Validity of fish, birds and mammals as surrogates for amphibians and reptiles in pesticide toxicity assessment.

Amphibians and reptiles are the two most endangered groups of vertebrates. Environmental pollution by pesticides is recognised as one of the major factors threatening populations of these groups. However, the effects of pesticides on amphibians and reptiles have been studied for few substances, which is partly related to the fact that these animals are not included in the mandatory toxicity testing conducted as part of environmental risk assessments of pesticides. Whether risks of pesticides to amphibians and reptiles are addressed by surrogate taxa used in risk assessment is currently under debate. In order to develop a scientifically sound and robust risk assessment scheme, information needs to be gathered to examine whether fish, birds and mammals are valid surrogates for amphibians and reptiles. We updated a systematic review of scientific literature that was recently published compiling toxicity data on amphibians and reptiles. The outcome of this review was analysed with the purposes to (1) compare endpoints from amphibians and reptiles with the available information from fish, birds and mammals, and (2) develop species sensitivity distributions (SSDs) for those substances tested in at least six amphibian species (no substances were found tested in at least six reptile species) to identify a candidate amphibian model species to be used as surrogate in risk assessment. A positive correlation was found between toxicity recorded on fish and amphibians, the former revealing, in general, to be more sensitive than the latter to waterborne pollutants. In the terrestrial environment, although birds and mammals were more sensitive than amphibians and reptiles to at least 60% of tested substances, just a few weak significant correlations were observed. As a general rule, homoeothermic vertebrates are not good surrogates for reptiles and terrestrial amphibians in pesticide risk assessment. However, some chemical-dependent trends were detected, with pyrethroids and organochlorine insecticides being more toxic to amphibians or reptiles than to birds or mammals. These trends could ultimately help in decisions about protection provided by surrogate taxa for specific groups of substances, and also to determine when risk assessment of pesticides needs to pay special consideration to amphibians and reptiles. The outcome of this review reflects that there is still much information needed to reduce uncertainties and extract relevant conclusions on the overall protection of amphibians and reptiles by surrogate vertebrates.

A new species of Hepatozoon Miller, 1908 (Apicomplexa: Adelerina) from the snake Philodryas nattereri Steindachner (Squamata: Dipsadidae) in northeastern Brazil.

Based on both unique morphological characteristics of the gamont, distinct changes caused to the host erythrocyte and analysis of partial 18S rRNA gene sequences, a new parasite of the genus Hepatozoon Miller, 1908 is described from the snake Philodryas nattereri Steindachner (Squamata: Dipsadidae) in northeastern Brazil. The new species, Hepatozoon musa n. sp., is characterized by large and curved mature gamonts (18.9 ± 0.9 µm in length and 3.8 ± 0.3 µm in width) that considerably engorge infected host erythrocytes and displace the nucleus laterally, which become longer and thinner. Phylogenetic estimates indicate the new species is more closely related to the recently described Hepatozoon cuestensis O'Dwyer, Moço, Paduan, Spenassatto, Silva & Ribolla, 2013, from Brazilian rattlesnakes. These recent findings highlight the need for further studies of Hepatozoon to better determine the biodiversity of this common but poorly-studied parasite group.

Assessing the diversity, host-specificity and infection patterns of apicomplexan parasites in reptiles from Oman, Arabia.

Understanding the processes that shape parasite diversification, their distribution and abundance provides valuable information on the dynamics and evolution of disease. In this study, we assessed the diversity, distribution, host-specificity and infection patterns of apicomplexan parasites in amphibians and reptiles from Oman, Arabia. Using a quantitative PCR approach we detected three apicomplexan parasites (haemogregarines, lankesterellids and sarcocystids). A total of 13 haemogregarine haplotypes were identified, which fell into four main clades in a phylogenetic framework. Phylogenetic analysis of six new lankesterellid haplotypes revealed that these parasites were distinct from, but phylogenetically related to, known Lankesterella species and might represent new taxa. The percentage of infected hosts (prevalence) and the number of haemogregarines in the blood (parasitaemia) varied significantly between gecko species. We also found significant differences in parasitaemia between haemogregarine parasite lineages (defined by phylogenetic clustering of haplotypes), suggesting differences in host-parasite compatibility between these lineages. For Pristurus rupestris, we found significant differences in haemogregarine prevalence between geographical areas. Our results suggest that host ecology and host relatedness may influence haemogregarine distributions and, more generally, highlight the importance of screening wild hosts from remote regions to provide new insights into parasite diversity.

