Novel insights into the genetics, morphology, distribution and hosts of the global fish parasitic digenean Proctoeces maculatus (Looss, 1901) (Digenea: Fellodistomidae).

Larval stages of the widely distributed digenean species Proctoeces maculatus (Looss, 1901) were reported 40 years ago from South Africa in the common octopus, Octopus vulgaris Cuvier (Octopodidae). However, the absence of adult specimens and molecular data from this region has hindered a comprehensive understanding of its distribution. In this study, we collected three species of intertidal and near-shore marine fishes [Clinus superciliosus (L.) (Clinidae), Diplodus capensis (Smith) (Sparidae) and Sparodon durbanensis (Castelnau) (Sparidae)] along the South African coast and discovered adult specimens of P. maculatus at five localities. By employing a combination of morphological and molecular techniques, including 28S rDNA, 18S rDNA and COI mtDNA analyses, the first report of adult P. maculatus from South Africa is presented. The findings encompass a comprehensive morphological description and molecular data, illuminating the true distribution of this species in the region.

A stable isotope analysis of the dietary patterns of the aquatic apex predator, the African tigerfish (Hydrocynus vittatus).

Stable isotope analyses, specifically δ13 C and δ15 N, are useful tools increasingly used to understand ecosystem function, food web structures, and consumer diets. Although the iconic tigerfish Hydrocynus vittatus is regarded as an apex predator in southern African freshwater systems, little information is available regarding their feeding behavior and how this may change with growth or differ between ecosystems, with most information stemming from stomach content analyses (SCA). The aim of the present study was to address this lack of information through a baseline study of the diet of large and small tigerfish in various lentic and lotic ecosystems in South Africa using stable isotope methods. Fish and various food web components and food sources were collected from two river and two lake ecosystems in South Africa. The δ13 C and δ15 N values for all samples were determined and multivariate analyses and Bayesian analytical techniques applied to determine the feeding ecology of H. vittatus and how this may differ with size and habitat type. Analyses revealed a substantial difference in the type and abundance of food sources contributing to the diet of H. vittatus between ecosystems, most prominently between the lotic systems, where less dietary specialization was observed, and lentic systems where more specialization was observed. Furthermore, there was a distinct difference in diet between small and large tigerfish, especially in the lotic system, indicating an ontogenetic diet shift as tigerfish grow and further supporting previous SCA studies. This is the first study of its kind on the African continent for H. vittatus and the findings illustrate the value of stable isotope analysis in providing in-depth information into the feeding ecology of consumers and how this may differ between size classes and habitat types.

A new species of Phoreiobothrium Linton, 1889 (Cestoda: Onchoproteocephalidea) from the spinner shark, Carcharhinus brevipinna (Valenciennes) off the coast of KwaZulu-Natal, South Africa.

The extreme sparsity of collection efforts in many parts of the word, including southern Africa, leaves a vast hidden diversity of marine cestode species, such as species of Phoreiobothrium. The examination of a common South African coastal-pelagic shark species, Carcharhinus brevipinna (Valenciennes), resulted in the discovery of a new species of Phoreiobothrium Linton, 1889. Phoreiobothrium martini sp. n. is undoubtedly different from all other congeners in several morphological characteristics, however, it is most easily distinguished from other species by its total length, the size of its hooks, and the number of post vaginal testes. The addition of P. martini sp. n. increases the total number of valid species of Phoreiobothrium to 20 worldwide. Furthermore, P. martini sp. n. marks the description of representatives of only the second onchoproteocephalidean genus from southern Africa, therefore expanding the generic biogeographical representation and introducing new host associations. Apart from the description of this new species of cestode, the discovery of additional species of Phoreiobothrium will be beneficial regarding future ecological investigations. With the high degree of host-specificity found amongst species of Phoreiobothrium, species such as P. martini sp. n. could be used as sentinel species for the accurate identification, separation and diagnosis of commonly misidentified shark species.

Morphological and molecular characterisation of Alella igillimpethu n. sp. (Copepoda: Siphonostomatoida: Lernaeopodidae) parasitising the southern African endemic intertidal klipfish, Clinus superciliosus.

