INTRODUCTION
Birds, mammals and reptiles harbour intracellular haemoparasites (i.e. plasmodiids, haemogregarines), which can cause serious damages to their hosts (Davies and Johnston, Reference Davies and Johnston2000; Goater et al. Reference Goater, Goater and Esch2014). However, in comparison with other vertebrates, the amount of studies on blood parasites of reptiles is comparatively small (Telford, Reference Telford2008). In many reptiles, it is common to find infection by haemoparasitic apicomplexan, a group of blood-borne intracellular sporozoans that includes, among others, the genera Hepatozoon, Plasmodium and Haemoproteus (Jacobson, Reference Jacobson2007; Telford, Reference Telford2008).
Although several studies have shown that many reptiles may tolerate haemoparasite infection and suffer little or no pathogenic effect (e.g. Wozniak et al. Reference Wozniak, Kazacos, Telford and Mclaughlin1996; Amo et al. Reference Amo, Fargallo, Martínez-Padilla, Millán, López and Martín2005; Vardo-Zalik and Schall, Reference Vardo-Zalik and Schall2008), heavily blood infected lizards and snakes may show anaemia and immunosuppression, resulting in impaired growth, lower body condition, and decreased reproductive output and juvenile survival (e.g. Schall, Reference Schall1990; Amo et al. Reference Amo, López and Martín2004). However, in comparison with lizards or snakes, other major groups of reptiles, such as amphisbaenians, have received very little attention in the study of their blood parasites (Telford, Reference Telford, Hoff, Frye and Jacobson1984, Reference Telford2008).
Amphisbaenians are one distinctive major group of fossorial reptiles with important morphological and functional adaptations to the underground life (e.g. reduced vision, elongated body and loss of limbs in most species) (Gans, Reference Gans1978, Reference Gans2005) and a suite of original responses to ecological demands (e.g. Papenfuss, Reference Papenfuss1982; Martín et al. Reference Martín, López and García2013a , Reference Martín, Ortega, López, Pérez-Cembranos and Pérez-Mellado b ). In addition, conditions of the fossorial environment might also affect host-parasite relationships. However, prevalence and genetic diversity of haemoparasites remain almost unknown for amphisbaenians (Smith, Reference Smith1996; Gans, Reference Gans2005; Telford, Reference Telford2008). Here, we present the results of a survey for blood parasites in a North African population of the amphisbaenian Trogonophis wiegmanii.
MATERIALS AND METHODS
We conducted field work during two weeks in March 2014 (spring) and two weeks in September 2014 (autumn) at the Chafarinas Islands (Spain), a small archipelago located in the southwestern area of the Mediterranean Sea (35°11′N, 02°25′W), 2·5 nautical miles off the northern Moroccan coast (Ras el Ma, Morocco). This archipelago consists of three islands: Congreso (25·6 ha), Isabel II (15·1 ha) and Rey Francisco (13·9 ha). Vegetation is conditioned by the aridity of the warm, Mediterranean climate (i.e. average annual precipitation is 300 mm), the high soil salinity, and the guano accumulation from numerous seabird colonies (García et al. Reference García, Marañón, Ojeda, Clemente and Redondo2002). Thus, current vegetation is dominated by bushes adapted to salinity and drought, such as species of Salsola, Lycium and Suaeda genera (García et al. Reference García, Marañón, Ojeda, Clemente and Redondo2002). In general, the soils are shallow and immature, characterized by a thin A horizon over the original volcanic rock, and their characteristics have a deep effect on amphisbaenians condition (Martín et al. Reference Martín, López and García2013a , Reference Martín, López, Gutiérrez and García2015). In these islands, amphisbaenians are abundant and are always found buried in the substrate or under rocks (Martín et al. Reference Martín, Polo-Cavia, Gonzalo, López and Civantos2011, Reference Martín, López and García2013a , Reference Martín, Ortega, López, Pérez-Cembranos and Pérez-Mellado b ).
We searched for amphisbaenians in the three islands by lifting all stones found. We captured adult amphisbaenians (n = 75) by hand, gathered morphological measurements and took blood samples in situ, and released animals at their exact point of capture in less than 5 min. To avoid sampling the same individual twice, we avoided sampling the same area twice. We collected blood samples from the caudal sinus at the base of the tail by gently piercing the skin with a 23 G, 1″, 0·60 × 25 mm needle for each amphisbaenian and taking a droplet of blood with a heparinized capillary. We made blood smears by blowing a drop of blood onto the microscope slide. Smears were air-dried until coagulation. In the laboratory, the smears were fixed in absolute methanol for 10 min, and then stained in Giemsa diluted 1:9 with phosphate buffer (pH 7·2) for 40 min before their examination for parasites. On mounted slides, we scanned smears entirely at 200× along the length of the slide, looking for haemoparasites. Further, we looked for potential intraerythrocytic parasites at 400× by looking for the number of parasites in several random fields until totally about 2000 erythrocytes (Amo et al. Reference Amo, López and Martín2004, Reference Amo, Fargallo, Martínez-Padilla, Millán, López and Martín2005). Blood smears were checked under the microscope twice by two different researchers (J. M. and M. G.), who had previous experience with haemoparasites of reptiles (e.g. Amo et al. Reference Amo, López and Martín2004, Reference Amo, Fargallo, Martínez-Padilla, Millán, López and Martín2005; Garrido and Pérez-Mellado, Reference Garrido and Pérez-Mellado2013).