Comments on the Systematic Revision of Adeleid Haemogregarines: Are More Data Needed?

Haemogregarines are a group of apicomplexan parasites composed of 3 families that infect a wide range of hosts. Many species within these families have been subjected to reclassifications and reassignments, especially because the use of molecular tools to estimate their phylogenetic relationships became more widespread. The 18S rRNA gene has been the only widely used gene for studying the diversity of haemogregarines and recent phylogenetic analyses of this gene have indicated incongruences with the current taxonomy, such that a new genus Bartazoon has recently been proposed. To investigate the current taxonomic situation further, we conducted an overview of all published 18S rRNA sequence data for haemogregarines. We highlight that our understanding of the real diversity and phylogenetic relationships of haemogregarines is still limited, which undermines the proposed systematic revision. Notably all the molecular evidence comes from a single gene, and many studies have shown that single-gene trees often do not reflect species trees. Combined with doubts over the relationships of Hemolivia, the recent identification of a new lineage that could also warrant creation of a new genus, and issues with the type species for Hepatozoon, we suggest that any taxonomic changes now would be premature. In our opinion, type species need to be assessed, sampling across hosts improved, and multiple genes employed prior to taxonomic alterations. Otherwise taxonomic instability will be likely.

Molecular detection of Anaplasma platys, Ehrlichia canis, Hepatozoon canis and Rickettsia monacensis in dogs from Maio Island of Cape Verde archipelago.

Tick-borne diseases are emerging worldwide and have an important zoonotic relevance. Dogs play an important role in the epidemiology of several zoonotic tick-borne pathogens acting as sentinels and/or reservoirs. This study focused on the molecular identification of tick-borne pathogens in blood samples of 153 autochthonous asymptomatic dogs in Maio Island, Cape Verde archipelago. Eighty-four (54.9%) dogs were positive for one or more pathogens. Fifty-five (35.9%) dogs were infected with Hepatozoon canis, 53 (34.6%) with Anaplasma platys, five (3.3%) with Ehrlichia canis and Rickettsia monacensis, an emerging human pathogen, was also identified in a single dog (0.7%). The former three pathogens cause important canine tick-borne diseases that are transmitted or potentially transmitted by Rhipicephalus sanguineus s.l., the only hard tick identified in Cape Verde. Furthermore, Wolbachia spp. was amplified from the blood of one dog. None of the dogs were positive for Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, Midichloria mitochondrii, Bartonella spp., Babesia spp. or Theileria spp. Fifty-four (35.3%) animals showed single infections and 30 (19.6%) co-infections, with A. platys and H. canis co-infection being the most frequent (28 dogs, 18.3%). The frequency of E. canis infection was statistically different among age groups (P=0.017), being higher among dogs older than 4 years compared to younger dogs. Infection by A. platys was also statistically different among age groups (P=0.031), being higher in dogs younger than 2 years compared to older dogs. The statistical analyses showed no significant association of PCR positivity with gender or location. The frequency of tick-borne pathogens detected in dogs in Maio Island, including R. monacensis, highlights the need to improve diagnosis and control in order to prevent the risk of transmission of these pathogens among dogs and humans living in or travelling to this touristic island.

Buffy coat smear or Knott's test: which to choose for canine microfilaria screening in field studies?

