Molecular characterization of haemosporidian and haemogregarine diversity in southwestern Iberian amphibians and reptiles

doi:10.21203/rs.3.rs-2468577/v1

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Molecular characterization of haemosporidian and haemogregarine diversity in southwestern Iberian amphibians and reptiles

https://doi.org/10.21203/rs.3.rs-2468577/v1

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published 18 Mar, 2023

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The knowledge of the diversity and geographic distribution of parasite species is the first step towards understanding processes of global epidemiology and species conservation. Despite recent increases in research on reptiles and amphibians haemosporidian and haemogregarine parasites, we still know little about their diversity and parasite-host interactions, especially in the Iberian Peninsula, where a few studies have been conducted. In this study, the haemosporidian and hemogregarine diversity and phylogenetic relationships of southwestern Iberian amphibians and reptiles were assessed using PCR approaches on 145 blood samples. The amphibians did not present any of both groups of parasites studied. Regarding the reptile species, six Hepatozoon and one Haemocystidum haplotypes were found infecting four different species, revealing new host records for these parasites. Among them, we found one new isolate Haemocystidium haplotype and three new isolates and a previously reported Hepatozoon haplotype from a north African snake. This finding suggests that some Hepatozoon parasites may not be host-specific and have large geographic ranges even crossing geographical barriers. These results increased the geographic distribution and the number of known host species of some reptile apicomplexan parasites, highlighting the great unexplored diversity of them in this region.

Haemocystidium sp.

Hepatozoon sp.

Malpolon monspessulanus

phylogenetic relationship

Podarcis virescens

Tarentola mauritanica

Parasites are one of the most ubiquitous and abundant organisms in the world having a wide range of negative effects on their host life-histories (Schmid-Hempel 2011). Moreover, parasites play key roles not only on individual hosts, but also on many ecology interactions, host population dynamics and community structure in many ecosystems (Price et al. 1986; Wood et al. 2007). In this scenario, the knowledge of the diversity and geographic distributions of parasite species at various spatial scales and host groups is the first step towards understanding processes of global epidemiology and species conservation (du Toit et al. 2013; Wells et al. 2015). The phylum Apicomplexa is a highly diverse protist group of obligatory parasitic organisms. The medical and veterinary importance of some of its genera (e.g. Plasmodium, Toxoplasma and Babesia, among others), has contributed to the increase in the number of the studies on this group of parasites in the last two decades (Yabsley and Shock 2012; Santiago-Alarcón and Marzal 2020). Even so, the number of formally described species is far from the existing reality (Votýpka et al. 2016). In addition, many of these species were described on mammal and avian hosts, while studies of amphibians’ and reptiles’ apicomplexan diversity are even scarcer (Telford 2009).

Amphibians and reptiles are hosts to a wide variety of hemoparasitic apicomplexan groups, including hemococcidians, haemogregarines, haemosporidians, piroplasms and Sarcocystidae (Davies and Johnston 2000; Telford 2009; Muriel et al. 2021). Haemogregarines of the genus Hepatozoon sp. are the most common apicomplexans reported infecting these animals, although other hemogregarine genera such as Haemogregarina sp., Hemolivia sp. and Karyolysus sp. can be also found (Smith 1996; Davies and Johnston 2000; Telford 2009). Haemogregarines have a heteroxenous life cycle with two different hosts: a vertebrate host in which asexual reproduction occurs, and an invertebrate vector in which sexual reproduction happens (Telford 2009). Recorded invertebrate vectors of apicomplexan parasites of reptiles and amphibians include ticks, mites, biting flies, mosquitoes and leeches (Smith 1996). Likewise, haemosporidians of the genus Plasmodium sp., Haemoproteus sp. and Haemocystidium sp. are also heteroxenous parasites with similar life cycles transmitted by hematophagous dipterans (Garnham 1966; Valkiūnas 2005). Among them, Plasmodium sp. and Haemocystidium sp. have been reported infecting reptiles, whereas Haemoproteus sp. has been found in both amphibians and reptiles (Davies and Johnston 2000; Valkiūnas 2005; Telford 2009; Muriel et al. 2021). However, the genus Haemocystidium has not been fully accepted by the academic community due to its limited knowledge, and it was previously synonymized with Plasmodium genus first and later with the genus Haemoproteus (Javanbakht et al. 2015; Austen et al. 2020). Nevertheless, molecular studies have shown that Haemocystidium species reported from reptiles are a distinct group from avian Haemoproteus and thus all reptilian Haemoproteus should be reclassified within the genus Haemocystidium (Maia et al. 2016a; Pineda-Catalan et al. 2013).

The establishment of molecular techniques for the detection of blood parasites (Vilcins et al. 2009) has revealed unexpectedly high levels of apicomplexan parasite diversity in amphibians and reptiles worldwide (O’Dwyer et al. 2013; Tomé et al. 2014; Harris et al. 2015). For example, in some apicomplexan genera such as Hepatozoon of certain regions and host species (e.g. Maia et al. 2012; Rajabi et al. 2017; Gutiérrez-Liberato et al. 2021). In contrast, studies about amphibian and reptile haemosporidians are scarcer in the literature compared to those of haemogregarines (e.g. Fantham et al. 1942; Martinele et al. 2016; Matta et al. 2018). The pattern of distribution and genetic diversity of parasite species is shaped by the range of hosts in which they occur (Poulin et al. 2011). So that, the screening of different species and in different countries is needed in order to reveal new lineages and new parasite-host interactions. However, it is still unknown a large share of the diversity of amphibian and reptile haemogregarine and haemosporidian fauna from many regions and host species.