Lernaeopodidae Milne Edwards, 1840, is an ecological and economically important fish parasite family of copepods (Crustacea: Copepoda), consisting of 48 genera with 334 valid species. To date, approximately 17 genera have been documented from both teleost and elasmobranch hosts from South African marine waters. As part of parasitological surveys targeting parasites of the endemic intertidal klipfish, Clinus superciliosus (Linnaeus) (Clinidae) along the South African coast, a species of Lernaeopodidae was discovered on the gills of this host. Alella igillimpethu n. sp. is described from Clinus superciliosus from Langebaan marina on the west coast of South Africa, with a prevalence of 42.1% and mean intensity of 2.9 (ranging from 1-13). Morphological identification was done using light and scanning electron microscopy and the species description was generated with the aid of DEscription Language for TAxonomy (DELTA) software. The mitochondrial cytochrome c oxidase subunit 1 (COI) region, as well as partial 18S and 28S ribosomal RNA genes confirmed the classification within the family Lernaeopodidae. This is the first report of Lernaeopodidae from C. superciliosus, or in fact any member of Clinidae, as well as the first molecular characterisation of any marine lernaeopodid infecting teleost fish from South Africa. This study contributes valuable genetic and morphological data for this copepod family, as well as new host and distribution records.

A new species of Siphoderina Manter, 1934 (Digenea: Cryptogonimidae) infecting the Dory Snapper Lutjanus fulviflamma (Teleostei: Lutjanidae) from the east coast of South Africa.

Parasitological assessment of marine fishes at Sodwana Bay in the iSimangaliso Marine Protected Area on the east coast of South Africa revealed a new species of cryptogonimid trematode infecting the pyloric caeca of the Dory Snapper, Lutjanus fulviflamma (Forsskål) (Lutjanidae). The new species is morphologically consistent with the concept of the large genus Siphoderina Manter, 1934; its phylogenetic position within this genus was validated through molecular sequencing of the ITS2 and partial 28S ribosomal DNA sub-regions. We name this species Siphoderina nana n. sp. and comment on the current state of understanding for this genus of cryptogonimids.

Using Confocal Microscopy and Pigment Analyses to Detect Adverse Insecticide Effects in non-target Freshwater Diatom species - a proof-of-concept Using Nitzschia palea.

The persistence of insecticides in aquatic environments is a cause of concern and to date hardly any studies have focused on the effects that DDT and deltamethrin have on non-target freshwater diatom communities. The application of diatoms in ecotoxicological studies is well acknowledged and therefore this study used laboratory bioassays to determine the effects that DDT and deltamethrin have on a monoculture of a diatom indicator species, Nitzschia palea. The insecticides affected the morphology of chloroplasts at all exposure concentrations. These effects were a maximum reduction in chlorophyll concentrations (4.8% and 2.3%), cell viability (51% and 42%), and increases in cell deformities (3.6% and 1.6%) following exposure to DDT and deltamethrin respectively. Based on the results we propose that methods, such as confocal microscopy, chlorophyll-α analysis and cell deformities are useful tools in assessing the effects of insecticides on diatoms.

A first for Southern Africa: description of a new Heterobothrium (Monogenea: Diclidophoridae) parasitizing the evileye pufferfish Amblyrhynchotes honckenii (Tetraodontiformes: Tetraodontidae).

The monogenean genus Heterobothrium (Monogenea: Diclidophoridae) currently consists of 12 accepted species described globally from various pufferfishes (Tetraodontidae). This includes the economically important Heterobothrium okamotoi Ogawa 1991 that causes severe disease in the cultured tiger puffer Takifugu rubripes in Japan. During parasitological surveys targeting near shore marine fishes of South Africa, a new Heterobothrium was discovered on the gills of five evileye pufferfish Amblyrhynchotes honckenii with a prevalence of 100% and mean intensity of 23 (4-72). Specimens were morphologically studied using both light and scanning electron microscopy and molecularly characterized by sequencing the partial ribosomal gene 28S. Heterobothrium victorwepeneri n. sp. differs from its 12 congeners by a combination of morphological characters, such as fourth clamp pair (anteriormost, 180° inverted) as the smallest with some of the sclerites differing in shape from the other pairs, 8 to 9 genital hooks, number of testes 40-50, and absent isthmus. The 28S tree depicted two main branches, one clustering together species of mazocraeidean families while the other clustered together only species of the Diclidophoridae. Heterobothrium victorwepeneri n. sp. is the first species of its genus to be recorded and described from South Africa and from the tetraodontid A. honckenii. This study also provides for the first time 28S sequence for a species of this highly host-specific genus.

