First report of Hepatozoon (Apicomplexa: Adeleorina) in caecilians, with description of a new species.

Hepatozoon spp. are identified for the first time in the amphibian order Gymnophiona, or caecilians, from the Seychelles island of Silhouette. Estimate of relationships derived from partial 18S rRNA gene sequences indicate these are not related to Hepatozoon spp. from frogs or to other Hepatozoon spp. from reptiles in the Seychelles. Assessment of mature gamonts from blood smears indicate that these can be recognized as a new species, Hepatozoon seychellensis n. sp.

Molecular survey of parasites in introduced Pelophylax perezi (Ranidae) water frogs in the Azores.

Water frogs, Pelophylax perezi, that are introduced in the Azores, were screened for parasites using PCR primers known to amplify Apicomplexa parasites, and using nematode-specific primers. With the former, three different organisms were detected: Hepatozoon, a trichodinid protozoan ciliate and a possible Stramenopile. Using the latter set of primers, a single unknown spirurid nematode was also detected. Phylogenetic analyses indicate that Hepatozoon detected within amphibian hosts appear to form a clade, although relationships of these parasites do not match the vertebrate intermediate host phylogeny. Regarding the possible Stramenopile, it is unclear whether this organism was actually present on the amphibian or in the water on the surface of the tissue sample. Our findings highlight that many different organisms can be detected with these primers and that they can be used to screen introduced host populations to detect parasites that have been brought with them.

Molecular assessment of apicomplexan parasites in the snake Psammophis from North Africa: do multiple parasite lineages reflect the final vertebrate host diet?

The Apicomplexa are intracellular pathogens of animals, with the Coccidia being the largest group. Among these are the hemogregarines, which include some of the most common hemoparasites found in reptiles. Several studies have reported a possible pattern of prey-predator transmission for some of these parasites. Snakes from the Mediterranean region have been found to be parasitized with Hepatozoon spp. similar to those in lacertids and gekkonids, supporting the prey-predator transmission hypothesis. Here we analyzed specimens of the saurophagous genus Psammophis from North Africa, an ecologically different region. Through molecular analysis of tissue samples we detected 3 different apicomplexan parasites: Caryospora, Sarcocystis, and Hepatozoon. Caryospora was detected in a Forskål's sand snake Psammophis schokari from Algeria, constituting the first time these parasites have been detected from a tissue sample through molecular screening. The obtained Sarcocystis phylogeny does not reflect the relationships of their final hosts, with the parasites identified from snakes forming at least 3 unrelated groups, indicating that it is still premature to predict definitive host based on the phylogeny of these parasites. Three unrelated lineages of Hepatozoon parasites were identified in Psammophis, each closely related to lineages previously identified from different lizard groups, on which these snakes feed. This once again indicates that diet might be a key element in transmission, at least for Hepatozoon species of saurophagous snakes.

Apicomplexa primers amplify Proteromonas (Stramenopiles, Slopalinida, Proteromonadidae) in tissue and blood samples from lizards.

Microscopy has traditionally been the most common method in parasitological studies, but in recent years molecular screening has become increasingly frequent to detect protozoan parasites in a wide range of vertebrate hosts and vectors. During routine molecular screening of apicomplexan parasites in reptiles using the 18S rRNA gene, we have amplified and sequenced Proteromonas parasites from three lizard hosts (less than 1% prevalence). We conducted phylogenetic analysis to confirm the taxonomic position and infer their relationships with other stramenopiles. Although our phylogeny is limited due to scarcity of molecular data on these protists, our results confirm they are closely related to Proteromonas lacertae. Our findings show that unexpected parasites can be amplified from host samples (blood and tissue) using general procedures to detect hemoparasites, and stress that positive PCR amplifications alone should not be considered as definitive proof of infection by particular parasites. Further validation by sequence confirmation and thorough phylogenetic assessment will not only avoid false positives and biased prevalence estimates but also provide valuable information on the biodiversity and phylogenetic relationships of other parasitic organisms. More generally, our results illustrate the perils of general diagnosis protocols in parasitological studies and the need of cross-validation procedures.

Molecular survey of Apicomplexa in Podarcis wall lizards detects Hepatozoon, Sarcocystis, and Eimeria species.