In the Iberian Peninsula, haemogregarine genus Hepatozoon sp. has been reported from 13 species of reptiles such as Algyrodes marchi, Psammodromus algirus or Timon lepida (Amo et al. 2005a; Amo et al. 2007; Maia et al. 2012), and to a greater extent in species of the genus Podarcis sp. (Amo et al. 2005b; Roca and Galdón 2010; Maia et al. 2012). In contrast, to the best of our knowledge no studies have been conducted there on amphibian haemogregarines or amphibian and reptile haemosporidians. This study aimed to characterize the diversity of haemosporidian and haemogregarine parasites by using molecular approaches in some species of amphibians and reptiles in the southwestern Iberian Peninsula. Furthermore, the phylogenetic relationships of the haemosporidian and haemogregarine species detected with those found to date are analysed.

Sample collection

The study was carried out across eight localities in the Extremadura region (southwestern Iberian Peninsula) (Fig. 1) in 2021. These areas were mainly composed of holm oak woodlands (Quercus ilex L.), grasslands and farmlands of typically Mediterranean climate, characterized by temperate, rainy winters and dry, hot summers (average annual temperature and rainfall: 16°C, 615 mm) (García and Mateos 2010). Amphibians and reptiles were handled by experienced staff and held in plastic containers for processing immediately in the field in aseptic conditions. Blood sample of each individual was taken using heparinized needles from different body areas depending on the species. Amphibian blood samples were taken from the ventral tail vein in newts and the ventral abdominal vein in frogs and toads (Allender and Fry 2008). Blood from all reptiles was removed from caudal tail vein (Sykes and Klaphake 2008), except for turtles which was removed from the caudal sinus at the base of the tail (Polo-Cavia et al. 2010). Samples were later stored in 500 µl of SET buffer (0.015 M NaCl, 0.05 M Tris, 0.001 M EDTA, pH 8.0) (Sambrook et al. 1989) for subsequent molecular analysis. All the individuals were safely released after ensuring that they were in perfect condition and at the same point of capture.

Molecular detection of blood parasite infections

Total genomic DNA from blood samples was extracted with the Thermo KingFisher purification system™, using the MagMAX™Cell-Free DNA Isolation kit (Thermo Fisher, Ref. A32700) and PK buffer for MagMAX™-96 DNA Multi sample kit (Thermo Fisher, Ref. 4489111). DNA samples were screened for haemosporidian parasites (genera Haemocystidium) by using a nested polymerase chain reaction (PCR) that targets a 478 bp fragment (excluding primers) of the mitochondrial cytochrome b gene of these parasites following Hellgren et al. (2004). This procedure is based on a first PCR using primers HaemNFI (5′-CAT ATA TTA AGA GAA ITA TGG AG-3′) and HaemNR3 (5′-ATA GAA AGA TAA GAA ATA CCA TTC-3′), followed by a second PCR using primers HaemF (5′-ATG GTG CTT TCG ATA TAT GCA TG-3′) and HaemR2 (5′-GCA TTA TCT GGA TGT GAT AAT GGT-3′). To detect the presence of haemogregarine parasites, a nested PCR amplification was also performed from each DNA sample initially using the primers HEMO1 (5′-TAT TGG TTT TAA GAA CTA ATT TTA TGA TTG-3′) and HEMO2 (5′-CTT CTC CTT CCT TTA AGT GAT AAG GTT CAC-3′) (Perkins and Keller 2001) and then primers HepF300 (5′-GTT TCT GAC CTA TCA GCT TTC GAC G-3′) and HepR900 (5′-CAA ATC TAA GAA TTT CAC CTC TGA C-3′) (Ujvari et al. 2004), targeting part of the 18S rRNA gene of apicomplexan parasites. Negative and positive controls were run with each reaction. Parasites detected by a positive amplification were purified using the Genejet PCR Purification Kit (Thermo-Fisher Scientific) and then sequenced in both directions on an ABI 3130 genetic analyser (provided by the Service of Bioscience Applied Techniques of the University of Extremadura, SAIUEx). A consensus sequence was created by combining the two partially overlapping regions of the forward and reverse sequences of each sample with Geneious Prime 2019.2.3 (Kearse et al. 2012). Obtained consensus sequences were then aligned using the program MEGA11 v. 11.0.11 (Tamura et al., 2021) and compared against published ones to find the best match on GenBank using BLAST. The new sequences found, were deposited in GenBank under the accession numbers ON458135, ON332722, ON332723 and ON332724.

The genetic relationships between the Haemocystidum and Hepatozoon parasites were investigated separately by analysing the haemosporidian cytochrome b and the 18S rDNA sequence divergence using the program MEGA11. The AIC criterion was used to choose the best model of sequence evolution and the parameters employed for our set of sequences. Maximum likelihood analyses were performed using a General Time Reversible model (GTR) with a Gamma distribution and Invariant sites for the Haemocystidium sequences and a Tamura 3-parameter model (T92) with a Gamma distribution for the Hepatozoon sequences. Support for nodes was estimated using the bootstrap technique (Felsenstein 1985) with 1000 replicates. All available Haemocystidium sequences from GenBank were included to build the phylogenetic tree. For the genus Hepatozoon, only those found in Mediterranean hosts were selected. The Haemocystidium and Hepatozoon phylogenetic trees were rooted with a sequence from Plasmodium falciparum (GeneBank M76611) and Adelina bambarooniae (GeneBank AF494059) respectively. Estimates of evolutionary divergences were also calculated (Supplementary material).

