansion of the geographical distribution of this species. In the present study, a high proportion of birds -over 50% -were parasitized by helminth parasites, a situation that is aligned with the findings of previous studies of other species of raptors (Illescas Gomez et al., 1993;Krone, 2000;San Martín et al., 2006;Oyarzún-Ruiz et al., 2016;Komorová et al., 2017). In relation to the geographical expansion of parasites and the additional host-parasite associations reported here, the ectoparasites C. nanum, N. chanabense, and Pseudalloptinus sp., and helminths P. alata, Microtetrameres sp., C. (H.) americana and N. travassosi were recorded for the first time in Chile. Also, C. nanum, N. chanabense, and C. (H.) americana are recorded for the first time in Neotropical birds of prey. On the other hand, the specific status of Craspedorrhynchus sp., Pseudalloptinus sp., Microtetrameres sp., and Cladotaenia sp. is required to determine if they belong to new scientific taxa.
Most of the isolated helminths in the present study, e.g. P. depressum, P. alata, Microtetrameres sp., C. (H.) americana, Centrorhynchus sp., N. travassosi, and Cladotaenia sp., have indirect life cycles, using invertebrates such as earthworms and arthropods or rodents as intermediate hosts; others, such acanthocephalans, use amphibians and reptiles as paratenic hosts (Yamaguti, 1959;Anderson, 2000;Atkinson et al., 2008;Richardson & Kinsella, 2010). The Harris's hawk is an opportunist, consuming not only medium and small mammals, reptiles, or birds, but also invertebrates (Pavez, 2019). Thus, this dietary behavior may explain the presence of a diversity of heteroxenous helminths reported here.
Raptors seem to be tolerant to the presence of helminth parasites; however, under certain circumstances, such as stress, there could be severe health impairment on these hosts (Illescas Gomez et al., 1993;Krone, 2000;Krone & Cooper, 2002;Santoro et al., 2010;Komorová et al., 2017). Although some isolated helminths have reportedly been the cause of disease (e.g., Cyathostoma, Cladotaenia, and Centrorhynchus) with evident lesions (Lavoie et al., 1999;Krone, 2000;Atkinson et al., 2008;Santoro et al., 2010;Vaughan-Higgins et al., 2013), the birds necropsied in our study did not show any signs of pathological lesions associated with the parasites. This finding could have been associated with the low parasitic load recorded here. On the other hand, the highest parasitic load recorded was for N. travassosi. Although there are no data on the pathological consequences of this fluke species, these parasites are generally considered non-pathogenic for birds (Atkinson et al., 2008).
The inclusion of deposited specimens from a museum (MNHN) in this study demonstrated the utility of such collaboration, as two additional ectoparasitic species, which were not found on the necropsied birds nor those from CR, were collected from these samples. Thus, future studies on the parasites obtained from wild animals should include deposited specimens. However, caution must be considered because of the possibility of contamination with ectoparasites from other bird skins is possible.