Polystoma nacialtuneli n. sp. (Monogenea: Polystomatidae) from the eastern spadefoot, Pelobates syriacus (Pelobatidae) in Turkey.

Polystoma nacialtuneli n. sp. is described from the urinary bladder of the eastern spadefoot, Pelobates syriacus from Turkey. This is the fifth polystome species known from Turkey and the third species in Pelobates. We show that this new parasite species can be distinguished from other polystome species in the area by a combination of characteristics, including parasite size and the shape and size of the hamuli. Polystoma pelobatis from Pelobates cultripes has a pair of well-developed hamuli, while P. fuscus from Pe. fuscus characteristically has a pair of underdeveloped hamuli barely larger than the marginal hooklets. Polystoma nacialtuneli n. sp. has well-developed hamuli that vary significantly in shape. Phylogenetic relationships of P. nacialtuneli n. sp. within Polystoma, supplemented with molecular divergences estimated from internal transcribed spacer 1 (ITS1) sequences, indicate that they are well separated from their closest relatives, i.e. P. fuscus and P. pelobatis from Pe. fuscus and Pe. cultripes, respectively.

Invasive species threat: parasite phylogenetics reveals patterns and processes of host-switching between non-native and native captive freshwater turtles.

One of the major threats to biodiversity involves biological invasions with direct consequences on the stability of ecosystems. In this context, the role of parasites is not negligible as it may enhance the success of invaders. The red-eared slider, Trachemys scripta elegans, has been globally considered among the worst invasive species. Since its introduction through the pet trade, T. s. elegans is now widespread and represents a threat for indigenous species. Because T. s. elegans coexists with Emys orbicularis and Mauremys leprosa in Europe, it has been suggested it may compete with the native turtle species and transmit pathogens. We examined parasite transfer from American captive to the two native species that co-exist in artificial pools of a Turtle Farm in France. As model parasite species we used platyhelminth worms of the family Polystomatidae (Monogenea) because polystomes have been described from American turtles in their native range. Phylogenetic relationships among polystomes parasitizing chelonian host species that are geographically widespread show patterns of diversification more complex than expected. Using DNA barcoding to identify species from adult and/or polystome eggs, several cases of host switching from exotic to indigenous individuals were illustrated, corroborating that parasite transmission is important when considering the pet trade and in reintroduction programmes to reinforce wild populations of indigenous species.

Alternative parasite development in transmission strategies: how time flies!

Among parasitic platyhelminths with complex life cycles, it has been well documented that transmission opportunities are the main forces shaping the diversity of life-history traits and parasite developmental strategies. While deviations in the development pathway usually involve shortening of life cycles, their extension may also occur following perception of remaining time by parasites. Polystoma gallieni, the monogenean parasite of Hyla meridionalis, is able to trigger two alternative developmental strategies depending on the physiological stage of the tadpoles upon which larvae attach. The distribution and reproductive outputs of both resulting phenotypes were surveyed to address questions about the dynamics of transmission in natural environments. Because modifications in the completion of life cycles can have drawbacks which may perturb the dynamic equilibrium of the resulting host-parasite systems, experimental infestations were also performed to assess parasite-parasite interactions. Our results suggest that the bladder adult phenotype, which involves transmission between frogs and tadpoles, is supplied secondarily by the branchial phenotype which involves transmission between tadpoles and metamorphs. They also support the occurrence of finely tuned trade-offs between hosts and parasites and highlight positive trends behind the extension of direct life cycles, in which host-derived signals account for the remaining time to achieve parasitic transmission.

Host specificity is linked to intraspecific variability in the genus Lamellodiscus (Monogenea).