We obtained blood samples of 86 amphibians belonging to five species and 59 reptiles belonging to 13 species (Table 1). Of the 86 amphibians analysed, we did not find any infected individual neither by haemosporidian nor haemogregarine parasites. On the other hand, from the 59 screened reptiles, two individuals were infected by Haemocystidium and six individuals were infected by Hepatozoon (Table 1).

Table 1

Samples analysed for apicomplexan parasites (haemosporidians and haemogregarines) in amphibians and reptiles from southwestern Iberian Peninsula. “Haem” and “Hep” represent the number of *Haemocystidium* and *Hepatozoon* positive sequences respectively. Shown are also accession numbers of positive sequences in GenBank.
Host species	N	Haem	Hep	Accession number
Amphibians
Epidalea calamita	41
Hyla meridionalis	1
Pelophylax perezi	27
Pleurodeles waltl	9
Triturus pygmaeus	8
Reptiles
Acanthodactylus erythrurus	2
Blanus cinereus	3
Chalcides bedriagai	1
Chalcides striatus	3
Hemidactylus turcicus	1
Malpolon monspessulanus	6		3	ON332723, ON332724, KC696567
Mauremys leprosa	3		1	KJ740753
Natrix maura	13
Podarcis virescens	3		2	ON332722, JX531954
Psammodromus algirus	6
Psammodromus occidentalis	3
Tarentola mauritanica	10	2		ON458135
Timon lepidus	5

After comparing our sequences with those published in GenBank, we found seven parasite lineages infecting sampled individuals: one Haemocystidum and six Hepatozoon isolates. From them, four parasite sequences had not been previously detected in prior studies, and subsequently were considered as new haplotypes: one Haemocystidium sequence was detected in Tarentola mauritanica (ON458135), two isolates of Hepatozoon were found infecting M. monspessulanus individuals (ON332723 and ON332724), and one Hepatozoon lineage was detected in P. virescens (ON332722). The other three haplotypes (KC696567, JX531954 and KJ740753) had been previously found in prior studies infecting reptiles.

The Haemocystidium phylogenetic analyses revealed the affiliation of the new haplotype from T. mauritanica together with other Haemocystidium sequences from geckos and snakes, and separately from the chelonian clade (Fig. 2). The Hepatozoon phylogenetic analyses revealed that the new Hepatozoon isolate from M. monspessulanus were closely related to other Hepatozoon sequences from Iberian and North African snakes forming an individual clade. The new isolate obtained from P. virescens formed a group with other Hepatozoon lineages from Iberian and North African Podarcis sp. (Fig. 3).

The knowledge of amphibian and reptile apicomplexan parasite diversity has increased notably in the last decade due to the use of molecular methods (O’Dwyer et al. 2013; Tomé et al. 2014; Harris et al. 2015). Here we analysed 145 individuals from 18 amphibian and reptile species from Iberian Peninsula to characterize the genetic diversity of haemosporidian and haemogregarine parasites. None of 86 individuals from five amphibian species were infected by these blood parasites. In contrast, six Hepatozoon and one Haemocystidum lineages were found infecting four reptile species. Notably, more than a half of these haplotypes had not been previously recorded in former studies. Moreover, our outcomes also revealed six new reptile – parasite records, thus identifying new host records for these parasites. These findings highlight the great unexplored diversity of apicomplexan parasites in reptiles from this region. Next, we will discuss these outcomes in detail.

Amphibians are hosts of a wide variety of hemoparasites including Haemogregarina, Hepatozoon, Lankesterella or Schellackia among others (Muriel et al. 2021). The presence of Haemoproteus in these animals is very unusual, having been identified only three species to date (Fantham et al. 1942), while haemogregarine parasites, and Hepatozoon species particularly, are more frequently recorded (e.g. Leal et al. 2009; Netherlands et al. 2015; Maia et al. 2016b). This is the first work studying apicomplexan parasites in Iberian amphibians, so although some infections have been found in reptiles from nearby localities (e.g. Marzal et al. 2017; this study), the absence of infected individuals could be explained by different factors. Firstly, these parasites normally have mosquito, leeches and ticks as principal vectors (Bardsley and Harmsen 1973; Desser 2001). However, while reptiles are mostly terrestrial, amphibians spend much of their time into the water. The absence of infected individuals in our samples could be due to the absence of appropriate aquatic vectors infecting amphibians in the study area or to the short exposure time to terrestrial vectors (Ball 1967; Barta and Desser 1984; Bennet et al. 1992). In addition, the available vectors in the area may have very specific feeding habits and amphibians would not be part of it or even a certain specialization of the parasite haplotypes infecting reptile species instead of amphibians (Apperson et al. 2004; Maia et al. 2016b; Fecchio et al. 2019). Another possible explanation is that the effects of the infection will result in the death of individuals, and only those whose good condition allowed them to survive were captured in this study (Bensch et al. 2007). Finally, amphibians can recognize both physical and chemical cues from some potential parasites, avoiding sites where vector or parasite densities are high (Kiesecker and Skelly 2000; Ferguson and Smith 2012).

