Molecular Survey and Microscopic Examination of Haemoparasites Infecting Lizards from Morocco.

PURPOSE: Understanding the factors influencing parasite diversification, their distribution and infection patterns among hosts provides valuable information on the impact of parasitism on natural populations and the dynamics and evolution of disease outbreaks. In this study, we investigated prevalence and intensity of infection by blood parasites, the diversity, distribution, host specificity and infection patterns of apicomplexan parasites in lizards of Morocco. METHODS: Samples from lizard specimens were collected between 2019 and 2021 from different prospected localities in Morocco. In total, 1500 lizards were collected, and their blood and tail tips were screened for blood parasites. RESULTS: Using a PCR approach, we detected two distinct apicomplexan parasite groups (haemogregarines and haemococcidians) and also Stramenopiles (Proteromonas). A significant difference was identified between hosts families, with no agamids or skinks infected and with particularly high infection rates in lacertids. Twenty-four samples were sequenced and included in a phylogenetic assessment. Two samples of Atlantolacerta andreanskyi were infected with unidentified species of Proteromonas. Three samples were infected with haemococcidians, including two samples with Schellackia and another sample with Lankesterella, more closely related to others isolated from bird hosts. These are the first reports of Lankesterella and Proteromonas in these lizards from Morocco. Nineteen different isolates of the complex of Hepatozoon/Karyolysus were identical or very similar to previously sequenced parasites from North Africa and Iberian Peninsula and included new host species. CONCLUSION: Our results highlight the need for future research on the vectors involved in transmission that may influence the ecology and distribution patterns of these parasites. Given that Morocco is a hot spot of endemicity and reptile richness, our results are relevant not only in the context of their taxonomy, but can also have implications for conservation.

Parthenogenesis in Darevskia lizards: A rare outcome of common hybridization, not a common outcome of rare hybridization.

Hybridization is a common evolutionary process with multiple possible outcomes. In vertebrates, interspecific hybridization has repeatedly generated parthenogenetic hybrid species. However, it is unknown whether the generation of parthenogenetic hybrids is a rare outcome of frequent hybridization between sexual species within a genus or the typical outcome of rare hybridization events. Darevskia is a genus of rock lizards with both hybrid parthenogenetic and sexual species. Using capture sequencing, we estimate phylogenetic relationships and gene flow among the sexual species, to determine how introgressive hybridization relates to the origins of parthenogenetic hybrids. We find evidence for widespread hybridization with gene flow, both between recently diverged species and deep branches. Surprisingly, we find no signal of gene flow between parental species of the parthenogenetic hybrids, suggesting that the parental pairs were either reproductively or geographically isolated early in their divergence. The generation of parthenogenetic hybrids in Darevskia is, then, a rare outcome of the total occurrence of hybridization within the genus, but the typical outcome when specific species pairs hybridize. Our results question the conventional view that parthenogenetic lineages are generated by hybridization in a window of divergence. Instead, they suggest that some lineages possess specific properties that underpin successful parthenogenetic reproduction.

An unexpected tenant: contamination in a Physeter catodon (Physeteridae, Artiodactyla) genome indicates undescribed species of Sarcocystis Lankester, 1882 (Sarcocystidae, Eucoccidiorida) in the marine environment.

Data mining animal of genomes has been used before to identify endoparasites, and may be a particularly useful tool to surpass some difficulties faced by studies in the marine environment. We detected a species of Sarcocystis Lankester, 1882, contamination in the sperm whale (Physeter catodon Linnaeus) reference genome available in the GenBank database. We identified and extracted multiple gene fragments and placed the sequences in a phylogenetic framework. Our results indicate that the sequences of Sarcocystis sp. found in the genome do not correspond to any currently described species, despite a few other similar sequences having been identified in fur seals (Pinnipedia) and another sperm whale. Including data from previous studies, we suggest there is enough evidence to support the occurrence of at least four species of Sarcocystis in marine mammals. We also demonstrate that the term "S. canis-like" has been used for samples not closely related to Sarcocystis canis Dubey et Speer, 1991.

Diversity of microorganisms in Hyalomma aegyptium collected from spur-thighed tortoise (Testudo graeca) in North Africa and Anatolia.

