The freshwaters of Iraq harbour a high diversity of endemic and phylogenetically unique species. One of the most diversified fish groups in this region is cyprinoids, and although their distribution is relatively well known, their monogenean parasites have only rarely been investigated. Herein, we applied an integrative approach, combining morphology with molecular data, to assess the diversity and phylogeny of cyprinoid-associated monogenean parasites. A total of 33 monogenean species were collected and identified from 13 endemic cyprinoid species. The highest species diversity was recorded for Dactylogyrus (Dactylogyridae, 16 species) and Gyrodactylus (Gyrodactylidae, 12 species). Four species of Dactylogyrus and 12 species of Gyrodactylus were identified as new to science and described. Two other genera, Dogielius (Dactylogyridae) and Paradiplozoon (Diplozoidae), were represented only by 4 and 1 species, respectively. Phylogenetic analyses of the Dactylogyrus and Gyrodactylus species revealed that the local congeners do not form a monophyletic group and are phylogenetically closely related to species from other regions (i.e. Europe, North Africa and Eastern Asia). These findings support the assumption that the Middle East served as an important historical crossroads for the interchange of fauna between these 3 geographic regions.

We describe two new species of monogenean parasites of the genus Gyrodactylus von Nordmann, 1832 infecting Neotropical catfishes (Siluriformes) in southern Mexico: Gyrodactylus chulini n. sp. from ‘chulín’, Rhamdia laticauda collected in Oaxaca; and Gyrodactylus juili n. sp. from ‘juil’, Rhamdia guatemalensis from Veracruz. Morphologically, both new taxa are similar to Gyrodactylus spp. infecting catfishes (Siluriformes) in South America. Sequences of the internal transcribed spacers (ITS1-5.8S–ITS2 rDNA), the D2+D3 domains of the large ribosomal subunit (28S rDNA) and the cytochrome oxidase II (COII) gene were obtained from multiple parasite specimens and analysed using Bayesian inference. Phylogenetic hypotheses using ITS rDNA and COII genes, recovered two new Gyrodactylus species from Rhamdia spp.: G. chulini n. sp.; and Gyrodactylus juili n. sp., which are sister species to Gyrodactylus lilianae, a parasite of Rhamdia quelen in Brazil, and show strong affinity to other gyrodactytlids infecting Neotropical catfishes. This suggests that these new taxa, the first gyrodactylids described from Rhamdia spp. in Mexico, co-migrated to Tropical Middle America with their Neotropical catfish hosts, after the emergence of the Isthmus of Panama.

Gyrodactylus sphinx Dmitrieva & Gerasev, 2000 is the only species of Gyrodactylus originally described from Aidablennius sphynx (Valenciennes) in the Black Sea. In the present study, monogeneans similar to G. sphinx are reported from the same host and from two other species of Blenniidae from the Black Sea, as well as from the Mediterranean Sea. This study aims to verify the taxonomic status of the specimens found in different hosts and localities, other than the type ones of G. sphinx. Twenty-two measurements of the haptoral structures of 169 gyrodactylids were used for the morphological study. Morphometric variability between different samples was analysed using analysis of variance, multivariate analysis of variance (MANOVA) and principal component analysis (PCA). Molecular studies were carried out using the nuclear internal transcribed spacer 2 and 5.8S ribosomal DNA regions. Network, Bayesian phylogenetic and species delimitation analyses were performed to infer the number of taxonomic units and the phylogeographic relationships occurring within and among them. MANOVA revealed a significant dependence of the morphometry of hamuli and marginal hooks on host species and regions, but a clear differentiation between samples was not confirmed by PCA. Moreover, the ranges of all dimensions overlapped between samples. However, molecular analyses suggested the occurrence of at least two taxonomic entities. The most common entity was present in individuals of the Black and Mediterranean seas, and is described here as Gyrodactylus gerasevi n. sp., whereas a second entity recognized as a G. sphinx was found only in individuals from two localities off Crimea. The monophyletic cluster grouping of these two species was placed within a large clade that also included a separate sister cluster with seven other species of the Gyrodactylus orecchiae cross-ocean species group.