The genus Plasmodium was not found in this study, in contrast with other studies in reptiles (e.g. Matta et al. 2018; Harris et al. 2019). On the contrary, two positive samples of Haemocystidium sp. and six of Hepatozoon sp. were found among reptiles studied. The reports of Haemocystidium sp. worldwide are still limited in literature, being found almost exclusively infecting some gecko and turtle species (Telford 2005; Javanbakh et al. 2015; Maia et al. 2016a) Thus, there is much work to be done about this controversial genus. Thus, to our knowledge, it represented the first report of Haemocystidium in western Europe and the first in the species Tarentola mauritanica. The phylogenetic analysis remained like those previously described (González et al. 2019; Austen et al. 2020) with a clade formed by chelonians and other by lacertids and snakes. The new isolate found from T. mauritanica could be placed together with this second clade, closely related to haplotypes from other gecko and a snake species. The presence of a Haemocystidium haplotype from a snake predator in a potential prey clade like the geckos, could be explained by the snake`s diet. It is suggested that certain Hepatozoon sp. could be transferred to a predator when consuming preys harbouring the parasite (Allen et al. 2011; Tomé et al. 2012, 2013). In this case, Naja annulifera could have acquired the Haemocystidium parasite from an infected gecko, explaining why the sequence is so close to that found in the geckos.

The haemogregarine genus Hepatozoon was the most time detected parasite found in this study with six haplotypes from six infected individuals. Hepatozoon is frequently reported infecting reptiles worldwide, with usually high prevalence (e.g. Ujvari et al. 2004; Maia et al. 2012, 2016b). Our results showed that the occurrence of Hepatozoon varied among species, as we only detected parasites in three of the thirteen species studied. Differences in susceptibility to infection among species could be associated with host immune defences, that can lead to prevent or better tolerate the infection (Klein 2004; Lindström et al. 2004). Alternatively, both the parasites and the vectors that transmit them may have a high degree of affinity for certain species of reptiles (Maia et al. 2016b; Fecchio et al. 2019). This may explain why some host species were more likely to be infected than others. Moreover, the host related variables, for example age or size (Brown et al. 2006; Salkeld and Schwarzkopf 2005), methodological aspects (Harris et al. 2011) or ecological factors (Sehgal et al. 2011; Gupta et al. 2013) must be also considered when analysing infection levels from different species and from different geographical areas.

Among the infected species, we found two Hepatozoon haplotypes infecting the lacertid Podarcis virescens in this study. One of them (JX531954) was previously reported infecting P. bocagei from Tanes (Spain) (Maia et al. 2012). Thus, this haplotype could be specific from Podarcis sp. and widespread distributed in western Spain. Phylogenetic analyses revealed that the haplotype was closely related to other North African lacertids haplotypes. The other new isolate (ON332722) fell in a group with other Podarcis species hosts from North Africa and the Iberian Peninsula (P. hispanica and P. vaucheri). Our findings represented the first report of Hepatozoon parasites infecting the species P. virescens.

Moreover, the haplotypes found infecting Malpolon monspessulanus in this study also represented the first assessment of the occurrence of Hepatozoon sp. in this species. According to the phylogenetic analyses, the two new isolates appeared to be closely related to each other forming part of a group with other snake haplotypes from North Africa and the Iberian Peninsula. The third haplotype identified was previously reported by Tomé et al. (2013) from Psammophis sibilans in North Africa and was also included in the snake group previously mentioned. This result followed the idea that some Hepatozoon parasites are not host-specific (Maia et al. 2016b; Tomé et al. 2021). The apparent switch of host genus may be facilitated by the hosts similarity (both are snakes), and therefore it is assumed that they will have more similar immune defences to deal with (Medeiros et al. 2013; Maia et al. 2016b; Clark and Clegg 2017). Furthermore, this may be more likely to occur when these hosts share a habitat (Maia et al. 2016b). However, they belong to different countries separated by a geographical barrier. In this sense, other studies with avian haemosporidians have reported the same patterns across the Strait of Gibraltar, finding the Plasmodium lineage LK6 (H158) in birds of Spain and Africa (Matta et al. 2015).

Finally, the Hepatozoon haplotype from Mauremys leprosa found here belonged to a previously reported lineage from the same species in a nearby locality (Marzal et al. 2017) and was very closely related to another M. leprosa haplotype of the same locality.

In conclusion, despite the low number of individuals infected by Haemocystidium sp. and Hepatozoon sp., a high percentage of new "lineages" have been detected, pointing out the lack of knowledge about the diversity of these parasites in the Iberian Peninsula. It is for this reason that much information is still required. Further studies should focus on the transmission and ecological factors influencing the infection patterns by these parasites and the effects on the host fitness. The knowledge of this information can be critical to understand their impact on biodiversity and can also have implications for conservation, especially in host groups as threatened and vulnerable as amphibians and reptiles.

Supplementary Information

Estimates of evolutionary divergences between Haemocystidium mitochondrial cytochrome b and 18S rDNA Hepatozoon sequences amplified from southwestern Iberian reptiles marked in blue and bold letters and other sequences available in GenBank.

