Parasites of the Neotropic cormorant Nannopterum (Phalacrocorax) brasilianus (Aves, Phalacrocoracidae) in Chile

Braz J Vet Parasitol 2020; 29(3): e003920 | https://doi.org/10.1590/S1984-29612020049 This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Parasites of the Neotropic cormorant Nannopterum (Phalacrocorax) brasilianus (Aves, Phalacrocoracidae) in Chile


Introduction
The Neotropic cormorant Nannopterum (Phalacrocorax) brasilianus (Suliformes: Phalacrocoracidae) is a widely distributed bird of Central and South America, reaching Mexico and the southern areas of the United States (Kennedy & Spencer, 2014;IUCN, 2017). It feeds on crustaceans, small fish, amphibians, and insects in different aquatic habitats with fresh, brackish, or saltwater (IUCN, 2017). In Chile, the Neotropic cormorant is distributed in marine coasts, lakes, and rivers from Arica (18°28'30"S, 70°18'52"W) to Tierra del Fuego (53°36'00"S, 69°23'00"W). It comprises two subspecies N. (P.) b. brasilianus and N. (P.) b. hornensis, with the latter being distributed along the Beagle Channel and Cape Horn in southern Chile (Chester, 2008). As established by the Chilean Hunting Law N° 19.473, the Neotropic cormorant is a pest bird in some regions of northern Chile (SAG, 2018).
Currently, the Neotropic cormorant does not face conservation issues, having an increasing population of more than 2 million individuals and a large distributional range (IUCN, 2017). However, it is still important to elucidate parasite species that might be affecting the health of Neotropic cormorants, particularly younglings, which are more susceptible to develop disease due to parasitism (Kuiken et al., 1999;Torres et al., 2005). Most surveys of the parasitic fauna of the Neotropic cormorant have been performed in Brazil, Argentina, Mexico, and the United States (Vicente et al., 1996;Fedynich et al., 1997;Drago et al., 2011;Monteiro et al., 2011). Information about its parasites in Chile is restricted to studies performed by Torres et al. (1982Torres et al. ( , 1991Torres et al. ( , 1992Torres et al. ( , 1993Torres et al. ( , 2000Torres et al. ( , 2005 and Garbin et al. (2011).
There are no reports of protozoa parasites in the Neotropic cormorants or any other cormorant species in Chile. However, there are records of Toxoplasma gondii, Cryptosporidium sp., Giardia sp., Entamoeba sp., Eimeria sp., and Sporozoa (Apicomplexa) oocyst in the great (Phalacrocorax carbo) and flightless cormorants (Phalacrocorax harrisi) (Medema, 1999;Plutzer & Tomor, 2009;Deem et al., 2010;Carrera-Jativa et al., 2014;Víchová et al., 2016;Rzymski et al., 2017). Certain fish species act as reservoirs of Cryptosporidium sp. (Gabor et al., 2011), and Giardia sp. (Ghoneim et al., 2012), underlining the role that cormorants might play as reservoirs of protozoan parasites due to their elevated fish consumption (Carss, 1997). Nonetheless, the importance that the Neotropic cormorant has in the maintenance of parasites in terrestrial and aquatic environments remains to be determined.
Palavras-chave: Helmintos, parasitas externos, parasitas internos, pássaro, piolhos. Garbin et al. (2011), in which only the presence of nematodes and other helminths was reported. Due the current lack of knowledge about the parasitic fauna of Neotropic cormorants in Chile, this study aimed to document new records of the ecto-, endoparasites, and protozoa of this widely distributed bird in the country. Additionally, most cormorants included in this study were residents of Talcahuano and San Vicente Bay, both areas characterized by the presence of estuarine wetlands and marshes, which are usually visited by resident and migratory birds to rest and feed (León & Benítez-Mora, 2005;García-Walther et al., 2017). Encounters among diverse bird species in these areas provide opportunities for the transmission of different parasite groups (Bjoersdorff et al., 2001), highlighting the importance of determining parasitic organisms in these birds to better understand parasite-host interactions in the established ecological communities.  (Figure 1). Cormorants collected from Talcahuano and San Vicente (Chile) in 2007 died because of an oil spill event that occurred in the area during the same year. All other carcasses examined in this study were donated by hunters. The Chilean Hunting Law N° 19.473, Article N 5°, Supreme decree N°5, allows hunting of Neotropic cormorants along its distributional range from April to August. All carcasses were brought to the Department of Animal Science, Universidad de Concepción, Chillán, and kept stored at -40 °C for future examination. Cormorants were externally inspected with a magnifier in search of ectoparasites. Feathers of the wings, tail, head, neck, flanks, back, and abdomen were closely examined. As birds were dead when they were collected, it is possible that ectoparasites may have abandoned their hosts after their death. Following collection, lice were cleared using 20% KOH and ascending concentrations of ethanol solutions (40%, 70%, and 96%) and were subsequently mounted using Canada balsam, as indicated in Palma (1978) and Price et al. (2003). Ectoparasites were identified using the keys provided in Giebel (1866), Keler (1938), Ryan & Price (1969), Clay (1973), Price et al. (2003), and Kuabara & Valim (2017).