Ticks carry a diverse community of microorganisms including non-pathogenic symbionts, commensals, and pathogens, such as viruses, bacteria, protozoans, and fungi. The assessment of tick-borne microorganisms (TBM) in tortoises and their ticks is essential to understand their eco-epidemiology, and to map and monitor potential pathogens to humans and other animals. The aim of this study was to characterize the diversity of microorganisms found in ticks collected from the spur-thighed tortoise (Testudo graeca) in North Africa and Anatolia. Ticks feeding on wild T. graeca were collected, and pathogens were screened by polymerase chain reaction using group-specific primers. In total, 131 adult Hyalomma aegyptium ticks were collected from 92 T. graeca in Morocco (n = 48), Tunisia (n = 2), Algeria (n = 70), and Turkey (n = 11). Bacteria and protozoa detected included Hemolivia mauritanica (22.9%), Midichloria mitochondrii (11.4%), relapsing-fever borreliae (8.4%), Ehrlichia spp. (7.6%), Rickettsia spp. (3.4%), Borrelia burgdorferi s.l. (0.9%), Francisella spp. (0.9%), and Wolbachia spp. (0.8%). The characterization of Rickettsia included R. sibirica mongolitimonae (Algeria), R. aeschlimannii (Turkey), and R.africae (Morocco). Hemolivia mauritanica and Ehrlichia spp. prevalence varied significantly with the sampling region/country. We did not detect significant associations in microorganism presence within ticks, nor between microorganism presence and tick mitochondrial DNA haplogroups. This is the first report of Francisella persica-like, relapsing fever borreliae, M. mitochondrii, and Wolbachia spp. in H. aegyptium ticks collected from wild hosts from the South and Eastern Mediterranean region, and of R. sibirica mongolitimonae and R. africae in H. aegyptium from Algeria and Morocco, respectively. Given that T. graeca is a common species in commercial and non-commercial pet trade, the evaluation of the role of this species and its ticks as hosts for TBM is particularly relevant for public health.

Assessing the influence of geographic distance in parasite communities of an exotic lizard.

The decay of similarity between biological communities with increasing geographical distance is a well-established pattern in ecology, but there are more complex factors acting on host population connections that influence this association for parasite communities, such as parasites' colonization ability and degree of connectivity between host populations. Here we aim to determine the helminth communities associated with different populations of the host lizard Hemidactylus mabouia, testing if the similarity of parasite communities decreases as the distance between them increases. For this, we collected samples of lizard populations in seven sites from Northeastern coast of Brazil and identified parasite species of helminths and pentastomids in each host, calculated the Sørensen indices of presence/absence and abundance of each pair of communities and related them to the geographical distance. We did not find a relationship of decaying similarity with increasing distance between the parasite communities of the host populations. This can be explained by factors such as the characteristics of the contact between the host populations, and by modes of transmission of most parasite species. Furthermore, it may be related to the exotic nature of the host in Brazil so that parasite communities have not reached equilibrium.

The taxonomy of the Tarentola mauritanica species complex (Gekkota: Phyllodactylidae): Bayesian species delimitation supports six candidate species.

The lack of morphological diagnosable characters typical of cryptic species, poses a particular problem to taxonomists. This is especially true when taxa are closely related, sharing considerable amounts of ancestral polymorphism. Phylogenetic studies on the Moorish gecko species-complex, Tarentola mauritanica, uncovered extremely high levels of mtDNA diversity with six identified clades, including one from the Canary Islands identified as T. angustimentalis. Because of the conserved morphology of this species and its paraphyletic status with respect to T. angustimentalis, it was suggested that T. mauritanica is a cryptic species complex. Nevertheless, none of the nuclear loci used were reciprocally monophyletic regarding the mitochondrial lineages due to retention of ancestral polymorphism. In this study, we added three new intron markers to the already available dataset and used additional tools, namely phylogenetic gene trees, species tree and species limits within a Bayesian coalescent framework to confirm the support of the main lineages. Bayesian clustering analysis supports all six mtDNA lineages as independent groups, despite showing signs of ancestral polymorphism or possibly gene flow between the Maghreb/South Iberia and Central Morocco clades. The species tree recovered two major groups; one clustering taxa from Europe and Northern Maghreb and another one encompassing the lineages from Central/Southern Morocco, Central Morocco and Canary Islands, indicating that the ancestor of T. angustimentalis came from the Central/Southern Morocco region. Finally, Bayesian coalescent species delimitation analysis supports all six mitochondrial clades as "unconfirmed candidate species", pending morphological data to define them.

A Combination of Divergence and Conservatism in the Niche Evolution of the Moorish Gecko, Tarentola mauritanica (Gekkota: Phyllodactylidae).

The quantification of realized niche overlap and the integration of species distribution models (SDMs) with calibrated phylogenies to study niche evolution are becoming not only powerful tools to understand speciation events, but can also be used as proxies regarding the delimitation of cryptic species. We applied these techniques in order to unravel how the fundamental niche evolved during cladogenesis within the Tarentola mauritanica species-complex. Our results suggest that diversification within this complex, during the Miocene and Pleistocene, is associated with both niche divergence and niche conservatism, with a pattern that varies depending on whether the variables involved are related to the mean or seasonality of temperature and humidity. Moreover, climatic variables related to humidity and temperature seasonality were involved in the niche shift and genetic diversification of the European/North African clade during the Pleistocene and in its maintenance in a fundamental niche distinct from that of the remaining members of the group. This study further highlights the need for a taxonomic revision of the T. mauritanica species-complex.