Acknowledgments

We are grateful to technical and human support provided by the Faculty of Bioscience Applied Techniques of SAIUEx (financed by UEX, Junta de Extremadura, MICINN, FEDER and FSE). We thank A. Sánchez-Cano (IREC) for confirming the results of some of the haemosporidian PCR tests and assuring us of their reliability. We also thank S. Casquero-Sánchez, J. Jiménez-Peñuela and E. de la Peña for their constructive comments. Finally, we are also grateful to many people who collaborated during the fieldwork (F. A. Parejo, A. M. Pulido, A. Parejo, J. Cendrero, J. M. Paisano, J. F. Castillo, M. Espinosa, A. Robustillo and D. Prado) and specially to D. Fernández-Guiberteau, who also taught us how to take blood samples from amphibians and reptiles.

Funding

This work was funded by “Consejería de Economía e Infraestructura of the Junta de Extremadura” and the European Regional Development Fund, a Way to Make Europe, through the research project (grant numbers IB20089 and IB16135).

Conflicts of interest

The authors declare that they have no conflict of interest.

Competing interests

The authors have no competing interests.

Availability of data and material

Not applicable.

Author’s contributions

Daniel Parejo-Pulido and Sergio Magallanes contributed to the study conception and design. Alfonso Marzal and Sergio Magallanes obtained the funding and resources for the study. Material preparation, data collection and molecular analysis were performed by Daniel Parejo-Pulido, Sergio Magallanes and Carlos Mora-Rubio. The first draft of the manuscript was written by Daniel Parejo-Pulido and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Ethics approval