BACKGROUND: In recent years, an increasing number of cases of canine dirofilariasis have been reported worldwide. However, the rate of infection in dogs is largely unknown in many remote areas, and the importance of field studies for determination of the prevalence of canine dirofilariasis in such areas is well recognized. The detection of microfilariae by the modified Knott's test (MKT) is a recommended screening method for canine dirofilariasis. OBJECTIVES: The purposes of this study were to compare the diagnostic sensitivity of the MKT with the buffy coat smear method (BCS), and to evaluate the utility of these 2 methods under field study conditions. METHODS: One hundred and fifty dogs of the Maio Island of Republic of Cabo Verde, were screened for microfilariae using MKT and BCS. The results of the 2 methods were generated in a blinded manner and statistically compared. RESULTS: The detection rate was 4.67% with the MKT and 5.33% with BCS, which is statistically not different. The latter allowed a morphologic identification of Dirofilaria repens (later confirmed by molecular biology methods) and an estimation of parasite load, which varied from 15 to 185 microfilariae/mL. CONCLUSIONS: The methods MKT and BCS were comparable in terms of diagnostic sensitivity. However, the BCS was technically less demanding and produced permanent preparations, in which co-infection with other hematologic pathogens can easily be assessed. Overall, this method is well suited to assess microfilariae in a large number of animals, and it could replace the MKT in studies devoted to dirofilariasis.

Microscopic and molecular characterization of Hepatozoon domerguei (Apicomplexa) and Foleyella furcata (Nematoda) in wild endemic reptiles from Madagascar.

Madagascar is one of the world's top twelve "megadiversity" hot spots hosting unique and threatened flora and fauna. Parasites are a major component of biodiversity but remain largely uncharacterized in wildlife. In this study we combine microscopic and molecular assessment of hemoparasites in endemic reptile species from Madagascar. We detected three distinct parasites: the apicomplexans Hepatozoon and Sarcocystis, and filarial nematodes. The prevalence and intensity of these apicomplexans were low overall, while microfilarial infections in chameleons were relatively high. We detected mixed infections of two Hepatozoon haplotypes in Madagascarophis colubrinus, and of Hepatozoon and microfilariae in a Furcifer sp. Phylogenetic analyses of Hepatozoon showed evidence of prey-predator transmission, with identical sequences found in the snakes M. colubrinus and Ithycyphus oursi, and their prey Furcifer sp. Based on previous studies regarding the life cycle of Hepatozoon domerguei Landau, Chabaud, Michel, and Brygoo, 1970 in these hosts and due to their morphological similarity, we propose that this Hepatozoon haplotype is Hepatozoon domerguei. Future studies, including the examination of invertebrate hosts, are needed to verify this preliminary taxonomic identification. A distinct hemogregarine haplotype was found in Oplurus sp., which displayed morphologically different gametocytes, some of which were apparently inside leukocytes. The Sarcocystis identified from Tracheloptychus petersi was identical to that reported in a North African snake, indicating that the same lineage is found in geographically distinct regions. By combining morphological and genetic information, Foleyella furcata (Linstow, 1899) filarial nematodes were identified in several Furcifer chameleons. This study provides insights into the distribution, diversity and host-parasite interactions of hemoparasites in wild reptile populations from Madagascar.

Molecular assessment of Hepatozoon (Apicomplexa: Adeleorina) infections in wild canids and rodents from north Africa, with implications for transmission dynamics across taxonomic groups.

Parasites play a major role in ecosystems, and understanding of host-parasite interactions is important for predicting parasite transmission dynamics and epidemiology. However, there is still a lack of knowledge about the distribution, diversity, and impact of parasites in wildlife, especially from remote areas. Hepatozoon is a genus of apicomplexan parasites that is transmitted by ingestion of infected arthropod vectors. However, alternative modes of transmission have been identified such as trophic transmission. Using the 18S rRNA gene as a marker, we provide an assessment of Hepatozoon prevalence in six wild canid and two rodent species collected between 2003 and 2012 from remote areas in North Africa. By combining this with other predator-prey systems in a phylogenetic framework, we investigate Hepatozoon transmission dynamics in distinct host taxa. Prevalence was high overall among host species (African jerboa Jaculus jaculus [17/47, 36%], greater Egyptian jerboa Jaculus orientalis [5/7, 71%], side-striped jackal Canis adustus [1/2, 50%], golden jackal Canis aureus [6/32, 18%], pale fox Vulpes pallida [14/28, 50%], Rüppell's fox Vulpes rueppellii [6/11, 55%], red fox Vulpes vulpes [8/16, 50%], and fennec fox Vulpes zerda [7/11, 42%]). Phylogenetic analysis showed further evidence of occasional transmission of Hepatozoon lineages from prey to canid predators, which seems to occur less frequently than in other predator-prey systems such as between snakes and lizards. Due to the complex nature of the Hepatozoon lifecycle (heteroxenous and vector-borne), future studies on these wild host species need to clarify the dynamics of alternative modes of Hepatozoon transmission and identify reservoir and definitive hosts in natural populations. We also detected putative Babesia spp. (Apicomplexa: Piroplasmida) infections in two canid species from this region, V. pallida (1/28) and V. zerda (1/11).