The occurrence of apicomplexan parasites in Podarcis sp. wall lizards from the Iberian Peninsula and Balearic islands was studied by amplification and sequencing of the 18S rRNA gene. Species from 3 genera, Hepatozoon , Sarcocystis , and Eimeria , were found. The phylogenetic analysis of the 18S rRNA gene provides unexpected insights into the evolutionary history of these parasites. All Hepatozoon spp. specimens were recovered as part of a clade already identified in lizards from North Africa. The Sarcocystis species, detected in Podarcis lilfordi from Cabrera Island in the Balearic Islands, appears related to Sarcocystis gallotiae , known only from endemic Gallotia sp. lizards from the Canary Islands. Based on the lack of snake predators on this island, this parasite presumably presents an atypical transmission cycle that uses the same host species as both intermediate and final host through cannibalism, like S. gallotiae . Eimeria sp. is reported for the first time from Podarcis spp. lizards. This study shows the power of detecting multiple different apicomplexan parasites through screening of tail tissue samples and blood drops that are often collected in reptiles for other purposes.

Hepatozoon infection prevalence in four snake genera: influence of diet, prey parasitemia levels, or parasite type?

Hepatozoon spp. (Apicomplexa: Haemogregarinidae) are the most commonly reported hemoparasites from snakes. Of over 300 Hepatozoon species identified, more than 120 were described from snakes. However, recent genetic assessments have found Hepatozoon lineages recovered from both prey and predators, indicating that diet may play an important role in the infection of final vertebrate hosts. Here 4 different snake genera with different diets were assessed. Hepatozoon spp. prevalence varied greatly between the genera, but only lineages already identified from potential prey, i.e., gecko and lacertid lizards, were recovered from the snakes. Interestingly, the Hepatozoon spp. lineage known from geckos was the most common in the snakes, but this does not reflect their diet. Higher parasitemia levels, reported for some geckos relative to lacertid lizards, may play a role. Alternatively, this lineage may be more effective at parasitizing snakes or may occur, despite being unrecorded, in other vertebrate groups consumed by snakes.

Molecular survey and microscopic examination of Hepatozoon Miller, 1908 (Apicomplexa: Adeleorina) in lacertid lizards from the western Mediterranean.

The genus Hepatozoon Miller, 1908 (Apicomplexa: Adeleorina) is composed of intracellular haemogregarine parasites that are widely distributed among all tetrapod groups. The present study combines microscopic and molecular data on haemogregarine parasites from lizards in the western Mediterranean. We screened tissue samples and examined blood smears for the presence of species of Hepatozoon from four lizards, namely Algyroides marchi Valverde, endemic to Southeast Spain, Podarcis bocagei Seoane from Spain and Portugal, P hispanica Steindachner from Spain, and P lilfordi Günther from Cabrera, Balearic Islands (Spain). Our results show that prevalence and intensity of Hepatozoon parasites vary between and within lizard species from different regions. Algyroides marchi and P bocagei from Spain had the lowest values, whereas P hispanica had the highest. Phylogeny based on 18S rRNA gene sequences indicates that most of the new Hepatozoon sequences are part of a clade exclusive from North African and Iberian lizards, except for a single P bocagei isolate that is found related to another clade including isolates from other reptile host species and rodents. Interestingly, isolates from Algyroides form a distinct monophyletic subgroup, which could be a signal of strict host-specificity within this host genus.

Molecular survey of Hepatozoon species in lizards from North Africa.

The prevalence of Hepatozoon parasites in 460 lizards from North Africa was studied by amplification and sequencing of the 18S rRNA gene. The phylogenetic analysis of the 18S rRNA gene provides new insights into the phylogeny of these parasites with multiple genetically distinct lineages recovered. Parasite prevalence differed significantly between lacertid lizards and geckos. Our results show that there is limited host specificity and no clear relation to the geographical distribution of Hepatozoon parasites.

Molecular characterization of Hepatozoon species in reptiles from the Seychelles.

Hepatozoon parasites were examined for the first time in reptiles from the Seychelles Islands. Although both prevalence and intensity were low, Hepatozoon species were detected in individuals from 2 endemic species, the lizard Mabuya wrightii and the snake Lycognathophis seychellensis. This was confirmed using visual identification and through sequencing part of the 18s rRNA gene. Phylogenetic analysis indicates that the Hepatozoon on the Seychelles form a monophyletic lineage, although more data are clearly needed to stabilize estimates of relationships based on this marker.