All applicable international, national and/or institutional guidelines for the care and use of animals were followed. Capture and manipulation of birds were authorized by the “Consejería para la Transición Ecológica y Sostenibilidad, Junta de Extremadura” (CN0043/20/ACA). Daniel Parejo-Pulido (functions a, b, c, Junta de Andalucía) and Sergio Magallanes (category C, Junta de Extremadura) had the permits for animal experimentation that guarantee animal handling and taking biological samples.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Allen KE, Yabsley MJ, Johnson EM, Reichard MV, Panciera RJ, Ewing SA, Little, SE (2011) Novel Hepatozoon in vertebrates from the southern United States. J Parasitol 97: 648-653. doi: 10.1645/GE-2672.1
Allender MC, Fry, MM (2008) Amphibian haematology. Vet Clin North Am Exot Anim Pract 11: 463-480. doi: 10.1016/j.cvex.2008.03.006
Amo L, Fargallo JA, Martínez-Padilla J, Millán J, López P, Martín, J (2005a) Prevalence and intensity of blood and intestinal parasites in a field population of a Mediterranean lizard, Lacerta lepida. Parasitol Res 96: 413-417. doi: 10.1007/s00436-005-1355-1
Amo L, López P and Martín J (2005b) Prevalence and intensity of haemogregarine blood parasites and their mite vectors in the common wall lizard, Podarcis muralis. Parasitol Res 96: 378-381. doi: 10.1007/s00436-005-1354-2
Amo L, López P, Martín, J (2007) Habitat deterioration affects antipredatory behavior, body condition, and parasite load of female Psammodromus algirus lizards. Can J Zool 85: 743-751. https://doi.org/10.1139/Z07-052
Apperson CS, Hassan HK, Harrison BA, Savage HM, Aspen SE, Farajollahi A, Crans W, Daniels TJ, Falco RC, Benedict M, Anderson M, McMillen L, Unnasch, TR (2004) Host feeding patterns of established and potential mosquito vectors of West Nile virus in the eastern United States. Vector Borne Zoonotic Dis 4: 71-82. https://doi.org/10.1089/153036604773083013
Austen JM, Hall J, Zahedi A, Goften A, Ryan U (2020) Further characterisation of Haemocystidium chelodinae-like Haemoproteidae isolated from the Bellinger River snapping turtle (Myuchelys georgesi). Parasitol Res 119: 601-609. doi: 10.1007/s00436-019-06547-9
Ball GH (1967) Blood sporozoans from East African amphibia. J Protozool 14: 521-527. doi: 10.1111/j.1550-7408.1967.tb02037.x
Bardsley JE, Harmsen, R (1973) The trypanosomes of anura. Adv Parasitol 11: 1-73. doi: 10.1016/s0065-308x(08)60184-0
Barta JR, Desser SS (1984) Blood parasites of amphibians from Algonquin Park, Ontario. J Wildl Dis 20: 180-189. doi: 10.7589/0090-3558-20.3.180
Bennett GF, Montgomerie R, Seutin, G (1992) Scarcity of haematozoa in birds breeding on the arctic tundra of North America. Condor 94: 289-292. https://doi.org/10.2307/1368821
Bensch S, Waldenström J, Jozén N, Westerdahl H, Hansson B, Sejberg D, Hasselquist D (2007) Temporal dynamics and diversity of avian malaria parasites in a single host species. J Anim Ecol 76: 112-122.
Brown GP, Shilton CM, Shine R (2006) Do parasites matter? Assessing the fitness consequences of haemogregarine infection in snakes. Can J Zool 84: 668-676. https://doi.org/10.1139/z06-044
Clark NJ, Clegg SM (2017) Integrating phylogenetic and ecological distances reveals new insights into parasite host specificity. Mol Ecol 26(11), 3074-3086. https://doi.org/10.1111/mec.14101
Davies AJ, Johnston MRL (2000) The biology of some intraerythrocytic parasites of fishes, amphibia and reptiles. Adv Parasitol 45: 1-107. doi: 10.1016/s0065-308x(00)45003-7
Desser SS (2001) The blood parasites of anurans from Costa Rica with reflections on the taxonomy of their trypanosomes. J Parasitol 87: 152-60. doi: 10.1645/0022-3395(2001)087[0152:TBPOAF]2.0.CO;2
du Toit N, van Vuuren BJ, Matthee S, Matthee CA (2013). Biogeography and host-related factors trump parasite life history: limited congruence among the genetic structures of specific ectoparasitic lice and their rodent hosts. Mol Ecol 22: 5185-5204. doi: 10.1111/mec.12459
Fantham HB, Porter A, Richardson LH (1942) Some haematozoa observed in vertebrates in eastern Canada. Parasitology 34: 199-226. doi:10.1017/S0031182000016176.
Fecchio A, Wells K, Bell JA, Tkach VV, Lutz HL, Weckstein JD, Clegg SM, Clark N (2019) Climate variation influences host specificity in avian malaria parasites. Ecol Lett 22: 547-557. https://doi.org/10.1111/ele.13215
Felsenstein J (1985) Confidence and phylogenies: an approach using the bootstrap. Evolution 39: 783-791. https://doi.org/10.2307/2408678
Ferguson LV, Smith TG (2012) Reciprocal trophic interactions and transmission of blood parasites between mosquitoes and frogs. Insects 3: 410-23. doi: 10.3390/insects3020410
García R, Mateos AB (2010) El Clima de Extremadura. In: Schnabel S, Lavado JF, Gómez A, García R (ed) Aportaciones a la Geografía Física de Extremadura, Cáceres, Asociación Profesional para la Ordenación del Territorio, el Ambiente y el Desarrollo Sostenible (Fundicotex), pp 25-52.
Garnham PCC (1966) Malaria parasites and other haemosporidian. Blackwell Scientific Oxford, UK.
González LP, Pacheco MA, Escalante AA, Maldonado ADJ, Cepeda AS, Rodríguez-Fandiño OA, Vargas-Ramírez M, Matta NE (2019) Haemocystidium spp., a species complex infecting ancient aquatic turtles of the family Podocnemididae: first report of these parasites in Podocnemis vogli from the Orinoquia. Int J Parasitol 10: 299-309. doi: 10.1016/j.ijppaw.2019.10.003
Gupta N, Bhaskar M, Gupta DK (2013) Ecological attributes of Hepatozoon lacertilis susceptibility in Indian lizards, Hemidactylus flaviviridis (Gekkonidae) and Calotes versicolor (Agamidae). Trop Biomed 30: 97-104.
