168 SHORT NOTE HERPETOZOA 27 (3/4) Wien, 30. Jänner 2015 SHORT NOTE data would be preferable given time (Will et al. 2005), but molecular examination has many advantages, besides being relatively quick and easy. For a start, sequence data is invaluable for placing parasites in a phyloge- netic framework. Furthermore, many times host samples are collected for other purpos- es, such as host genetic assessments, but these same samples are available for the study of parasites. Apicomplexan blood parasites are a prime example of a group where this molec- ular approach can be useful. Assessments of these have in the past focused on groups with strong anthropogenic interests due to health reasons, such as Plasmodium, or groups with significant economic impact. Others, such as Hepatozoon, despite being the most common blood parasite of reptiles (TElFORD 2009) gained little attention. Molecular analysis however, using primers specific for a section of the 18S rRNA gene, has greatly clarified phylogeny (e.g., BARTA et al. 2012; HARRiS et al. 2012), identified infections in new host orders (e.g., PiNTO et al. 2012), and indicated that predator-prey Scanning for apicomplexan trophic pathways may be widespread in parasites (Suborder Adeleorina) some cases, such as between lizards and in five Holarctic anuran species snakes (TOMé et al. 2013). At the same time, other parasites such as Stramenopiles Parasites are ubiquitous, but a poorly were detected (MAiA et al. 2012a). Tests known component of biodiversity, with esti- using specific primers can be misleading as mates of 0.1 % of species described for some some parasites may not be detected (ZEH- groups (MORRiSON 2009), while other scien- TiNDJiEv et al. 2012). Thus, it is necessary tists simply note “we have no credible way to investigate this aspect in different host of estimating how many parasitic protozoa groups. … exist” (DOBSON et al. 2008). yet parasites Amphibians as a whole have suffered are of dual interest for conservation biolo- global declines, and parasites are a key gists, both for their adverse impact on hosts driving factor (BEEBEE & gRiFFiTHS 2005). with parasite-driven declines in wildlife Although the role of the fungus Batracho - becoming increasingly common (PEDERSEN chytrium dendrobatidis is widely accepted & FENTON 2007), but also because, in mono - (e.g., DASZAk et al. 2003), testing for other phagous parasites, this relationship with the parasites is needed. various infections by hosts increases their risk of co-extinction. Hepatozo on species have been identified in indeed, models suggest co-extinction may amphibians using microscopy (e.g., STEN- be the most common form of biodiversity BERg & BOWERMAN 2010), but also molec- loss (DuNN et al. 2009). given the difficul- ular examination of an introduced popula- ties in alpha-taxonomy of most groups, and tion of frog, Pelophylax perezi (lóPEZ- the lack of parasitologists (ŠlAPETA 2013), SEOANE, 1885), from the Azores islands molecular analyses, much like the common (HARRiS et al. 2013b). On the other hand, ‟DNA barcoding” approach, may be an ex - examination of Bufo calamita lAuRENTi, tremely valuable first assessment for some 1768, from the iberian Peninsula did not parasites. Clearly, integrative approaches detect apicomplexan parasites (HARRiS et combining morphological and molecular al. 2013a). SHORT NOTE HERPETOZOA 27 (3/4) Wien, 30. Jänner 2015 SHORT NOTE 169 Table 1: Species analyzed for parasites, the number tested using alternative source material (tissue or blood), and the number examined on slides under the microscope. Species Tissue (toe) Blood Slides Pelobates cultripes (C uviER , 1829) 45 --- --- Pelophylax saharicus (B OulENgER , 1913) 52 30 26 Hyla meridionalis BOETTgER , 1874 --- 11 7 Amietophrynus mauritanicus (S CHlEgEl , 1841) 30 8 10 Bufotes boulengeri (l ATASTE , 1879) 47 8 Bufo bufo (l iNNAEuS , 1758) 5 --- --- The aim of the present study was to Olympus CX41 microscope with a built-in scan for apicomplexan parasites a number digital camera (SC30) (Olympus, Hamburg, of amphibians from Europe and North germa ny). Several photomicrographs per Africa, using samples that had been prima - slide were taken at 400 fold magnification rily collected for studies of the host. Be - and stitched using Cell^B software (basic cause of this, in most cases blood smears to image-acquisition and archiving software, assess the prevalence under the microscope Olym pus, Münster, germany). in case para - were not available. An established PCR pro - sites were not detected after ca. 10 minutes tocol amplifying a region of the 18S rRNA of examination, the slides were considered gene, successfully applied to amphibians negative. When parasites were identified, from the Azores, and reptiles from this even in very low numbers, slides were region (e.g., MAiA et al. 2012b) was adopt - scored as positive. For some examples, in - ed in the present study. it is known that the tensity of infection was estimated based on primers used can also detect organisms numbers of parasites per 3,000 red blood other than Apicomplexa (e.g., MAiA et al. cells. 