Eighty
Birds were necropsied using the modified Withlock technique (Cattán & Tagle, 1974). Digestive and respiratory organs were inspected in search of endoparasites and some segments of the intestine were observed with the stereomicroscope (20x and 40x) in order to collect parasites that could have been adhered to the mucous membrane. Endoparasites were stored following methods in Pritchard & Kruse (1982). Trematoda were preserved in 70% ethyl alcohol and stained with carmine and alum carmine stains for identification. Acanthocephalans and cestodes were maintained in water for 10 min and then kept in 10% buffered formalin and a mix of 70% ethyl alcohol and 10% lactic acid, respectively. Nematoda were preserved in 70% ethyl alcohol and then fixated with glycerin in 50-100 mL flasks (Oyarzún-Ruiz & González-Acuña, 2020). Endoparasites were identified using keys and descriptions in Skrjabin et al. (1954), Skrjabin (1961Skrjabin ( , 1969, Yamaguti (1958Yamaguti ( , 1959Yamaguti ( , 1961, and Khalil et al. (1994). A fecal sample was obtained from the distal rectum of each bird in order to search for intestinal protozoa using the flotation technique described in Boch & Supperer (1982).
The terminology used to describe parasitological assemblage descriptors (prevalence, mean intensity, and mean abundance) follows that of Bush et al. (1997). 'Range' displays the minimum and maximum number of individuals of a parasite species collected from the least and most infested hosts, respectively.

Results and Discussion
Ectoparasites Thirty-four (42.5%) birds were parasitized by at least one species of ectoparasite. Eidmaniella pelucida Rudow, 1869 ( Figure 2a) and Pectinopygus gyroceras ( Figure 2b) were identified as lice species in the Neotropic Cormorant. The ratios of nymph/adult and male/female for P. gyroceras were 0.81 and 1.67, respectively (70 females, 42 males and 91 nymphs). Only a single individual E. pelucida was collected. Information about population parameters for P. gyroceras can be found in Table 1. Information about both ectoparasite species found in this study is extremely sparse. Eidmaniella pelucida was recorded in Phalacrocorax capensis and P. carbo in North America, P. carbo in Spain, and Leucocarbo bougainvillii (Phalacrocoracidae) in Peru (Emerson, 1947;Dale, 1970;Mateo, 2006). Similarly, P. gyroceras has been previously described in N. (P.) brasilianus in the United States and Brazil (Malcomson, 1960;Kuabara & Valim, 2017). This is the first time that E. pelucida is described in the Neotropic cormorant and expands the distributional range of P. gyroceras to Chile.
It is important to highlight that pollution with hydrocarbons could have resulted in a less intense infestation with ectoparasites in cormorants from Talcahuano and San Vicente Bay. This, because hydrocarbons adhere to feathers and cause a should be disruption of the water repellent properties of birds' plumage (Jenssen, 1994), allowing polluted water to cover feathers and remove ectoparasites. Moreover, affected feathers lose their ability to provide insulation and leads to hypothermia in birds, which might have caused ectoparasites to abandon their now dead hosts before carcasses arrived at the university for inspection. Parasites could have also been washed away when cleaning birds affected by the oil. All these effects of hydrocarbons could have led to a sub-estimation of ectoparasite abundance, intensity, and prevalence.

Endoparasites
From the 80 birds examined, 77 (96.25%) were infected with at least one species of endoparasite. A total of 9566 parasites were collected in total: 5455 Trematoda (57.02%), 2979 Nematoda (31.14%), 911 Acanthocephala (9.52%), and 221 Cestoda (2.31%). No protozoan parasites were detected in this study during faecal examination. Previously, protozoan from the genera Giardia and Cryptosporidium have been informed in feces from P. carbo through microscopy (Medema, 1999;Rzymski et al., 2017) and PCR (Plutzer & Tomor, 2009). It is important to consider that the absence of protozoa oocysts and eggs observed in this study does not necessarily mean that birds were free of these parasites, but could be a result of factors associated with the low sensitivity of the flotation technique and/or low infestation rate (Zajac & Conboy, 2012). It is recommended for future studies to employ more sensitive techniques, such as molecular methods, to precisely diagnose protozoan parasite infection (Plutzer & Tomor, 2009).

Trematoda
Trematode species Hysteromorpha triloba (Figure 3a) and Ascocotyle felippei (Figure 3b) were identified and were isolated from two and ten cormorants, respectively. Hysteromorpha triloba was the most abundant parasite with 4784 individuals found in a single bird. Information about population parameters for Trematoda recorded in this study can be found in Table 1.