Additionally, we stored a few drops of blood in 500 µL SET buffer (0·015 m NaCl, 0·05 m Tris, 0·001 m EDTA, pH 8·0) at room temperature for molecular analyses. Genomic DNA was extracted from blood samples in the laboratory using a standard chloroform/isoamylalcohol method (Sambrook et al. Reference Sambrook, Fritch and Maniatis2002). To detect the presence of Hepatozoon spp. haemoparasites, we used a polymerase chain eaction (PCR) to amplify the 18S RNA gene of Hepatozoon spp. as described previously (Harris et al. Reference Harris, Maia and Perera2011). Briefly, a nested PCR reaction with primers HEMO1 and HEMO2 (Perkins and Keller, Reference Perkins and Keller2001), and then primers HEPF300 and HEPR900 (Ujvari et al. Reference Ujvari, Madsen and Olsson2004), were used for the identification of Hepatozoon infection. Additionally, detection of haemosporidian parasites was made using primers and PCR protocol designed to amplify a portion of the cytochrome b gene of avian Plasmodium and Haemoproteus (Hellgren et al. Reference Hellgren, Waldenström and Bensch2004). This latter protocol has been successfully used for testing Plasmodium infection in reptiles (Davis et al. Reference Davis, Benz, Ruyle, Kistler, Shock and Yabsley2013). Briefly, diluted genomic DNA (25 ng µL−1) was used as a template in every PCR assay for detection of the parasites using nested-PCR protocols. The amplifications were evaluated by running 2·5 µL of the final PCR on a 2% agarose gel. All PCR experiments contained one negative control for every eight samples. In the very few cases of negative controls showing signs of amplification (never more than faint bands in agarose gels), the whole PCR-batch was run again to make sure that all potential positives were true.
RESULTS
We extracted blood from 75 adult amphisbaenian individuals. Visual careful examination at the microscope of the blood smears in two different scans made by two different observers did not result in the finding of any haemoparasite. To confirm these negative results, molecular analyses of 30 blood samples did not either yield any positive result for genomic DNA of either Hepatozoon, Plasmodium or Haemoproteus.
DISCUSSION
This is one of the very few studies that focused on the detection of haemoparasite infection in amphisbaenians (Pessoa, Reference Pessoa1968; Telford, Reference Telford, Hoff, Frye and Jacobson1984; Lainson, Reference Lainson2003). Up to our knowledge, within fossorial reptiles, just a single amphisbaenian species, Amphisbaena alba, was found infected by haemogregarines (Haemogregarina amphisbaenae) in Brazil (Pessoa, Reference Pessoa1968) and Venezuela (Telford, Reference Telford, Hoff, Frye and Jacobson1984). A review of the species of the genus Hepatozoon that infect reptiles (Smith, Reference Smith1996) cited the mentioned H. amphisbaenae infecting A. alba as the only case of an intracellular protozoan infecting a fossorial reptile. However, Lainson (Reference Lainson2003) was unable to encounter H. amphisbaenae in 43 blood samples of A. alba collected in various localities from Brazil. Lainson (Reference Lainson2003) concluded that most probably the haemogregarine described by Pessoa (Reference Pessoa1968) did not belong to Haemogregarina genus. Likewise, Lainson (Reference Lainson2003) suggested that the intracellular parasite that Telford (Reference Telford, Hoff, Frye and Jacobson1984) found in just a single specimen of A. alba from Venezuela was probably the same species as Pessoa (Reference Pessoa1968) did. Our results, based on a wide survey of Trogonophis wiegmanni, indicate that there is a lack of haemoparasite infection in this amphisbaenian, at least in these island populations.