This study assessed the role of historical processes on the geographic isolation, molecular evolution, and morphological diversification of host-parasite populations from the southern Brazilian coast. Adult specimens of Scleromystax barbatus and Scleromystax macropterus were collected from the sub-basin of the Nhundiaquara River and the sub-basin of the Paranaguá Bay, state of Paraná, Brazil. Four species of Gyrodactylus were recovered from the body surface of both host species. Morphometric analysis of Gyrodactylus spp. and Scleromystax spp. indicated that subpopulations of parasites and hosts could be distinguished from different sub-basins and locations, but the degree of morphological differentiation seems to be little related to geographic distance between subpopulations. Phylogenetic relationships based on DNA sequences of Gyrodactylus spp. and Scleromystax spp. allowed distinguishing lineages of parasites and hosts from different sub-basins. However, the level of genetic structuring of parasites was higher in comparison to host species. Evidence of positive selection in mtDNA sequences is likely associated with local adaptation of lineages of parasites and hosts. A historical demographic analysis revealed that populations of Gyrodactylus and Scleromystax have expanded in the last 250 000 years. The genetic variation of parasites and hosts is consistent with population-specific selection, population expansions, and recent evolutionary co-divergence.

While group formation provides antipredatory defences, increases foraging efficiency and mating opportunities, it can be counterintuitive by promoting disease transmission amongst social hosts. Upon introduction of a pathogen, uninfected individuals often modify their social preferences to reduce infection risk. Infected hosts also exhibit behavioural changes, for example, removing themselves from a group to prevent an epidemic. Conversely, here we show how Trinidadian guppies infected with a directly transmitted ectoparasite, Gyrodactylus turnbulli, significantly increase their contact rates with uninfected conspecifics. As uninfected fish never perform this behaviour, this is suggestive of a parasite-mediated behavioural response of infected hosts, presumably to offload their parasites. In the early stages of infection, however, such behavioural modifications are ineffective in alleviating parasite burdens. Additionally, we show that fish exposed to G. turnbulli infections for a second time, spent less time associating than those exposed to parasites for the first time. We speculate that individuals build and retain an infection cue repertoire, enabling them to rapidly recognize and avoid infectious conspecifics. This study highlights the importance of considering host behavioural modifications when investigating disease transmission dynamics.

To better understand the spread of disease in nature, it is fundamentally important to have broadly applicable model systems with readily available species which can be replicated and controlled in the laboratory. Here we used an experimental model system of fish hosts and monogenean parasites to determine whether host sex, group size and group composition (single-sex or mixed-sex) influenced host-parasite dynamics at an individual and group level. Parasite populations reached higher densities and persisted longer in groups of fish compared with isolated hosts and reached higher densities on isolated females than on isolated males. However, individual fish within groups had similar burdens to isolated males regardless of sex, indicating that females may benefit more than males by being in a group. Relative condition was positively associated with high parasite loads for isolated males, but not for isolated females or grouped fish. No difference in parasite dynamics between mixed-sex groups and single-sex groups was detected. Overall, these findings suggest that while host sex influences dynamics on isolated fish, individual fish in groups have similar parasite burdens, regardless of sex. We believe our experimental results contribute to a mechanistic understanding of host-parasite dynamics, although we are cautious about directly extrapolating these results to other systems.

Parasites are detrimental to host fitness and therefore should strongly select for host defence mechanisms. Yet, hosts vary considerably in their observed parasite loads. One notable source of inter-individual variation in parasitism is host sex. Such variation could be caused by the immunomodulatory effects of gonadal steroids. Here we assess the influence of gonadal steroids on the ability of guppies (Poecilia reticulata) to defend themselves against a common and deleterious parasite (Gyrodactylus turnbulli). Adult male guppies underwent 31 days of artificial demasculinization with the androgen receptor-antagonist flutamide, or feminization with a combination of flutamide and the synthetic oestrogen 17 β-estradiol, and their parasite loads were compared over time to untreated males and females. Both demasculinized and feminized male guppies had lower G. turnbulli loads than the untreated males and females, but this effect appeared to be mainly the result of demasculinization, with feminization having no additional measurable effect. Furthermore, demasculinized males, feminized males and untreated females all suffered lower Gyrodactylus-induced mortality than untreated males. Together, these results suggest that androgens reduce the ability of guppies to control parasite loads, and modulate resistance to and survival from infection. We discuss the relevance of these findings for understanding constraints on the evolution of resistance in guppies and other vertebrates.

Parasites typically have low reproductive fitness on paratenic hosts. Such hosts offer other significant inclusive fitness benefits to parasites, however, such as increased mobility and migration potential. The parasite fauna of the guppy (Poecilia reticulata) is dominated by the directly transmitted ectoparasites Gyrodactylus bullatarudis and Gyrodactylus turnbulli. In the wild, close predatory and competitive interactions occur between the guppy and the killifish Rivulus hartii. Previous observations suggest that these fish can share gyrodactylids, so we tested experimentally whether these parasites can use R. hartii as an alternative host. In aquaria, G. bullatarudis was the only species able to transmit from prey to predator. Both parasite species transferred equally well to prey when the predator was experimentally infected. However, in semi-natural conditions, G. bullatarudis transmitted more successfully to the prey fish. Importantly, G. bullatarudis also survived significantly longer on R. hartii out of water. As R. hartii can migrate overland between isolated guppy populations, G. bullatarudis may have an enhanced ability to disperse and colonize new host populations, consistent with its wider distribution in the wild. To our knowledge, this is the first empirical study demonstrating a predator acting as a paratenic host for the parasites of its prey.