Gutiérrez-Liberato GA, Lotta-Arévalo IA, Rodríguez-Almonacid CC, Vargas- Ramírez M, Matta NE (2021) Molecular and morphological description of the first Hepatozoon (Apicomplexa: Hepatozoidae) species infecting a neotropical turtle, with an approach to its phylogenetic relationships. Parasitology 148: 747-759. doi: 10.1017/S0031182021000184
Harris DJ, Maia JP, Perera A (2011) Molecular characterization of Hepatozoon species in reptiles from the Seychelles. J Parasitol 97: 106-10. doi: 10.1645/GE-2470.1
Harris DJ, Borges-Nojosa DM, Maia JP (2015) Prevalence and diversity of Hepatozoon in Native and Exotic Geckos from Brazil. J Parasitol 101: 80-85. doi: 10.1645/14-522.1
Harris DJ, Santos JL, Borges-Nojosa DM, Castro DP (2019) Molecular screening of Plasmodium (Haemosporidia: Plasmodiidae) parasites from reptiles in Brazil. J Parasitol 105: 913-917. https://doi.org/10.1645/18-149
Hellgren O, Waldenström J, Bensch S (2004) A new PCR assay for simultaneous studies of Leucocytozoon, Plasmodium, and Haemoproteus from avian blood. J Parasitol 90: 797-802. doi: 10.1645/GE-184R1
Javanbakht H, Kvičerová J, Dvořáková N, Mikulíček P, Sharifi M, Kautman M, Maršíková A, Široký P (2015) Phylogeny, diversity, distribution, and host specificity of Haemoproteus spp. (Apicomplexa: Haemosporida: Haemoproteidae) of palaearctic tortoises. J Eukaryot Microbiol 62: 670-678. doi: 10.1111/jeu.12227
Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A (2012) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28: 1647-1649. doi: 10.1093/bioinformatics/bts199
Kiesecker JM, Skelly DK (2000) Choice of oviposition site by gray treefrogs: The role of potential parasitic infection. Ecology 81: 2939-2943. https://doi.org/10.2307/177354
Klein SL (2004) Hormonal and immunological mechanisms mediating sex differences in parasite infection. Parasite Immunol 26: 247-264. doi: 10.1111/j.0141-9838.2004.00710.x
Lindström KM, Foufopoulos J, Pärn H, Wikelski M (2004) Immunological investments reflect parasite abundance in island populations of Darwin’s finches. Proc R Soc B: Biol Sci 271: 1513-1519. doi: 10.1098/rspb.2004.2752
Leal DDM, O’Dwyer LH, Ribeiro VC, Silva RJ, Ferreira VL, Rodrigues RB (2009) Hemoparasites of the genus Trypanosoma (Kinetoplastida: Trypanosomatidae) and hemogregarines in anurans of the São Paulo and Mato Grosso do Sul States-Brazil. An Acad Bras Cienc 81: 199-206. doi: 10.1590/s0001-37652009000200006
Maia JP, Harris DJ, Perera A (2011) Molecular survey of Hepatozoon species in lizards from North Africa. J Parasitol 97: 513-517. doi: 10.1645/GE-2666.1
Maia JP, Perera A, Harris DJ (2012) Molecular survey and microscopic examination of Hepatozoon Miller, 1908 (Apicomplexa: Adeleorina) in lacertid lizards from the western Mediterranean. Folia Parasitol 59: 241-248. doi: 10.14411/fp.2012.033
Maia JP, Harris DJ, Carranza S (2016a) Reconstruction of the evolutionary history of Haemosporida (Apicomplexa) based on the cytbgene with characterization of Haemocystidium in geckos (Squamata: Gekkota) from Oman. Parasitol Int 65: 5-11. doi: 10.1016/j.parint.2015.09.003
Maia JP, Harris DJ, Carranza S, Goméz-Díaz E (2016b) Assessing the diversity, host-specificity and infection patterns of apicomplexan parasites in reptiles from Oman, Arabia. Parasitology 143: 1730–1747. doi: 10.1017/S0031182016001372
Martinele I, Tostes R, Castro R, D’Agosto M (2016) Prevalence of Haemoproteus spp. (Apicomplexa: Haemoproteidae) in tortoises in Brazil and its molecular phylogeny. Parasitol Res 115: 249-254. doi: 10.1007/s00436-015-4741-3
Marzal A, Ibáñez A, González-Blázquez M, López P, Martín J (2017) Prevalence and genetic diversity of blood parasite mixed infections in Spanish terrapins, Mauremys leprosa. Parasitology 144: 1449-1457. doi: 10.1017/S0031182017000889
Matta VA, da Silva LP, Lopes RJ, Drovetski SV (2015) The Strait of Gibraltar poses an effective barrier to host-specialised but not to host-generalised lineages of avian Haemosporidia. Int J Parasitol 45: 711-719. doi: 10.1016/j.ijpara.2015.04.006
Matta NE, González LP, Pacheco MA, Escalante AA, Moreno AM, González AD, Calderón-Espinosa ML (2018) Plasmodium parasites in reptiles from the Colombia Orinoco-Amazon basin: A re-description of Plasmodium kentropyxi Lainson R, Landau I, Paperna I, 2001 and Plasmodium carmelinoi Lainson R, Franco CM, da Matta R, 2010. Parasitol Res 117: 1357-1370. doi: 10.1007/s00436-018-5815-9. doi: 10.1007/s00436-018-5815-9
Medeiros MCI, Hamer GL, Ricklefs RE (2013) Host compatibility rather than vector-host-encounter rate determines the host range of avian Plasmodium parasites. Proc R Soc B: Biol Sci 280(1760): 20122947. https://doi.org/10.1098/rspb.2012.2947
Muriel J, González-Blázquez M, Matta NE, Vargas-León CM, Marzal A (2021) Parasitas Sanguíneos de Anfíbios. Editora da Universidade Federal do Piauí, Teresina, Brazil.
Netherlands EC, Cook CA, Kruger DJ, du Preez LH, Smit NJ (2015) Biodiversity of frog haemoparasites from sub-tropical Northern Kwazulu-Natal, South Africa. Int J Parasitol Parasites Wildl 4: 135-141. doi: 10.1016/j.ijppaw.2015.01.003
O’Dwyer LH, Moço TC, Paduan KDS, Spenassatto C, da Silva RJ, Ribolla PEM (2013) Description of three new species of Hepatozoon (Apicomplexa, Hepatozoidae) from Rattlesnakes (Crotalus durissus terrificus) based on molecular, morphometric and morphologic characters. Exp Parasitol 135: 200-207. doi: 10.1016/j.exppara.2013.06.019
