INTRODUCTION
Magellanic penguins (Spheniscus magellanicus) are native to Argentina, Chile and the Falkland (Malvinas) Islands. The species’ population size is estimated between 1·2 and 1·6 million breeding pairs distributed in a minimum of 138 colonies (Boersma et al. Reference Boersma, Frere, Kane, Pozzi, Pütz, Raya-Rey, Rebstock, Simeone, Smith, Van Buren, Yorio, García-Borboroglu, García-Borboroglu and Boersma2014). The Argentinean Patagonian coast is the most critical habitat for the species, concentrating approximately 75% of its population in 63 breeding colonies (Birdlife International, 2012).
Blood parasites are relevant pathogens to penguins, with a potential to cause substantial morbidity and mortality (Vanstreels et al. Reference Vanstreels, Braga and Catão-Dias2016a ). Because of their obligatory heteroxenous life cycle, the spatiotemporal distribution of protozoan blood parasites is inherently related to that of their invertebrate vectors and, as a result, is strongly influenced by climatic factors (Rogers and Randolph, Reference Rogers and Randolph2000; Harvell et al. Reference Harvell, Mitchell, Ward, Altizer, Dobson, Ostfeld and Samuel2002; Garamszegi, Reference Garamszegi2011). In particular, the mosquito-borne Plasmodium spp. (avian malaria) are considered significant pathogens to Magellanic penguins due to their well-documented ability to cause rapid outbreaks with high mortality in individuals held in captivity (Vanstreels et al. Reference Vanstreels, Braga and Catão-Dias2016a ).
Plasmodium spp. have been extensively reported infecting Magellanic penguins in captivity worldwide (Fix et al. Reference Fix, Waterhouse, Greiner and Stoskopf1988; Tollini et al. Reference Tollini, Brocksen and Sureda2000; Ko et al. Reference Ko, Kang, Jung, Bae and Kim2008; Bueno et al. Reference Bueno, Lopez, Menezes, Costa-Nascimento, Lima, Araújo, Guida and Kirchgatter2010; Vanstreels et al. Reference Vanstreels, Capellino, Silveira, Braga, Rodríguez-Heredia, Loureiro and Catão-Dias2016b ) and in wild specimens undergoing rehabilitation in Chile (Carvajal and Alvarado, Reference Carvajal and Alvarado2009) and Brazil (Silveira et al. Reference Silveira, Belo, Lacorte, Kolesnikovas, Vanstreels, Steindel, Catão-Dias, Valkiūnas and Braga2013; Cabana et al. Reference Cabana, Vanstreels, Xavier, Osório, Adornes, Leite, Soares, Silva-Filho, Catão-Dias and Meireles2014; Campos et al. Reference Campos, Pires, Nascimento, Dutra, Torres-Filho, Toma, Brener and Almosny2014; Vanstreels et al. Reference Vanstreels, Kolesnikovas, Sandri, Silveira, Belo, Ferreira-Junior, Epiphanio, Steindel, Braga and Catão-Dias2014, Reference Vanstreels, Silva-Filho, Kolesnikovas, Bhering, Ruoppolo, Epiphanio, Amaku, Ferreira-Junior, Braga and Catão-Dias2015). However, Plasmodium has not been detected in wild Magellanic penguins, nor have any of the other blood parasites that infect other species of penguins, which comprise Babesia, Borrelia, Haemoproteus, Leucocytozoon, Trypanosoma and nematode microfilariae (Vanstreels et al. Reference Vanstreels, Braga and Catão-Dias2016a ). It is not clear whether the absence of records of blood parasites in wild Magellanic penguins is reflective of their true absence or is instead related to an insufficiency in sampling effort and/or a failure of the diagnostic methods employed to detect them.
In this study, we investigate the occurrence of blood parasites in Magellanic penguins on the northern Argentinean Patagonian coast, combining traditional (blood smear examination) and molecular methods [nested polymerase chain reactions (PCR) targeting Babesia, Haemoproteus, Plasmodium and Leucocytozoon]. Furthermore, we evaluate the climatogeography of the natural distribution of Magellanic penguins in relation to studies on the occurrence of blood parasites, aiming to identify climatic patterns that can be used to predict the occurrence of such parasites.