SUMMARYWe investigated whether host specificity is linked to variability within species of Lamellodiscus monogeneans, which are gill ectoparasites of the Sparidae. We sampled fish parasites in the northeastern part of the Mediterranean Sea: 4 specialist species, using 1 single host species, and 3 generalist species, using 2 distinct host species. Intraspecific variability was assessed from 2 different datasets. Morphometric variability of the attachment organ, called the haptor, was estimated first from measurements of several sclerified haptoral parts on 102 individuals. Genetic variability was calculated based on comparisons of sequences derived from the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA of 62 individuals. Morphometric variances in the specialist versus generalist species were compared via principal component analysis and F-tests, and uncorrected genetic distances (p-distances) were estimated within each species. We showed that the inter-individual variance of morphometric characters, as well as p-distances, are clearly greater within generalist species than specialist ones. These findings suggest that a relative increase in morphological and molecular variability enhances the possibility to colonize new host species in monogeneans, and supports the hypothesis that intraspecific variability could be a potential determinant of host specificity.

The influence of intensity of infection by a trematode parasite on the reproductive biology of Gammarus insensibilis (Amphipoda).

Following the behavioural alterations induced by the trematode Microphallus papillorobustus Rankin 1940 (Trematoda, Microphallidae) on its second intermediate host, the amphipod Gammmarus insensibilis, infected individuals are likely to mate among themselves. We investigated the influence of parasite intensity on the reproductive biology of infected hosts. In the mating system of amphipods, males compete severely for access to females and large males have greater ability to obtain large and more fecund females. We showed that the null hypothesis of random pair formation according to parasite intensity could not be rejected. In addition, infected males obtained females of the expected size according to their own sizes, whatever their parasite intensities. However, in both males and females, the parasite intensity increased the intermoult duration. Because size and reproductive success are strongly correlated in amphipods, we discuss the influence of this process on host fitness.

Parasites as host [corrected] evolutionary prints: insights into host evolution from parasitological data.

The close relationships which link parasitic organisms to their hosts have led to the use of parasites as biological tags. Most studies on this topic refer to parasites as host ecological tags. Recent development of molecular methods which give access to the genomic structures of populations have provided new information on the evolutionary biology of parasites. In this paper, we have attempted to review whether parasites can be considered as "host evolutionary prints", and focus our discussion on host biodiversity and biogeography.

Ancient DNA from Ascaris: extraction amplification and sequences from eggs collected in coprolites.

On the Middle-Age site of Namur (Belgium) the analysis of coprolites revealed the presence of many well-preserved Ascaris eggs. Following rehydratation of the coprolite samples, 104 eggs were collected and extracted with an ultrasonication and phenol-chloroform based method. Three overlapping fragments of the 18S rRNA gene and one fragment of the cytochrome b gene have been reproducibly amplified, cloned and sequenced. The analysis of these sequences confirms the identification of the eggs as coming from Ascaris. Our study reveals that coprolites can be an interesting source of parasites that can be readily identified using molecular approaches. The study of ancient DNA from helminth parasites is of interest as it may answer long-standing questions in the history of infectious diseases and gives a possibility to compare these ancient sequences with those of modern populations.

Origin and evolution of African Polystoma (Monogenea: Polystomatidae) assessed by molecular methods.

Among Polystomatidae (Monogenea), the genus Polystoma, which mainly infests neobatrachian hosts, is the most diverse and occurs principally in Africa, from where half the species have been reported. Previous molecular phylogenetic studies have shown that this genus originated in South America, and later colonised Eurasia and Africa. No mention was made on dispersal corridors between Europe and Africa or of the origin of the African Polystoma radiation. Therefore, a molecular phylogeny was inferred from ITS1 sequences of 21 taxa comprising two species from America, seven representatives from Europe and 12 from Africa. The topology of the phylogenetic tree reveals that a single event of colonisation took place from Europe to Africa and that the putative host carrying along the ancestral polystome is to be found among ancestral pelobatids. Percentage divergences estimates suggest that some presumably distinct vesicular species in unrelated South African anurans and some neotenic forms found in several distinct hosts in Ivory Coast, could, in fact, belong to two single polystome species parasitising divergent hosts. Two main factors are identified that may explain the diversity of African polystomes: (i), we propose that following some degree of generalism, at least during the juvenile stages of both hosts and parasites, distinctive larval behaviour of polystomes engenders isolation between parasite populations that precludes sympatric speciations; (ii), cospeciation events between Ptychadena hosts and their parasites are another factor of diversification of Polystoma on the African continent. Finally, we discuss the systematic status of the Madagascan parasite Metapolystoma, as well as the colonisation of Madagascar by the host Ptychadena mascareniensis.