Pseudodactylogyrus anguillae (Yin & Sproston, 1948) from the giant mottled eel Anguilla marmorata Quoy & Gaimard, 1824, in the Phongolo River, South Africa: an invader on the African continent.

The global invasive anguillid gill parasite Pseudodactylogyrus anguillae (Yin and Sproston, 1948) has only recently been documented from eels in South Africa. As there is no known eel trade in South Africa, the source of introduction of this parasite has been debated, and its status as an alien parasite was rendered uncertain. We report on the first infection of Pseudodactylogyrus anguillae from the giant mottled eel Anguilla marmorata from the Phongolo River (South Africa) using classic morphological and molecular methodologies and clarify the introduction status category of this parasite as alien and invasive.

Regional Scale Risk to the Ecological Sustainability and Ecosystem Services of an African Floodplain System.

The Phongolo floodplain is one of southern Africa's most important systems. In this study, we carried out a regional scale ecological risk assessment to evaluate the risk of multiple stressors associated with the use of the aquatic resources in the floodplain to selected social and ecological endpoints representing its sustainability. The floodplain has undergone significant changes as a result of the impacts of multiple stressors. This includes high risk of impact and threatened sustainability between the Pongolapoort Dam and the Ndumo Game Reserve (NGR). This compares to relatively low risk to the maintenance of the endpoints within the NGR. The reserve provides a protection and refuge function for regional biodiversity maintenance and ecosystem sustainability processes. In the study a range of scenarios were considered and demonstrate that the system will respond to protection measures and or increased resource use options. Should flood reductions or water quality pollution drivers continue on increasing trajectories, the condition of the Phongolo River and floodplain will probably deteriorate into an unacceptable, unsustainable state. Removal of the protection services of the NGR would result in an unsustainable ecosystem and loss of ecosystem services for regional vulnerable African communities. Additional evidence should be obtained from monitoring and research to refine, validate, and update the assessment in an adaptive management context. The risk assessment framework approach implemented in the Phongolo floodplain can contribute to the management of other floodplains ecosystems and the sustainability management of social and ecological attributes and processes of these important ecosystems.

Sea lice (Copepoda: Caligidae) from South Africa, with descriptions of two new species of Caligus.

Thirteen species of sea lice (family Caligidae) are reported from a range of elasmobranch and actinopterygian fishes caught off South Africa or obtained from public aquaria in South Africa. Two new species of Caligus Müller, 1785 are described: C. linearis n. sp. from Pomatomus saltatrix (Linnaeus) and C. tumulus n. sp. from Chrysoblephus cristiceps (Valenciennes). A supplementary description is provided for both sexes of Caligus tetrodontis Barnard, 1948 taken from Amblyrhynchotes honckenii (Bloch) and previous records of this parasite from South African fishes are critically reviewed. It is concluded that Caligus material from Arothron hispidus Linnaeus was previously misidentified as C. tetrodontis and is in urgent need of re-examination. Morphological and molecular observations on Caligus furcisetifer Redkar, Rangnekar & Murti, 1949 indicate that this copepod is phenotypically and genetically identical to Lepeophtheirus natalensis Kensley & Grindley, 1973, and the latter becomes a junior subjective synonym of C. furcisetifer. We include new geographical distribution records for Caligus longipedis Bassett-Smith, 1898, C. rufimaculatus Wilson, 1905 and Lepeophtheirus spinifer Kirtisinghe, 1937, extending into South African waters, as well as both new distribution and host records for Alebion gracilis Wilson, 1905, Caligus dakari van Beneden, 1892 and Lepeophtheirus acutus Heegaard, 1943. The molecular analysis confirmed the monophyly of the genus Caligus. The South African species of Caligus did not cluster together, but the two included South African species of Lepeophtheirus were recovered as sister taxa.

The role of fish helminth parasites in monitoring metal pollution in aquatic ecosystems: a case study in the world's most productive platinum mining region.