METHODS
Sample collection
All procedures were authorized by local authorities (99/2011-DFyFS-SRRN, N° 083 SsCyAP/12) and approved by the Animal Ethics Committee of the University of São Paulo (CEUA-USP 601415). Sample collection occurred during two expeditions (24–27 January 2012 and 21–25 January 2014; i.e. late chick-rearing) to four breeding colonies in the Argentinean province of Chubut (Table 1). Two hundred and eighty-four non-moulting adult Magellanic penguins were caught at the colonies and manually restrained; sampled individuals presented no external signs of illness or lesions. Blood samples (<0·5% of body mass) were collected through venipuncture of the jugular vein with a heparinized syringe, then birds were marked with a temporary stain (to prevent recapture) and released back to where they had been caught. Blood was then transferred to heparin tubes and kept in a cool container.
Number of samples analysed in this study
Laboratory procedures
Within 3–6 h after collection, blood samples were homogenized then used to prepare two thin blood smears and 1 mL of heparinized blood was frozen in liquid nitrogen (−196 °C) and later transferred to a −80 °C freezer. Blood smears were air-dried, then fixed with methanol; within 1–4 days, one slide was stained with 8% Giemsa and another with Wright-Rosenfeld (Rosenfeld, Reference Rosenfeld1947). One slide from each individual was examined for intracellular and extracellular blood parasites in 150 fields under 1000× magnification (approx. 20–25 min per slide; field of view area = 0·126 mm2) by an experienced observer (R.E.T. Vanstreels).
Samples were pooled into 96 triplets, and DNA extraction was conducted using the DNEasy Blood and Tissue Kit (69506, Qiagen – Valencia, USA) and was verified and quantified through UV spectrophotometry (Nanodrop 1000, Thermo Fisher Scientific – Waltham, USA). Using the protocols detailed by Vanstreels et al. (Reference Vanstreels, Silva-Filho, Kolesnikovas, Bhering, Ruoppolo, Epiphanio, Amaku, Ferreira-Junior, Braga and Catão-Dias2015) with no modifications, we conducted nested PCR targeting: (a) the mitochondrial cyt-b gene of Haemoproteus and Plasmodium (using primers HaemNFI/HaemNR3 and HaemF/HaemR2 originally described by Hellgren et al. Reference Hellgren, Waldenström and Bensch2004 and Waldenström et al. Reference Waldenström, Bensch, Hasslequist and Östman2004), (b) the mitochondrial cyt-b gene of Leucocytozoon (using primers HaemNFI/HaemNR3 and HaemFL/HaemR2L originally described by Hellgren et al. Reference Hellgren, Waldenström and Bensch2004), and (c) the 18S rRNA gene of Babesia (using primers Bab5·1/BabB and RLBF/RLBR originally described by Medlin et al. Reference Medlin, Elwood, Stickel and Sogin1988 and Gubbels et al. Reference Gubbels, de Vos, van der Weide, Viseras and Schouls1999). Each reaction was conducted with positive controls for Plasmodium, Leucocytozoon and Babesia, as well as a negative control (chicken raised in an arthropod-free environment). Gel electrophoresis was conducted to visualize amplification products, using 2% agarose gel and SYBR Safe (Invitrogen S33102, Life Technologies – Carlsbad, USA).
Estimated true prevalence
Because sampling and diagnostic tests are imperfect, we estimated the highest bound of the true prevalence (i.e. the highest prevalence at which parasites could have occurred but gone undetected) for our sampling effort as well as previous studies on wild Magellanic penguins. For this purpose, the Blaker's exact confidence interval was calculated (Reiczigel et al. Reference Reiczigel, Földi and Ózsvári2010; Sergeant, Reference Sergeant2016) based on the sample size of each study and the diagnostic method employed. Because test sensitivity has been estimated between 72 and 81% for thin blood smears and between 64 and 89% for PCR (Richard et al. Reference Richard, Sehgal, Jones and Smith2002; Valkiūnas et al. Reference Valkiūnas, Zehtindjiev, Dimitrov, Križanauskienė, Iezhova and Bensch2008), we used the following values of test sensitivity as worst-case and best-case scenarios, respectively: 70 and 80% for studies employing only blood smears, 65 and 90% for studies employing only PCR, and 80 and 90% for studies combining both methods. Test specificity was fixed at 100% to produce the most conservative estimates, and confidence level was fixed at 95%.