Phylogenetic relationships and evolution of cleaning behaviour in the family Labridae: importance of body colour pattern.

The family Labridae contains numerous fishes known to act as cleaners in the wild. Previous studies suggested that a small body size and specific colour patterns may be prerequisites for cleaning. We investigated whether cleaning behaviour is linked to particular fish phenotypes. We first present a phylogeny based on partial 12S rRNA gene sequences of 32 wrasses sampled from different localities in the Indo-Pacific and Atlantic oceans, and in the Mediterranean Sea. Secondly, descriptive data (fish body size, fish body shape and fish body colour patterns) were analyzed in a phylogenetic context using comparative methods. We found no relationship between fish cleaning behaviour and fish body size and shape, but instead a correlation between cleaning behaviour and the presence of a dark lateral stripe within wrasses. Our results suggest that the evolution of cleaning depends upon the presence of a dark median lateral stripe on the fish body surface.

Evolutionary relationships of sibling tapeworm species (Cestoda) parasitizing teleost fishes.

DNA/DNA hybridization and sequencing of rDNA (partial 18S rDNA and ITS1) were used to investigate phylogenetic relationships among seven host-specific Bothriocephalus parasites (Cestoda, Pseudophyllidae). The small nucleotide divergence between six of the seven bothriocephalids suggests that isolation and differentiation of Bothriocephalus lineages in the different host species probably occurred recently and over a short time span. Comparison of the molecular phylogeny of the parasite species to the phylogeny of their hosts (teleostean fishes) revealed little congruence between the branching patterns of hosts and parasites, suggesting that bothriocephalids have not cospeciated with their hosts.

DNA reassociation kinetics and genome complexity of a fish (Psetta maxima: Teleostei) and its gut parasite (Bothriocephalus gregarius: Cestoda).

1. The genomic structure of a fish (Psetta maxima) and of a Tapeworm (Bothriocephalus), who form a close host-parasite association, was determined by reassociation kinetics experiments. 2. Spectrophotometric readings of single-stranded versus double-stranded DNA separated on hydroxylapatite columns after reassociation at Cot values ranging from 0.0001 to 10(5) allowed the drawing of the reassociation curves of both genomes. 3. Different fractions according to their degree of repetitivity were evidenced, and the relative amounts of repetitive versus single-copy sequences, as well as their complexity, were calculated. 4. It appears that the amount of non-repetitive DNA is lower in the Tapeworm than in its vertebrate host, although the complexity of these single-copy sequences is the same.

Determination of the evolutionary relationships in Rattus sensu lato (Rodentia : Muridae) using L1 (LINE-1) amplification events.

We determined approximately 215 bp of DNA sequence from the 3'-untranslated region (UTR) of 240 cloned L1 (LINE-1) elements isolated from 22 species of Rattus sensu lato and Rattus sensu stricto murine rodents. The sequences were sorted into different L1 subfamilies, and oligonucleotides cognate to them were hybridized to genomic DNA of various taxa. From the distribution of the L1 subfamilies in the various species, we inferred the partial phylogeny of Rattus sensu lato. The four Maxomys species comprise a well-defined clade separate from a monophyletic cluster that contains the two Leopoldamys and four Niviventer species. The Niviventer/Leopoldamys clade, in turn, shares a node with the clade that contains Berylmys, Sundamys, Bandicota, and Rattus sensu stricto. The evolutionary relationships that we deduced agree with and significantly extend the phylogeny of Rattus sensu lato established by other molecular criteria. Furthermore, the L1 amplification events scored here produced a unique phylogenetic tree, that is, in no case did a character (a given L1 amplification event) appear on more than one branch. The lack of homoplasy found in this study supports the robustness of L1 amplification events as phylogenetic markers for the study of mammalian evolution.