Perkins SL, Keller AK (2001) Phylogeny of nuclear small subunit rRNA genes of hemogregarines amplified with specific oligonucleotídeos. J Parasitol 87: 870-876. doi: 10.1645/0022-3395(2001)087[0870:PONSSR]2.0.CO;2
Pineda-Catalan O, Perkins SL, Peirce MA, Engstrand R, Garcia-Davila C, Pinedo-Vasquez M, Aguirre AA (2013) Revision of hemoproteid genera and description and redescription of two species of chelonian hemoproteid parasites. J Parasitol 99: 1089-1098. doi: 10.1645/13-296.1
Polo-Cavia N, Engstrom T, López P, Martín J (2010) Body condition does not predict immunocompetence of western pond turtles in altered versus natural habitats. Anim Conserv 13: 256-264. https://doi.org/10.1111/j.1469-1795.2009.00329.x
Poulin R, Krasnov BR, Mouillot D (2011) Host specificity in phylogenetic and geographic space. Trends Parasitol 27: 355-361. doi: 10.1016/j.pt.2011.05.003.
Price PW, Westoby M, Rice B, Atsatt PR, Fritz RS, Thompson JN, Mobley K (1986) Parasite mediation in ecological interactions. Annual Review of Ecology and Systematics 17: 487-505.
Rajabi F, Javanbakht H, Sajjadi SS (2017) A preliminary study of haemoparasites in marsh frogs, Pelophylax ridibundus (Ranidae) from Iran. J Entomol Zool Stud 5: 1314-1317.
Roca V, Galdón MA (2010) Haemogregarine blood parasites in the lizards Podarcis bocagei (Seoane) and P. carbonelli (Pérez-Mellado) (Sauria: Lacertidae) from NW Portugal. Syst Parasitol 75: 75-79. doi: 10.1007/s11230-009-9206-6
Salkeld DJ, Schwarzkopf L (2005) Epizootiology of blood parasites in an Australian lizard: A mark-recapture study of a natural population. Int J Parasitol 35: 11-18. doi: 10.1016/j.ijpara.2004.09.005
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Plainview, NY.
Santiago-Alarcón D, Marzal A (2020) Avian Malaria and Related Parasites in the Tropics: Ecology, Evolution and Systematics. Springer, Cham, Switzerland.
Schmid-Hempel P (2011) Evolutionary parasitology: the integrated study of infections, immunology, ecology and genetics. Oxford University Press, Oxford, UK.
Sehgal RNM, Buermann W, Harrigan RJ, Bonneaud C, Loiseau C, Chasar A, Sepil I, Valkiūnas G, Iezhova T, Saatchi S, Smith TB (2011) Spatially explicit predictions of blood parasites in a widely distributed African rainforest bird. P Proc R Soc B: Biol Sci 278: 1025-1033. doi: 10.1098/rspb.2010.1720
Smith TG (1996) The genus Hepatozoon (Apicomplexa: Adeleina). J Parasitol 82: 565-585.
Sykes IV JM, Klaphake E (2008) Reptile Hematology. Vet Clin North Am Exot Anim Pract 11: 481-500. doi: 10.1016/j.cvex.2014.09.011
Tamura K, Stecher G, Kumar S (2021) MEGA11: Molecular Evolutionary Genetics Analysis version 11. Molecular Biology and Evolution 33: 1870-1874. doi: 10.1093/molbev/msab120
Telford SR (2005) A Haemocystidium species from the east african gecko Lygodactylus capensis grotei. J Parasitol 91: 135-138. doi: 10.1645/GE-3395
Telford SR (2009) Hemoparasites of the Reptilia: Color Atlas and Text. CRC Press, Boca Raton, Florida.
Tomé B, Maia JP, Harris DJ (2012) Hepatozoon infection prevalence in four snake genera: influence of diet, prey parasitaemia levels or parasite type? J Parasitol 98: 913-917. doi: 10.1645/GE-3111.1
Tomé B, Maia JP, Harris DJ (2013) Molecular assessment of apicomplexan parasites in the snake Psammophis from North Africa: do multiple parasite lineages reflect the final vertebrate host diet? J Parasitol 99: 883-887. doi: 10.1645/12-95.1
Tomé B, Maia JP, Salvi D, Brito JC, Carretero MA, Perera A, Meimberg H, Harris DJ (2014) Patterns of genetic diversity in Hepatozoon spp. infecting snakes from North Africa and the Mediterranean Basin. Syst Parasitol 87: 249-258. doi: 10.1007/s11230-014-9477-4
Tomé B, Maia JP, Perera A, Carranza S, Vasconcelos R (2021) Parasites in a hotspot: diversity and specificity patterns of apicomplexans infecting reptiles from the Socotra Archipelago. Parasitology 148: 42-52. doi: 10.1017/S0031182020002000
Ujvari B, Madsen T, Olsson M (2004) High prevalence of Hepatozoon spp. (Apicomplexa, Hepatozoidae) infection in water pythons (Liasis fuscus) from tropical Australia. J Parasitol 90: 670-672. doi: 10.1645/GE-204R
Valkiūnas G (2005) Avian malaria parasites and other haemosporidia. CRC Press, Boca Raton, Florida.
Vilcins IE, Ujvari B, Old JM, Deane E (2009) Molecular and morphological description of a Hepatozoon species in reptiles and their ticks in the Northern Territory, Australia. J Parasitol 95: 434-442. doi: 10.1645/GE-1725.1
Votýpka J, Modrý D, Oborník M, Šlapeta J, Lukeš J (2016) Apicomplexa. In Archibald JM, Simpson AGB and Slamovits CH (ed) Handbook of the Protists, pp 1-58.
Wells K, O’Hara RB, Morand S, Lessard J-P, Ribas A (2015) The importance of parasite geography and spillover effects for global patterns of host–parasite associations in two invasive species. Divers Distrib 21: 477-486. https://doi.org/10.1111/ddi.12297
Wood CL, Byers JE, Cottingham KL, Altman I, Donahue MJ , Blakeslee AMH (2007) Parasites alter community structure. Proc Natl Acad Sci USA 104: 9335-9339. doi: 10.1073/pnas.0700062104
Yabsley MJ, Shock BC (2012) Natural history of Zoonotic Babesia: Role of wildlife reservoirs. Int J Parasitol Parasites Wildl 22: 18-31. doi: 10.1016/j.ijppaw.2012.11.003.

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Molecular characterization of haemosporidian and haemogregarine diversity in southwestern Iberian amphibians and reptiles

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Molecular detection of blood parasite infections

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