Geospatial and climate analyses
Maps and climatograms of the historical climate normals of South America (data from 1950 to 2000, depending on the region) were prepared using published data (Hajek and Di Castri, Reference Hajek and Di Castri1975; Fontannaz, Reference Fontannaz2001; Hijmans et al. Reference Hijmans, Cameron, Parra, Jones and Jarvis2005; Ramos et al. Reference Ramos, Santos and Fortes2009; SMN, 2016). The natural distribution of Magellanic penguins was based on Birdlife International (2012), but was extended to Rio de Janeiro and Espírito Santo states as ‘vagrant’ considering the several hundred individuals that have regularly stranded in that region in recent years (L.F.S.P. Mayorga, personal communication).
We compared the historical climate parameters between locations where Plasmodium was recorded in captive Magellanic penguins and locations where wild Magellanic penguins were studied for blood parasites. For this purpose, two-tailed Mann–Whitney tests were used to compare average annual highest temperature, mean daily temperature during hot semester (October–March), mean daily temperature during cold semester (April–September), average annual lowest temperature, and mean annual precipitation, as obtained from the historical climate normals datasets.
RESULTS
No blood parasites were seen in blood smears, and all samples were negative in the nested PCR tests targeting Haemoproteus/Plasmodium, Leucocytozoon or Babesia. Figures 1 and 2 compare the geographic distribution and the climate of locations where investigations on the occurrence of blood parasites in Magellanic penguins have been conducted. Table 2 summarizes the sampling and laboratory details of this and previous studies on wild specimens, providing the estimated true prevalence for each study.
Geographic distribution of studies investigating the occurrence of blood parasites in Magellanic penguins in relation to the species’ natural distribution and historical precipitation data.
Comparison of the historical climate normals of different sites in relation to the occurrence of Plasmodium in Magellanic penguins. Climatograms present the average maximum (red line), mean daily (dashed grey line) and average minimum (blue line) temperature (in Celsius degrees, left axis) and the average monthly precipitation (blue bars; in millimetres, right axis).
Summary of studies attempting to detect blood parasites in wild Magellanic penguins
TBS, thin blood smear; PCRHP, PCR targeting Haemoproteus and Plasmodium; PCRL, PCR targeting Leucocytozoon; PCRB, PCR targeting Babesia. References: (1) Hawkey et al. Reference Hawkey, Horsley and Keymer1989; (2) Keymer et al. Reference Keymer, Malcolm, Hunt and Horsley2001; (3) Jovani et al. Reference Jovani, Tella, Forero, Bertellotti, Blanco, Ceballos and Donázar2001; (4) Tella et al. Reference Tella, Forero, Bertellotti, Donázar, Blanco and Ceballos2001; (5) Moreno et al. Reference Moreno, Yorio, Garcia-Borboroglu, Potti and Villar2002; (6) Quillfeldt et al. Reference Quillfeldt, Martínez, Hennicke, Ludynia, Gladbach, Masello, Riou and Merino2010; (7) Sallaberry-Pincheira et al. Reference Sallaberry-Pincheira, González-Acuña, Herrera-Tello, Dantas, Luna-Jorquera, Frere, Valdés-Velasquez, Simeone and Vianna2015; (8) This study.
Locations where Plasmodium was recorded in captive Magellanic penguins differed from locations where studies failed to identify blood parasites with regards to: mean daily temperature during hot semester (respectively 21·4 vs 13·8 °C; P = 0·007), mean daily temperature during cold semester (16·6 vs 7·1 °C; P < 0·001), average annual lowest temperature (11·5 vs 1·0 °C; P < 0·001) and mean annual precipitation (1342·2 vs 300·2 mm; P < 0·001). On the other hand, no difference was identified in relation to average annual highest temperature (27·7 vs 21·7 °C; P > 0·9).
DISCUSSION
To date, the southernmost records of blood parasites in captive penguins in South America are cases of Plasmodium spp. infections in Valdivia, Chile (39°49′S) (Carvajal and Alvarado, Reference Carvajal and Alvarado2009), and San Clemente del Tuyú, Argentina (36°20′S) (Vanstreels et al. Reference Vanstreels, Capellino, Silveira, Braga, Rodríguez-Heredia, Loureiro and Catão-Dias2016b ), whereas all studies have failed to detect blood parasites in wild Magellanic penguins sampled south of 40°S (Fig. 1). One could therefore be led to suspect that a latitudinal temperature gradient might be the main constraint to the occurrence of blood parasites in wild Magellanic penguins. However, because haemosporidians (Haemoproteus, Plasmodium and Leucocytozoon) have been reported in South American forest birds as far south as Navarino Island (54°56′S) (Merino et al. Reference Merino, Moreno, Vásquez, Martínez, Sánchez-Monsálvez, Estades, Ippi, Sabat, Rozzi and McGehee2008), where daily mean temperatures range between 1·8 and 9·8 °C throughout the year (Hajek and Di Castri, Reference Hajek and Di Castri1975), it is clear that temperature is not per se the constraint to the occurrence of these parasites. Instead, the combination of strong winds and the scarcity of freshwater on the Atlantic Patagonian coast might be the key factor driving the lack of haemosporidian in Magellanic penguins at that region.