2012a; TOMé et al. 2012). Therefore, all suc - DNA was extracted using standard cessful PCR amplifications were sequenced, high salt methods ( SAMBROOk et al. 1989). since a positive PCR amplification cannot Detection of blood parasites was made be assumed to indicate the presence of a using PCR reactions with the primers particular parasite. Results based on molec - HepF300 and HepR900 ( uJvARi et al. ular and visual methods were compared to 2004), which were designed to amplify He - assess the efficiency of detection using patozoon parasites. Conditions of the PCR molecular methods. are detailed in HARRiS et al. (2011). A sub - Tissue samples (toe clips) were taken set of 47 samples was also tested with the from 136 amphibians belonging to six HEMO1 and HEMO2 primers ( PERkiNS & species, from various localities in the kEllER 2001). Although these are known iberian Peninsula, the Balearic islands and to be less efficient at detecting Hepatozoon Morocco, and stored in 96 % ethanol (Table relative to the Hep primers ( MAiA et al. 1). The taxonomy of many amphibians in 2012b), they were used and evaluated in this region is in a state of flux, but here the case they gave results in the present am - authors follow BEukEMA et al. (2013). For phibian study. Negative and positive con - a smaller number of specimens, blood drops trols were run with each reaction. PCR stored on Whatman paper (56 specimens, 4 products were analyzed by electrophoresis species) and blood smears (51 specimens, 4 in 2 % agarose and visualized by gel Red species) were also available (indicated in staining and uv transillumination. The Table 1). All samples are part of the DB positive PCR products obtained were puri - collection housed at CiBiO, uP. Blood fied and sequenced by a commercial smears were air-dried, fixed with methanol, sequencing facility (Macrogen Europe, The stained with diluted giemsa (one part Netherlands). Positive PCRs were com - giemsa solution, nine parts distilled water) pared against the public database genBank, for 55 minutes, and examined using an using a BlAST similarity search. 170 SHORT NOTE HERPETOZOA 27 (3/4) Wien, 30. Jänner 2015 SHORT NOTE 136) for apicomplexan parasites was found with the Hep primers, and the BlAST com - parison revealed 99.8 % similarity with Dactylosoma ranarum from a Pelophylax synkl . esculentus (liNNAEuS , 1758) host from Corsica, France (Accession numbers Hq224957 and Hq224958, BARTA et al. 2012). Only a single nucleotide differed from the genBank sequences over 613 bp of compared sequence data. Testing of blood drops with both primer sets also failed to detect any positive infections. However, when blood slides were visually examined, various positive samples infected with hemogregarines were identified (Fig. 1). These were found in two host species, Pelo - phylax saharicus (B OulENgER , 1913) and Amietophrynus mauritanicus (S CHlE gEl , 1841) , and at least for P. saharicus preva - lence was high (12 in 26 [= 46 %] of indi - viduals screened). With regard to the inten - sity of infections, samples visually diag - nosed positive for low parasitaemia levels of Dactylosoma (0.1 % infected red blood cells) were detected by the molecular method, whereas even heavier hemogrega - rine infections (up to 3 % infected cells in the sample of A. mauritanicus - DB15569) were scored as negative in the molecular ap - proach. Scanning for parasites using con - served primers has the potential to greatly improve knowledge on parasite diversity and distribution. like all molecular ap - proaches, it can be improved by adoption of an “integrated” approach, as barcoding alone can be misleading ( Will et al. 2005). For most studies of Hepatozoon , the Hep primers ( uJvARi et al. 2004) have proven to be efficient at detecting not only divergent Hepatozoon lineages (e.g., Figure 1:. Samples of red blood cells showing hemogregarine infections in A - Amietophrynus HARRiS et al. 2012), but also various other mauritanicus (S CHlEgEl , 1841) , and B - Pelophylax parasites (e.g., TOMé et al 2013). Most stud - saharicus (B OulENgER , 1913) , both of which failed ies indicated that using these primers, iden - to amplify hemogregarine DNA using the screening tification efficiency was at least as high, or protocol employed. C - infection by presumed Dactylosoma ranarum in Pelophylax saharicus . even higher, when compared to the visual length of the scale bar corresponds to 20 μm. assessment of blood smears (e.g., O’D WyER et al. 2013). yet, in the present study, they failed to amplify DNA of any hemogre - garines, which were clearly identified in at initially, analysis was carried out on least two of the host species examined. One the toe clips, as these are most readily avail - could argue that amphibian toe clips are not able from genetic studies of the vertebrate ideal sources of material for studies of these hosts. Only a single positive sample (out of parasites. Also, in blood drops stored in SHORT NOTE HERPETOZOA 27 (3/4) Wien, 30.