A comparison of multiple methods for estimating parasitemia of hemogregarine hemoparasites (apicomplexa: adeleorina) and its application for studying infection in natural populations.

Identifying factors influencing infection patterns among hosts is critical for our understanding of the evolution and impact of parasitism in natural populations. However, the correct estimation of infection parameters depends on the performance of detection and quantification methods. In this study, we designed a quantitative PCR (qPCR) assay targeting the 18 S rRNA gene to estimate prevalence and intensity of Hepatozoon infection and compared its performance with microscopy and PCR. Using qPCR, we also compared various protocols that differ in the biological source and the extraction methods. Our results show that the qPCR approach on DNA extracted from blood samples, regardless of the extraction protocol, provided the most sensitive estimates of Hepatozoon infection parameters; while allowed us to differentiate between mixed infections of Adeleorinid (Hepatozoon) and Eimeriorinid (Schellackia and Lankesterella), based on the analysis of melting curves. We also show that tissue and saline methods can be used as low-cost alternatives in parasitological studies. The next step was to test our qPCR assay in a biological context, and for this purpose we investigated infection patterns between two sympatric lacertid species, which are naturally infected with apicomplexan hemoparasites, such as the genera Schellackia (Eimeriorina) and Hepatozoon (Adeleorina). From a biological standpoint, we found a positive correlation between Hepatozoon intensity of infection and host body size within each host species, being significantly higher in males, and higher in the smaller sized host species. These variations can be associated with a number of host intrinsic factors, like hormonal and immunological traits, that require further investigation. Our findings are relevant as they pinpoint the importance of accounting for methodological issues to better estimate infection in parasitological studies, and illustrate how between-host factors can influence parasite distributions in sympatric natural populations.

Patterns of genetic diversity in Hepatozoon spp. infecting snakes from North Africa and the Mediterranean Basin.

Species of Hepatozoon Miller, 1908 are blood parasites most commonly found in snakes but some have been described from all tetrapod groups and a wide variety of hematophagous invertebrates. Previous studies have suggested possible associations between Hepatozoon spp. found in predators and prey. Particularly, some saurophagous snakes from North Africa and the Mediterranean region have been found to be infected with Hepatozoon spp. similar to those of various sympatric lizard hosts. In this study, we have screened tissue samples of 111 North African and Mediterranean snakes, using specific primers for the 18S rRNA gene. In the phylogenetic analysis, the newly-generated Hepatozoon spp. sequences grouped separately into five main clusters. Three of these clusters were composed by Hepatozoon spp. also found in snakes and other reptiles from the Mediterranean Basin and North Africa. In the other two clusters, the new sequences were not closely related to geographically proximate known sequences. The phylogeny of Hepatozoon spp. inferred here was not associated with intermediate host taxonomy or geographical distribution. From the other factors that could explain these evolutionary patterns, the most likely seems series of intermediate hosts providing similar ribotypes of Hepatozoon and a high prevalence of host shifts for Hepatozoon spp. This is indicated by ribotypes of high similarity found in different reptile families, as well as by divergent ribotypes found in the same host species. This potentially low host specificity has profound implications for the systematics of Hepatozoon spp.

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.