The combined study effort conducted along the Atlantic Patagonian coast, especially with the recent studies employing high-sensitivity nested PCR tests, provides strong basis to conclude that haemosporidians are absent or near-absent in wild Magellanic penguins in that region. However, it is worth noting that sampling efforts to date have been predominantly focused on the Argentinean Patagonia and the Magellan strait (Fig. 1), a remarkably arid region, with average rainfall generally lower than 300 mm per year, strong winds and scarce freshwater. The emphasis in sampling Magellanic penguins in this region is not surprising considering it is where most of the species’ population is concentrated (Birdlife International, 2012). However, the very limited sampling effort on the Pacific Patagonian coast and the Falkland Islands precludes a categorical conclusion that haemosporidian parasites do not infect Magellanic penguins in the wild.
The Chilean Patagonian coast has a remarkably diverse climate, with generally higher rainfall than its Argentinean counterpart (>1000 mm/year, see Figs 1 and 2), and ecological modelling indicates it provides a more favourable environment for dipteran vectors (WRBU, 2016). Albeit relatively small for Magellanic penguin standards, the breeding colonies in Chile congregate several hundreds to thousands of pairs (Boersma et al. Reference Boersma, Frere, Kane, Pozzi, Pütz, Raya-Rey, Rebstock, Simeone, Smith, Van Buren, Yorio, García-Borboroglu, García-Borboroglu and Boersma2014), representing a significant fraction of the species’ population. Similarly, the Falkland Islands are not as dry as the Argentinean Patagonian coast, with an average rainfall of 680 mm/year. Although the archipelago is reportedly mosquito-free (Medlock et al. Reference Medlock, Schaffner and Fontenille2010), a Plasmodium sp. infection was recently identified in a thin-billed prion (Pachyptila belcheri) breeding on New Island (Quillfeldt et al. Reference Quillfeldt, Martínez, Hennicke, Ludynia, Gladbach, Masello, Riou and Merino2010). Additional studies with extensive sampling of wild Magellanic penguins on the Chilean Patagonian coast and the Falkland Islands are therefore warranted.
With regards to Babesia, we did not find evidence of this parasite in wild Magellanic penguins. However, it is still early to conclude whether this parasite is present or absent on the Argentinean Patagonian coast, since this was the first study to employ molecular methods for its diagnosis. Ixodes spp. ticks are thought to be responsible for the transmission of Babesia to penguins (Cunningham et al. Reference Cunningham, Gibbs, Rogers, Spielman and Walraven1993; Earlé et al. Reference Earlé, Huchzermeyer, Bennett and Brossy1993; Montero et al. Reference Montero, González, Chaparro, Benzal, Bertellotti, Masero, Colominas-Ciuró, Vidal and Barbosa2016), and Ixodes uriae has been sporadically recorded on seabirds in Argentina, Chile and the Falkland Islands (Muñoz-Leal and González-Acuña, Reference Muñoz-Leal and González-Acuña2015). While I. uriae does not seem to be a common parasite of wild Magellanic penguins, it can clearly thrive in penguin colonies at harsh environmental conditions such as those of the southern tip of South America (Barbosa et al. Reference Barbosa, Benzal, Vidal, D'Amico, Coria, Diaz, Motas, Palacios, Cuervo, Ortiz and Chitimia2011; Muñoz-Leal and González-Acuña, Reference Muñoz-Leal and González-Acuña2015) and therefore the occurrence of tick-borne blood parasites should be further investigated throughout the breeding distribution of Magellanic penguins.
It is worth noting that besides these broader trends in climatic and geographic distribution, other factors may also affect the detection of blood parasites in seabirds. Because most studies on wild Magellanic penguins have targeted the sampling of non-moulting apparently healthy adults, it is possible that blood parasites were not detected because they are confined to other life stages. Previous studies have shown that blood parasite infections tend to be more frequent and more acute in penguin chicks as they approach fledging and in adult penguins during moult (Fallis et al. Reference Fallis, Bisset and Allison1976; Allison et al. Reference Allison, Desser and Whitten1978; Hill et al. Reference Hill, Howe, Gartrell and Alley2010; Argilla et al. Reference Argilla, Howe, Gartrell and Alley2013), and future studies on Magellanic penguins might therefore benefit from sampling these life stages.