Prevalence and diversity of Hepatozoon in native and exotic geckos from Brazil.

Hepatozoon is a genus of hemogregarines constituting the most widespread and common reptile hemoparasite. Although various molecular assessments of these parasites have been conducted in lizards from Africa and Europe, similar studies are needed for South American lizards. Through amplification and sequencing of fragments of the 18S rRNA gene, we assess the prevalence of Hepatozoon parasites in 230 geckos from South America, including the endemic species Hemidactylus agrius, Hemidactylus brasilianus, Lygodactylus klugei, Phyllopezus pollicaris, Phyllopezus periosus, and an exotic species, Hemidactylus mabouia . We found an overall low prevalence of Hepatozoon infection (7/230, 3%) with only 3 of the 6 host species infected with Hepatozoon ( Hemidactylus mabouia , P. pollicaris, and P. periosus). Within the 7 infected host samples, 5 genetically distinct lineages of Hepatozoon parasites were identified, only 1 of which was similar to previously published haplotypes. Thus, although prevalence is low, genetically based diversity of Hepatozoon in geckos from South America is very high. Three of these lineages appear basal to 1 of the major clades of Hepatozoon, suggesting that this clade might have originated in South America, and thereby indicating a potential phylogeographic pattern that had not been previously identified. Future studies should assess the distribution and competence of invertebrate hosts in the regions analyzed, and Hepatozoon diversity in other less well-known regions of the world.

Morphological and molecular characterization of Karyolysus--a neglected but common parasite infecting some European lizards.

BACKGROUND: Blood parasites of the genus Karyolysus Labbé, 1894 (Apicomplexa: Adeleida: Karyolysidae) represent the protozoan haemogregarines found in various genera of lizards, including Lacerta, Podarcis, Darevskia (Lacertidae) and Mabouia (Scincidae). The vectors of parasites are gamasid mites from the genus Ophionyssus. METHODS: A total of 557 individuals of lacertid lizards were captured in four different localities in Europe (Hungary, Poland, Romania and Slovakia) and blood was collected. Samples were examined using both microscopic and molecular methods, and phylogenetic relationships of all isolates of Karyolysus sp. were assessed for the first time. Karyolysus sp. 18S rRNA isolates were evaluated using Bayesian and Maximum Likelihood analyses. RESULTS: A total of 520 blood smears were examined microscopically and unicellular protozoan parasites were found in 116 samples (22.3% prevalence). The presence of two Karyolysus species, K. latus and K. lacazei was identified. In total, of 210 samples tested by polymerase chain reaction (PCR), the presence of parasites was observed in 64 individuals (prevalence 30.5%). Results of phylogenetic analyses revealed the existence of four haplotypes, all part of the same lineage, with other parasites identified as belonging to the genus Hepatozoon. CONCLUSIONS: Classification of these parasites using current taxonomy is complex - they were identified in both mites and ticks that typically are considered to host Karyolysus and Hepatozoon respectively. Furthermore although distortions to the intermediate host erythrocyte nuclei were observed, the defining characteristic of Karyolysus, the haplotypes were nearly identical to those reported from lizards in the Iberian Peninsula, where such distortions were not reported and which were thus identified as Hepatozoon. Based on the phylogenetic analyses, neither vertebrate host, nor geographical patterns of the studied blood parasites could be established.

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.

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.

Cyanobacterial hydrogenases: diversity, regulation and applications.

Cyanobacteria may possess two distinct nickel-iron (NiFe)-hydrogenases: an uptake enzyme found in N(2)-fixing strains, and a bidirectional one present in both non-N(2)-fixing and N(2)-fixing strains. The uptake hydrogenase (encoded by hupSL) catalyzes the consumption of the H(2) produced during N(2) fixation, while the bidirectional enzyme (hoxEFUYH) probably plays a role in fermentation and/or acts as an electron valve during photosynthesis. hupSL constitute a transcriptional unit, and are essentially transcribed under N(2)-fixing conditions. The bidirectional hydrogenase consists of a hydrogenase and a diaphorase part, and the corresponding five hox genes are not always clustered or cotranscribed. The biosynthesis/maturation of NiFe-hydrogenases is highly complex, requiring several core proteins. In cyanobacteria, the genes that are thought to affect hydrogenases pleiotropically (hyp), as well as the genes presumably encoding the hydrogenase-specific endopeptidases (hupW and hoxW) have been identified and characterized. Furthermore, NtcA and LexA have been implicated in the transcriptional regulation of the uptake and the bidirectional enzyme respectively. Recently, the phylogenetic origin of cyanobacterial and algal hydrogenases was analyzed, and it was proposed that the current distribution in cyanobacteria reflects a differential loss of genes according to their ecological needs or constraints. In addition, the possibilities and challenges of cyanobacterial-based H(2) production are addressed.