Molecular relationships between closely related species of Bothriocephalus (Cestoda:Platyhelminthes).

DNA/DNA hybridization has been carried out to establish the evolutionary relationships among several host-specific bothriocephalid tapeworms species (Cestoda: Platyhelminthes). Comparative anatomy is not informative for deciphering the relationships among these sibling morphological taxa, and clearcut genetic differences have previously identified seven biological species which are each highly specific to a single host. We show that two species (Bothriocephalus gregarius and B. renaudii) infesting the same host (turbot: Psetta maxima) in two different geographic areas are not sister-taxa. Moreover, a strong decrease in the amount of DNA change is observed in one species of tapeworm, documenting a marked heterogeneity of rates of nucleotidic substitution among these very closely related organisms. Based on these observations, different hypotheses are developed for understanding the evolutionary history of this assemblage of parasites, suggesting that host-switching has played an important role in the recent past.

Determining and dating recent rodent speciation events by using L1 (LINE-1) retrotransposons.

Phylogenies based on the inheritance of shared derived characters will be ambiguous when the shared characters are not the result of common ancestry. Such characters are called homoplasies. Phylogenetic analysis also can be problematic if the characters have not changed sufficiently, as might be the case for rapid or recent speciations. The latter are of particular interest because evolutionary processes may be more accessible the more recent the speciation. The repeated DNA subfamilies generated by the mammalian L1 (LINE-1) retrotransposon are apparently homoplasy-free phylogenetic characters. L1 retrotransposons are transmitted only by inheritance and rapidly generate novel variants that produce distinct subfamilies of mostly defective copies, which then "age" as they diverge. Here we show that the L1 character can both resolve and date recent speciation events within the large group of very closely related rats known as Rattus sensu stricto. This lineage arose 5-6 million years ago (Mya) and subsequently underwent two episodes of speciation: an intense one, approximately 2.7 Mya, produced at least five lineages in <0.3 My; a second began approximately 1.2 Mya and may still be continuing.

Characterization of cDNA encoding a L37a ribosomal protein from Taenia crassiceps and its potential use in phylogenetic reconstructions.

In this study, we characterized for the first time the complete sequence of a L37a cDNA from a cestode specie: Taenia crassiceps. A phylogenetic analysis of L37a ribosomal proteins from distant animal species is presented and the potential use of such proteins in molecule-based phylogeny is discussed.

[Africa or Asia, which is the evolutionary origin of human schistosomes?]. / Afrique ou Asie, quelle est l'origine évolutive des schistosomes humains?

The origin and the evolution of Schistosomatidae species, due to their medical importance (responsible of the second most important human parasitosis after malaria), arouse a great interest. A combination of phylogenetic studies using several molecular markers has provided support for the traditional grouping and evolutionary inferences derived from morphological and biological data. The genus Schistosoma, which comprises all species parasitizing Man, is generally split into four evolutionary lineages (mansoni, haematobium, indicum and japonicum lineages). The group of African schistosomes (including mansoni and haematobium lineages) appears very divergent from the japonicum lineage. Recent phylogenetic studies using partial 28S rDNA sequencing and including Orientobilharzia turkestanicum from Iran, an Asian parasite of livestock, found, unexpectedly, that this species nested among Schistosoma species, thus rendering the latter paraphyletic, and suggested an Asian origin for the Schistosoma genus. The present work re-examines the question of the geographical origin of human schistosomes by analysing a new genomic marker (ITS2) as well as by including the use of O. turkestanicum originating from northeastern China. Our results are in agreement with previous work using 28S, in demonstrating that Schistosoma is not monophyletic. However, O. turkestanicum, whatever the method of analysis used (distance or parsimony), was grouped with members of the japonicum group to the exclusion of African Schistosoma species. Then, our data argue strongly for the need for further phylogenetic study including new taxa and new genomic sequences before definitely concluding either an Asian or African origin for the genus Schistosoma.