Due to the increasing consumption of platinum (Pt), especially in automobile exhaust catalysts, environmental concentrations of Pt are of emerging concern worldwide. Limited information exists on environmental concentrations, particularly in Pt mining regions, while South Africa is the world's main supplier of Pt. Moreover, other metals are also released as by-products of Pt mining, which might also cause environmental concern. Certain fish parasite taxa have the ability to accumulate metals orders of magnitude higher than their hosts and can be used to reliably detect metals with naturally low abundance. Studies on Pt accumulation in parasite-host systems are limited. Therefore, the aims of the present study were (1) to determine the accumulation of a variety of metals (cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni), lead (Pb), platinum (Pt), and zinc (Zn)) in helminth fish parasites compared with their hosts from a reference site and an impoundment impacted by Pt mining activities; (2) to assess whether there is a difference between bioaccumulation of metals in infected and uninfected hosts, as well as between hosts with different infection intensities; and (3) to compare the biomarker responses (acetylcholine esterase activity (AChE), metallothionein content (MT), catalase activity (CAT), reduced glutathione content (GSH), malondialdehyde content (MDA), protein carbonyls induction (PC), superoxide dismutase activity (SOD), and cellular energy allocation (CEA)) between infected and uninfected hosts. The cestode Atractolytocestus huronensis accumulated significantly higher concentrations of Cr, Ni, and Pt than their host Cyprinus carpio, while the nematode Contracaecum sp. accumulated significantly higher concentrations of Pt and Zn than their host Clarias gariepinus. Infected fish showed lower metal concentrations compared to uninfected fish, while the parasites had no significant effects on their hosts' biomarker responses. The parasites demonstrated the bioavailability of metals derived from Pt mining activities and their ability to resist its toxic effects. Thus, these parasites are promising sensitive accumulation indicators for Cr, Ni, Pb, and Pt contaminations from Pt mining activities.

Biomarker Responses in the Freshwater Shrimp Caridina nilotica as Indicators of Persistent Pollutant Exposure.

Biomarkers are applied as early warning indicators of organisms' exposure to pollutants. The aim of this study was to utilise a multi-biomarker approach in the freshwater shrimp Caridina nilotica (Decapoda: Atyidae) as indicators of persistent pollutant exposure. A suite of biomarkers was selected to cover oxidative stress and damage, and energetics of the organisms. Five sites, representing an agricultural and pesticide application gradient, were sampled during two flow related hydro-periods in rivers of the Phongolo floodplain, north-eastern South Africa. Cytochrome P450 (CYP) activity was significantly higher in shrimp at sites directly adjacent to regions of increased human activity. Increased oxidative responses, i.e. catalase (CAT; p < 0.01) and protein carbonyl (PC, p < 0.01) were also found at these sites. The energetics biomarker did not show any influence of increased contaminant exposure. We demonstrated that the biomarkers of exposure (CYP) and effect (CAT, PC) were suitable to detect effects of stressors, probably persistent pollutants.

Hooked on you: shape of attachment structures in cymothoid isopods reflects parasitic strategy.

BACKGROUND: Parasite attachment structures are critical traits that influence effective host exploitation and survival. Morphology of attachment structures can reinforce host specificity and niche specialisation, or even enable host switching. Therefore, it is important to understand the determinants of variation in attachment structures. Cymothoid isopods are striking ectoparasites of fishes that include the infamous 'tongue-biters.' They are known to parasitise hosts in one of four qualitatively distinct anatomical regions. Here, we quantify variation in cymothoid attachment structures - hook-like appendages called dactyli - and test whether differences in dactylus shape are correlated with parasite mode (where they attach), allometry, or both, using multivariate ordinary least squares regression. We also assess the influence of shared ancestry on shape using a molecular phylogeny to weight our models using phylogenetic generalised least squares regression. RESULTS: We find clear differences in shape between externally-attaching and internally-attaching cymothoids but also between anterior and posterior dactyli across various species with the same attachment mode. Allometric effects are significant for anterior but not posterior dactyli. Mouth-attaching species show greater shape variability than gill- and mouth-attaching species. We find no evidence that there are clade-specific patterns of association between parasite mode and dactylus shape. CONCLUSIONS: Parasite mode appears to be the main driver of attachment morphology. This likely reflects several components of parasite ecology including feeding and functional demands of attachment in different microhabitats. Geometric morphometric approaches to the quantification of shape variation of simple structures is an effective tool that provides new insights into the evolvability of parasite attachment.

Monophyly of the species of Hepatozoon (Adeleorina: Hepatozoidae) parasitizing (African) anurans, with the description of three new species from hyperoliid frogs in South Africa.