Perspectives for climate change and distribution shifts
Even if future studies conclude that blood parasites are absent in wild Magellanic penguins throughout the species’ distribution, there is evidence to indicate this could change in the future. A recent northward expansion of the population of Magellanic penguins has been noted in Argentinean Patagonia, with new colonies being established and rapidly increasing (Gómez-Laich et al. Reference Gómez-Laich, Wilson, Sala, Luzenti and Quintana2015; Pozzi et al. Reference Pozzi, García-Borboroglu, Boersma and Pascual2015). For example, the Punta Norte/San Lorenzo colony (42°04′S) was founded in 1977 and rapidly escalated to become one of the species’ largest colonies, with more than 134 000 breeding pairs by 2008. Similarly, small northern colonies established in the last decade such as Complejo Islote Lobos (41°26′S, founded in 2002) and El Pedral (42°56′S, founded in 2009) have speedily grown, contrasting with the stable or declining trends of some of the larger breeding colonies in central Patagonia (Wilson et al. Reference Wilson, Scolaro, Grémillet, Kierspel, Laurenti, Upton, Gallelli, Quintana, Frere, Müller, Straten and Zimmer2005; Pozzi et al. Reference Pozzi, García-Borboroglu, Boersma and Pascual2015). This northward distributional shift of the Magellanic penguin, which is possibly linked to a decreased prey availability in the central Argentinean coast (Gómez-Laich et al. Reference Gómez-Laich, Wilson, Sala, Luzenti and Quintana2015; Pozzi et al. Reference Pozzi, García-Borboroglu, Boersma and Pascual2015), could lead the species to breed in areas that are suitable for mosquitoes and other vectors.
On the other hand, recent studies indicate a poleward extension of the distribution of dipteran insects, and there is evidence of a distribution shift of mosquito-borne pathogens in response to climate change (Rogers and Randolph, Reference Rogers and Randolph2000; Harvell et al. Reference Harvell, Mitchell, Ward, Altizer, Dobson, Ostfeld and Samuel2002; Garamszegi, Reference Garamszegi2011). In this scenario, the clash between the northward expansion of Magellanic penguins and the southward expansion of mosquitoes could provide novel opportunities for the transmission of blood parasites on the northern Argentinean Patagonian coast, with potentially grave consequences for this species’ conservation.
Additionally, even in regions with arid and mosquito-adverse climate it is possible that human presence near penguin colonies – in the form of towns, settlements, farms, ranches, tourist visitation centers, resorts, park ranger stations, etc. – may provide micro-environments of wind protection and freshwater availability that favour the thriving of dipteran vectors. A troubling example of this is the recent increase in mosquito and fly populations in the coastal Argentinean city of Puerto Madryn (42°46′S), which prompted a fumigation program to prevent outbreaks of vector-borne diseases that may pose a risk to public health (MPM, 2009, 2016). It is therefore urgent to conduct studies examining the presence of invertebrates and synanthropic birds in areas where Magellanic penguins breed within close proximity to human settlements or facilities, to evaluate the potential for blood parasite transmission.
ACKNOWLEDGEMENTS
We are grateful to Felix Capellino, Gastón Delgado, Sergio Heredia, Julio Loureiro, David Verón, Cecilia Decker, Mónica Jacobsen, Leonhard Schnittger, Marcos Amaku, Susan Kutz, Claudia Niemeyer, Luciana Gallo, Matias Di Martino, Lucas Beltramino, Jorge Oyakawa, Daniela Debone, Luana Ortolan, Michelle Sercundes, Luis Felipe S. P. Mayorga, Nola Parsons, Lisa Nupen for their valuable contributions. This study was supported by Laboratório de Patologia Comparada de Animais Selvagens da Universidade de São Paulo, Fundación Mundo Marino, Instituto de Patobiología del Instituto Nacional de Tecnología Agropecuária, Wildlife Conservation Society and the One Health Institute at University of California, Davis.
FINANCIAL SUPPORT
Financial support was provided by Fundação de Amparo à Pesquisa do Estado de São Paulo (grant numbers FAPESP 2009/53956-9 and 2010/51801-5) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