We propose several alternative hypotheses, not mutually exclusive, to explain the negative records of blood parasites in T. wiegmanni. First, the absence of blood parasites has been commonly attributed to the lack of appropriate vectors in the environment (Bennett et al. Reference Bennett, Montgomerie and Seutin1992; Martínez-Abraín et al. Reference Martínez-Abraín, Esparza and Oro2004). Haemoparasites recognized of reptiles can be transmitted either by the ingestion of an invertebrate vector (mainly mites, ticks or leeches) that previously fed on infected reptiles (Smith, Reference Smith1996; Telford, Reference Telford2008) or either by the bite of the arthropod vector (Telford, Reference Telford, Hoff, Frye and Jacobson1984, Reference Telford2008; Klein et al. Reference Klein, Young, Telford and Kimsey1987). While the former is considered the main transmission mechanism for haemogregarines (sensu lato), including all Hepatozoon spp., Plasmodium and Haemoproteus are most commonly infected through the bite of arthropod vectors, mainly flies and mosquitoes. Hence, either one or the other mechanism predominates, the inclusion of an arthropod vector is needed to complete the life cycle of the mentioned haemoparasites in reptiles. In this sense, fossorial habits of amphisbaenians might reduce the probability of a vector relationship evolving. This has been previously pointed out to explain why haemogregarines are found in all families of snakes except in those families containing species with strictly fossorial habitats, such as Typhlopidae, Leptotyphlopidae and Uropeltidae (Telford, Reference Telford, Hoff, Frye and Jacobson1984, Reference Telford2008; Smith, Reference Smith1996). The lack of infection by haemoparasites in our study population of amphisbaenians might also be due to the scarcity of suitable vectors (mites, ticks, mosquitoes, etc.) in the fossorial environment and the absence of these prey in the diet of amphisbaenians (Martín et al. Reference Martín, Ortega, López, Pérez-Cembranos and Pérez-Mellado2013b ). In fact, we have never observed mites or ticks as ectoparasites of T. wiegmanni in this population (Martín, unpublished results). Moreover, seabirds, which are very abundant in the study islands, are widely considered as potential dispersals of pathogens to remote islands, as they can act as dispersers of their arthropod vectors (Dietrich et al. Reference Dietrich, Gómez-Díaz and McCoy2011) and are infected by haemoparasites (Quillfeldt et al. Reference Quillfeldt, Arriero, Martínez, Masello and Merino2011). However, the absence of haemoparasites in T. wiegmanni suggests that the role of seagulls is negligible in this case.
Second, it has also been proposed that marine and arid habitat seem to represent an unsuitable environment for potential vectors of haematozoan parasites (Little and Earlé, Reference Little and Earlé1994; Piersma, Reference Piersma1997; Mendes et al. Reference Mendes, Piersma, Lecoq, Spaans and Ricklefs2005). Therefore, island isolation or extreme arid and salinity conditions in the Chafarinas archipelago (Martín et al. Reference Martín, López, Gutiérrez and García2015) might affect the presence of invertebrate hosts and therefore the prevalence of haemoparasites in amphisbaenians. However, we have found in the same study area blood parasites (Hepatozoon) in epigeal skinks (Fam. Scinicidae) and lizard (Fam Lacertidae) species that are sympatric with this amphisbaenian and share similar habitats but that are not fossorial (Martín, unpublished results).
Third, it might be possible that amphisbaenians could be free of blood parasites due to good immunological capabilities (Ricklefs, Reference Ricklefs1992). However, other types of internal parasites, such as coccidians and nematodes, have been often found in amphisbaenians, also in our study species and population with high prevalences (e.g. Lainson, Reference Lainson2003; Megía-Palma et al. Reference Megía-Palma, Martínez, Acevedo, Martín, García-Roa, Ortega, Peso-Fernández, Albaladejo, Cooper, Paranjpe, Sinervo and Merino2015).
Finally, the absence of blood parasites may also be attributed to lack of suitable host–parasite assemblage (Medeiros et al. Reference Medeiros, Hamer and Ricklefs2013), such as problems faced by parasites to complete their life cycles in reptile species that spend their lives underground, or absence of physiological compatibility between haemoparasites and amphisbaenians.
We conclude that haemoparasites are absent in this population of the amphisbaenian T. wiegmanni. Further studies should consider whether this is a particular situation restricted to this species or to the particular island conditions of this population, or whether this is a general characteristic, related to the fossorial environment, of amphisbaenians and other fossorial reptiles.
ACKNOWLEDGEMENTS
We thank two reviewers for helpful comments. The field station of the ‘Refugio Nacional de Caza de las Islas Chafarinas’ provided logistical support. We thank Juan Ignacio Montoya, Javier Díaz, Gonzalo Martínez, Angel Sanz, Francisco López, Alfredo Ruiz and Javier Zapata for friendship and help in the Islands. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
FINANCIAL SUPPORT
This work was supported by the ‘Organismo Autónomo de Parques Nacionales’ (J. M.), with additional financial support from Ministerio de Economía y Competitividad projects CGL2011-24150/BOS (J. M.), CGL2014-53523-P (J. M.) and CGL2015-64650 (A. M.).