Haemogregarines (Apicomplexa: Adeleiorina) are a diverse group of haemoparasites reported from almost all vertebrate classes. The most commonly recorded haemogregarines to parasitize anurans are species of Hepatozoon Miller, 1908. To date 16 Hepatozoon species have been described from anurans in Africa, with only a single species, Hepatozoon hyperolli (Hoare, 1932), infecting a member of the Hyperoliidae. Furthermore, only two Hepatozoon species are known from South African anurans, namely Hepatozoon theileri (Laveran, 1905) and Hepatozoon ixoxo Netherlands, Cook and Smit, 2014, from Amietia delalandii (syn. Amietia quecketti) and three Sclerophrys species, respectively. Blood samples were collected from a total of 225 individuals representing nine hyperoliid species from several localities throughout northern KwaZulu-Natal, South Africa. Twenty frogs from three species were found positive for haemogregarines, namely Afrixalus fornasinii (6/14), Hyperolius argus (2/39), and Hyperolius marmoratus (12/74). Based on morphological characteristics, morphometrics and molecular findings three new haemogregarine species, Hepatozoon involucrum Netherlands, Cook and Smit n. sp., Hepatozoon tenuis Netherlands, Cook and Smit n. sp. and Hepatozoon thori Netherlands, Cook and Smit n. sp., are described from hyperoliid hosts. Furthermore, molecular analyses show anuran Hepatozoon species to be a separate monophyletic group, with species isolated from African hosts forming a monophyletic clade within this cluster.

Polycyclic aromatic hydrocarbons (PAHs) in sediments from a typical urban impacted river: application of a comprehensive risk assessment.

Soweto and Lenasia, the most densely populated area of South Africa, is simultaneously a thriving metropolis, with a fair share of people still living in squalor conditions directly dependant on the natural resources. Because of industrialisation the populace and environment in this urban area are exposed to various pollutants. The aquatic environment was selected as a proxy to study the effect of industrial pollution in this area. The concentrations, source identification, and various environmental risks of polycyclic aromatic hydrocarbons (PAHs) were determined in sediments of the upper reaches of the Klip River. Composite sediment samples collected in low-flow conditions in 2013 and 2014 ranged from 270-5400 ng/g. The PAHs in this aquatic ecosystem were dominated by 4-ring congeners and could be attributed to combustion of organic fuels by chemical mass balance. Heavy traffic and industrial complexes in the northern part of the study area were responsible for the PAH fingerprints. Probable adverse effects such as toxicity to benthic biota were proven after comparison with international sediment quality guidelines (SQG) both survey years. Toxic equivalence quotients (TEQs) calculated for the sediments using fish potency factors (FPFs) were up to 30 times greater than the Canadian guideline for dioxin-like compounds, indicating high probability of carcinogenic effect to fish mediated through the aryl-hydrocarbon receptor. Finally, sediments in the area posed moderate to high ecological risk, which corroborates the other toxicity assessments. The advantage of investigating multiple risk endpoints, is the comprehensive results obtained that allows for a more realistic representation of the study area. Consequently more aspects are kept into account that results in better conclusions.

Linking organochlorine exposure to biomarker response patterns in Anurans: a case study of Müller's clawed frog (Xenopus muelleri) from a tropical malaria vector control region.

Organochlorine pesticides are highly persistent in aquatic ecosystems. Amphibians, specifically anurans, play an intricate part in the aquatic food web, and have very permeable skin which makes them prone to bioaccumulation of persistent pollutants. In this study the bioaccumulation of various legacy organochlorine pesticides (OCPs)-including dichlorodiphenyltrichloroethane (DDT), currently used for malaria vector control (MVC)-was assessed along with a set of biomarker responses in Müller's clawed frog Xenopus muelleri collected from the lower Phongolo River floodplain in South Africa. Possible relationships between bioaccumulation and biomarkers (of exposure, oxidative stress biomarkers, and cellular energy allocation) alongside their temporal changes were investigated. The OCP concentrations showed a significant increase over time for the duration of the study. The increase correlated negatively with rainfall from the region. DDT levels were well below expected effects levels with p,p-DDE being the main contributing metabolite. The results of this study indicate OCPs actively accumulate at sub-lethal levels in aquatic frogs from the study area, while showing possible relations towards some of the biochemical stress responses measured. Most notable were negative relationships indicated between p,p-DDE and acetylcholinesterase, malondialdehyde, and carbohydrates and protein energy availability. Levels of DDT were not found to be significantly higher than other legacy pesticides in the frog tissue, although evidence of newly introduced DDT in the frog tissue was found. Further investigation about sub-lethal effects of these pesticides on anurans is required to gain better insight into their full impact on animal livelihood.