Understanding disease transmission is important to species management and human health. Host body condition, nutrition and disease susceptibility interact in a complex manner, and while the individual effects of these variables are well known, our understanding of how they interact and translate to population dynamics is limited. Our objective was to determine whether host relative body condition influences epidemic dynamics, and how this relationship is affected by food availability. Poecilia reticulata (guppies) of roughly similar size were selected and assembled randomly into populations of 10 guppies assigned to 3 different food availability treatments, and the relative condition index (Kn) of each fish was calculated. We infected 1 individual per group (‘source’ fish) with Gyrodactyus turnbulli and counted parasites on each fish every other day for 10 days. Epidemic parameters for each population were analysed using generalized linear models. High host Kn—particularly that of the ‘source’ fish—exerted a positive effect on incidence, peak parasite burden, and the degree of parasite aggregation. Low food availability increased the strength of the associations with peak burden and aggregation. Our findings suggest that host Kn and food availability interact to influence epidemic dynamics, and that the condition of the individual that brings the parasite into the host population has a profound impact on the spread of infection.

The enemy release hypothesis proposes that in parasite depleted habitats, populations will experience relaxed selection and become more susceptible (or less tolerant) to pathogenic infections. Here, we focus on a population of guppies (Poecilia reticulata) that are found in an extreme environment (the Pitch Lake, Trinidad) and examine whether this habitat represents a refuge from parasites. We investigated the efficacy of pitch in preventing microbial infections in Pitch Lake guppies, by exposing them to dechlorinated water, and reducing gyrodactylid infections on non-Pitch Lake guppies by transferring them to Pitch Lake water. We show that (i) natural prevalence of ectoparasites in the Pitch Lake is low compared to reference populations, (ii) Pitch Lake guppies transferred into aquarium water develop microbial infections, and (iii) experimentally infected guppies are cured of their gyrodactylid infections both by natural Pitch Lake water and by dechlorinated water containing solid pitch. These results indicate a role for Pitch Lake water in the defence of guppies from their parasites and suggest that Pitch Lake guppies might have undergone enemy release in this extreme environment. The Pitch Lake provides an ideal ecosystem for studies on immune gene evolution in the absence of parasites and long-term evolutionary implications of hydrocarbon pollution for vertebrates.

The small subunit ribosomal RNA (srRNA) gene was amplified from Gyrodactylus salaris using the polymerase chain reaction (PCR), cloned, and the complete gene sequence of 1966 bp determined. The V4 region of the srRNA gene was identified and amplified from single specimens of G. salaris, G. derjavini and G. truttae. Comparison of the V4 sequences from these three species revealed sequence differences from which restriction fragment length polymorphisms (RFLPs) were predicted and an oligonucleotide probe (GsV4) specific to G. salaris designed. Digestion of the amplified V4 region of the srRNA gene with Hae III and either Alw I, BstY I, Dde I or Mbo I provided a means of discriminating between G. salaris, G. derjavini and G. truttae. The GsV4 probe was used to detect the srRNA gene from G. salaris in Southern and dot blots of the amplified V4 region. The nucleotide sequences reported in this paper have been submitted to the EMBL Data Library under accession numbers Z26942 (G. salaris), Z35128 (G. derjavini) and Z35129 (G. truttae).

The effect of waterborne zinc (Zn) on Gyrodactylus population dynamics was studied on isolated guppies maintained at concentrations ranging from 0 to 240 μg Zn/l. After 1 week pre-exposure to Zn, each fish was experimentally infected with 3 gyrodactylids and parasite numbers were recorded daily on each fish until the fish either died or recovered from infection. Parasite establishment was most successful at 0 and 240 μg Zn/l (97%) compared with the intermediate Zn concentrations. Low to moderate concentrations of Zn were beneficial to the parasite, as evidenced by the concentration-dependent increase in peak parasite burden on recovered fish up to 120 μg Zn/l. In contrast, 240 μg Zn/l may have been toxic to the parasite, as both peak parasite burden (in fish that recovered from infection), and maximum rate of increase of the parasite population (in fish that died) declined at this concentration. The combined effect of infection and Zn is harmful to the fish, because mortality of infected fish (but not uninfected fish) increased with increasing Zn concentrations. We suggest that the observed mortality occurs because of the inability of fish to continuously produce mucous that is a key factor for protecting fish from both waterborne Zn and ectoparasites.