Evidence of two genetic entities in Bothriocephalus funiculus (Cestoda) detected by arbitrary-primer polymerase chain reaction random amplified polymorphic DNA fingerprinting.

The genetic diversity of two samples of Cestoda (Bothriocephalus funiculus, Renaud and Gabrion, 1984) parasitizing two sympatric teleostean species was assessed using random amplified polymorphic DNA (RAPD). A total of 72 Bothriocephalus were analyzed individually, and electrophoretic analysis of the amplification products of 65 primers among the 68 tested revealed monomorphic patterns, reflecting the close genetic relatedness within and between the parasites of the two samples. However, 3 primers showed polymorphic patterns at 6 RAPD sites. Analysis of the distribution of these genomic fragments, assuming random mating, showed strong linkage disequilibria (only 8 genetic combinations were observed among the 32 expected). Two genetic entities displaying a high degree of host specificity were evidenced within our two samples of funiculus. This powerful molecular technique can be used as a diagnostic tool in studies concerning the biodiversity of related genetic entities and could have broad applications in parasitology.

Molecular phylogenetic analysis of the genus Gyrodactylus (Platyhelminthes: Monogenea) inferred from rDNA ITS region: subgenera versus species groups.

Analyses of small subunit ribosomal RNA gene sequences of representatives of major taxa of Monopisthocotylea were performed to identify the sister group of Gyrodactylus. Nuclear ribosomal DNA sequences from the complete internal transcribed spacer (ITS) region were used to infer phylogeny of 37 Gyrodactylus species and Gyrodactyloides bychowskii, Macrogyrodactylus polypteri and Gyrdicotylus gallieni, using maximum likelihood, parsimony and Bayesian inference. The genus Gyrodactylus appeared to be a monophyletic group in all analyses, based on the present data set. Within the genus, there were 3 major groups recognized by high bootstrap values and posterior probabilities. None of the 6 subgenera appeared to be monophyletic, and the most basal subgenus G. (Gyrodactylus) was paraphyletic. Characteristics of the excretory system of Gyrodactylus do not seem to be conservative enough to reveal subgenera within Gyrodactylus and we suggest abandoning existing subgenera as indicators of phylogeny. The grouping of species based on the morphology of the ventral bar and marginal hooks seems to have sufficient power to infer relationships between the Gyrodactylus species.

A Paedomorphic parasite associated with a neotenic amphibian host: phylogenetic evidence suggests a revised systematic position for Sphyranuridae within anuran and turtle Polystomatoineans.

The phylogenetic relationships of the families Polystomatidae and Sphyranuridae (subclass Polystomatoinea) within tetrapod monogenean parasites were investigated using partial 18S rDNA sequences. About 600 nucleotides of 11 species were sequenced, including 7 species of the most common subfamilies of Polystomatidae found in anurans and turtles, 1 species of the family Sphyranuridae parasitizing exclusively urodelans, and 3 species of the subclass Oligonchoinea infesting teleostean fishes. The phylogenetic analyses were performed using three reconstruction methods: neighbor-joining, maximum-parsimony, and maximum-likelihood. Polystomatoineans but not polystomatids were shown to be monophyletic. Within the polystomatoineans there are two clades: one includes the amphibian monogeneans (anuran polystomatids and urodelan sphyranurids) and the other includes the turtle polystomatids. Polystomatoineans may have coevolved with amphibian hosts, and an ancestral "polystome" dispersed at least 200 million years ago, either from the basal stem of lissamphibians or from an anuran ancestral stock, to freshwater turtles. Furthermore, the urodelan genus Sphyranura, initially assigned to the family Sphyranuridae on the basis of morphological and ontogenetic evidence, is clearly nested within polystomatids, suggesting that its systematic status must be revised. This supports recent findings which argue that species of the family Sphyranuridae may be paedomorphic parasites exclusively infesting neotenic mudpuppies.