Rediscovery of Chonopeltis meridionalis Fryer, 1964 (Crustacea: Branchiura) from Labeo rosae Steindachner in the River Olifants, Mpumalanga, and the taxonomic status of C. victori Avenant-Oldewage, 1991 and C. koki Van As, 1992.

Chonopeltis Thiele, 1900 presently comprises 14 species, it is endemic to Africa and its species show a high degree of host-specificity towards fish families and in some cases, individual fish species. Chonopeltis meridionalis Fryer, 1964 was originally described from Labeo rosae Steindachner collected in the River Nuanetsi in Rhodesia, now Zimbabwe, Limpopo River System. At the time of description C. meridionalis was the most southerly species of the genus. Chonopeltis victori Avenant-Oldewage, 1991 was described from the River Olifants, Mpumalanga, South Africa, which also forms part of the Limpopo River System. The host fish was Labeo rosae and Labeobarbus marequensis (A. Smith), whilst C. koki Van As, 1992 was described from Labeo cylindricus Peters, collected in the River Zambezi, Eastern Caprivi, Namibia. During surveys conducted in 2012, additional material of a species of Chonopeltis was collected from Labeo rosae in the River Olifants. Upon closer examination, the new material was identified as C. meridionalis. Further investigation revealed that C. victori and C. koki share a number of characteristics with C. meridionalis. This paper provides the first scanning electron microscopy of C. meridionalis, includes additional information on fully-mature as well as sub-adult males and females. Finally, it was concluded that C. victori and C. koki are junior synonyms of C. meridionalis.

Metal concentrations in Hydrocynus vittatus (Castelnau 1861) populations from a premier conservation area: Relationships with environmental concentrations.

Metals are known to have deleterious effects on aquatic ecosystems, so monitoring the bioaccumulation of these pollutants is important for the assessment of potential impacts. The aim of the study was therefore to determine the degree to which aquatic biota in selected rivers of the world renowned Kruger National Park (KNP) are exposed to metals entering from the catchments outside the KNP and to determine how these metal levels reflect what is occurring within the environment. Many of these rivers have never previously been assessed and those which have, have not been assessed within the past two decades. Tigerfish (Hydrocynus vittatus) is an economically important apex predator and was selected as the bioindicator for this study. Fish were sampled from the KNP sections of the Luvuvhu, Letaba and Olifants Rivers during the high and low flow periods from 2009 to 2011. The analysis of various metals from the aforementioned systems revealed spatial and temporal related variation in metal level patterns of the chosen bioindicator, with concentrations in fish from the Olifants River>Letaba River>Luvuvhu River and 2009>2010>2011. Although there were differences between rivers, metal concentrations were comparable and indicate that all of the studied KNP rivers, even those considered to be in a natural state are affected to some degree by anthropogenic activities. However, concentrations found during this study were much lower than previous studies in the area, as well as contaminated sites across the world, but were higher than concentrations in fish from contaminated sites in the Vaal River, South Africa. Bioaccumulated metals showed no correlation to metals in the water column, but some were correlated to sediment metal concentrations and the contamination and bioavailability of these metals within the sediments. This is of concern when managing the water resources of the conservation area since the contaminants enter the park from outside the borders and pose potential risks to the mandated conservation of aquatic biota within the KNP.

Trematode diversity in freshwater fishes of the Globe I: 'Old World'.

In this paper, we review, continent by continent, the trematode fauna of freshwater fishes of the 'Old World', a vast area consisting of the Palaearctic, Ethiopian, Oriental and Australasian zoogeographical regions. Knowledge of this fauna is highly uneven and clearly incomplete for almost all regions, sometimes dramatically so. Although the biggest problem remains the completion of the 'first pass' of alpha taxonomy, there are in addition great problems relating to biogeography and elucidation of life-cycles. For the latter, molecular data, i.e. matching DNA sequences of larval stages and corresponding adults, may represent a powerful tool that should be used in future studies. Another challenging problem represents the existence of cryptic species and, in particular, considerable decrease of experts in taxonomy and life-cycles of trematodes.