H. triloba
Hysteromorpha triloba is a cosmopolitan parasite that infects snails and fishes as first and second intermediate hosts, respectively, reaching piscivorous birds as their definitive hosts (Hugghins, 1954a, b). It is commonly found in cormorants, being described in different cormorant species and found in several countries. For instance, it has been reported in Nannopterum (Phalacrocorax) auritus in the United States and Canada (Chandler & Rausch, 1948), Microcarbo melanoleucos and Phalacrocorax fuscescens in Australia (Johnston, 1942), and P. carbo in Mongolia, Ukraine, Czech Republic, Curonian Lagoon Area (Russia and Lithuania), India, Japan, Australia, and Poland (Yamaguti, 1939;Johnston, 1942;Gupta, 1963;Našincová et al., 1993;Kanarek et al., 2003;Kornyushin, 2008;Švažas et al., 2011;Lebedeva & Chantuu, 2015). Hysteromorpha triloba has been previously recorded in the Neotropic cormorant in Argentina, Brazil, and the United States (Fedynich et al., 1997;Drago et al., 2011;Monteiro et al., 2011), however, this is the first time that it is reported in the Neotropic cormorant in Chile.

Cestoda
Paradilepis caballeroi (Figure 3c, d) was the only cestode isolated from cormorant carcasses. Information about population parameters for the species in this study can be found in Table 1.

Nematoda
Five nematode species were isolated from Neotropic cormorants' carcasses, Anisakis sp. (Figure 4a Anisakis sp. and C. rudolphii s. l. has been previously recorded in the Neotropic cormorant and C. (C.) phenisci has been described for the Imperial shag in Chile (Torres et al., 1982(Torres et al., , 2005Oyarzún-Ruiz & Muñoz-Alvarado, 2015). This is the first report of C. (C.) phenisci in the Neotropic cormorant. Information about population parameters for Nematoda recorded in this study can be found in Table 1.

B. carbonis
Baruscapillaria carbonis is a specialist parasite of cormorants and it is suggested that fish may play an important role in its development and transmission (Frantová, 2001). Baruscapillaria carbonis has been described in P. carbo and Microcarbo pygmaeus in the European continent (Baruš & Sergeeva, 1990;Frantová, 2001;Sitko & Okulewicz, 2010;Kanarek & Zaleśny, 2014). Baruscapillaria carbonis has been reported in other fish-eating hosts in the Palearctic region, but those records are considered dubious as they were not properly described (Baruš et al., 1978;Moravec et al., 1994). This study is the first record of B. carbonis in the Neotropic cormorant.
Species of the Avioserpens genus usually occur in the subcutaneous tissue of piscivorous birds (Gibson, 1973;Wang et al., 1983). Nine species have been recognized in different domestic and wild birds, most of them in the Northern Hemisphere. For instance, Avioserpens mosgovoyi Supryaga, 1965 was identified in Podiceps cristatus, Figure 4. (a) View of anterior end of Anisakis sp. larva showing the presence of boring tooth (arrow) and lips forming medial bilobed process without interlabia (250x magnification); (b, c) Photomicrographs of Avioserpens sp. displaying the (b) anterior view end bearing two double cephalic papillae (arrows) (40x magnification); (c) and magnification of a cephalic papillae (500x magnification); (d) View of an individual from Cyathostoma (Cyathostoma) phenisci showing its anterior end with a wide buccal capsule bearing six teeth at its basis (arrow) and its muscular esophagus dilated at its caudal third (arrowhead) (400x magnification); (e) View of posterior end from a male Contracaecum rudolphii s. l. displaying its long and thin pair of subequal spicules (arrow) (400x magnification).
In this study, a total of 13 parasite morphs were collected from Neotropic cormorant, 11 of which were described to the species level. Most of these descriptions are new records for Chile. Lice P. gyroceras and E. pellucida, trematodes A. felippei and H. triloba, nematodes C. rudolphii s. l., B. carbonis, and C. (C.) phenisci, cestode P. caballeroi, and acanthocephalans A. phalacrocoracis, C. arctocephali, and P. altmani are, in most cases, parasite species distributed in different geographical areas of the American continent. There are some species, such as H. triloba, that are cosmopolitan and have been recorded in a wide range of hosts. Although this study describes their presence in Chile for the first time, it is likely that they have been present in the country since the establishment of parasite-host associations with local wildlife. Conversely, it is also possible that some of these species were introduced into the country through parasitized migratory birds that arrive seasonally to spend the summer in the Southern Figure 5. (a) Andracantha phalacrocoracis showing its cylindrical proboscis swollen at its posterior half armed with several longitudinal rows of small hooks, neck lacking hooks (arrow heads), and trunk covered with small spines (asterisks) (400x magnification); (b) Profilicollis altmani with its typical spherical proboscis covered with several rows of small hooks and lacking hooks in the neck (400x magnification); (c) Corynosoma arctocephali showing its proboscis constricted at its middle area (arrow) and armed with larger hooks (asterisks) in comparison to hooks present in the anterior and posterior areas of the proboscis (400x magnification).
Hemisphere (Martínez & González, 2017). Nonetheless, molecular studies are necessary in order to test this latter hypothesis, which will most likely involve the application of molecular tools to study parasite populations in their different distribution areas.