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 sequencesNucleotide sequence data reported in this paper are available in the GenBank, DDBJ and EMBL databases under the accession numbers AJ566375-79, AJ566768, AJ567670-74, AJ581657. 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.

This paper adds new insight to a molecular phylogeny of Gyrodactylus, based on a complete sequence of the ITS rDNA region of 4 subgenera and a more detailed molecular analysis. We propose a hierarchical approach in elucidating the phylogeny of this species-rich genus. A total of 37 sequences (915–1239 bp) from 10 representative species from 4 out of 6 subgenera, as defined by Malmberg (1970), are included in the analysis. Genetic differences observed at the 5.8S locus provide objective criteria to separate (sub)genera, while deep genetic differences of the spacers form a sound basis for species-specific identification. We demonstrate that each Gyrodactylus subgenus possesses a unique sequence of the 5.8S gene. Thus, there is concordance between the 5.8S gene and the excretory system used by Malmberg (1970) as a diagnostic character of subgenus status. At the species level, there is a discrepancy between morphological and molecular variation. Whereas the morphological variation, expressed in the shape and size of the attachment apparatus, is very low, the molecular variation, expressed at the ITS1 and ITS2 regions, is very high. This can be attributed either to the fast evolving ITS region or to the fact that the genus consists of groups of a higher taxonomic level than previously recognized. Perspectives and limitations of using the ITS rDNA region for a molecular phylogeny of this genus are discussed.

The identification and discrimination of 2 closely related and morphologically similar species of Gyrodactylus, G. salaris and G. thymalli, were assessed using the statistical classification methodologies Linear Discriminant Analysis (LDA) and k-Nearest Neighbours (KNN). These statistical methods were applied to morphometric measurements made on the gyrodactylid attachment hooks. The mean estimated classification percentages of correctly identifying each species were 98·1% (LDA) and 97·9% (KNN) for G. salaris and 99·9% (LDA) and 73·2% (KNN) for G. thymalli. The analysis was expanded to include another 2 closely related species and the new classification efficiencies were 94·6% (LDA) and 98·0% (KNN) for G. salaris; 98·2% (LDA) and 72·6% (KNN) for G. thymalli; 86·7% (LDA) and 91·8% (KNN) for G. derjavini; and 76·5% (LDA) and 77·7% (KNN) for G. truttae. The higher correct classification scores of G. salaris and G. thymalli by the LDA classifier in the 2-species analysis over the 4-species analysis suggested the development of a 2-stage classifier. The mean estimated correct classification scores were 99·97% (LDA) and 99·99% (KNN) for the G. salaris–G. thymalli pairing and 99·4% (LDA) and 99·92% (KNN) for the G. derjavini–G. truttae pairing. Assessment of the 2-stage classifier using only marginal hook data was very good with classification efficiencies of 100% (LDA) and 99·6% (KNN) for the G. salaris–G. thymalli pairing and 97·2% (LDA) and 99·2% (KNN) for the G. derjavini–G. truttae pairing. Paired species were then discriminated individually in the second stage of the classifier using data from the full set of hooks. These analyses demonstrate that using the methods of LDA and KNN statistical classification, the discrimination of closely related and pathogenic species of Gyrodactylus may be achieved using data derived from light microscope studies.

Gyrodactylus arcuatus-like monogeneans collected from the gills of 3 goby species, Pomatoschistus minutus, P. lozanoi and P. pictus, from the Voordelta Area (SW Netherlands) were compared on a morphometric basis with G. arcuatus from its type host, the three-spined stickleback, Gasterosteus aculeatus. Univariate and multivariate analysis was carried out on measurements of 17 characters of the opisthaptoral hard parts of 268 specimens. The contribution of host-dependent and seasonal factors to the observed morphological variation are discussed. Univariate statistics (ANOVA) were used to detect features that were useful in separating the gyrodactylids from the different host groups. Subsequent factor analysis and discriminant analysis, combining all variables, led to the separation of 3 distinct forms (possibly species) dependent on the host species harbouring them. G. arcuatus (sensu stricto) was only present on G. aculeatus, while P. minutus and P. lozanoi carried a common species, Gyrodactylus sp. 1, and P. pictus hosted Gyrodactylus sp. 2. Seasonal variation in size of the sclerities was demonstrated on the Gyrodactylus species from P. minutus and P. lozanoi, and should be taken into consideration when specimens are compared. The findings are discussed in view of the possibility of phylogenetic host specificity of the gyrodactylids occurring on goby species that live sympatrically in the Voordelta area.