Phylogeny and paraphyly among tetrapod blood flukes (Digenea: Schistosomatidae and Spirorchiidae)

The blood flukes of turtles (Digenea: Spirorchiidae) and the blood flukes of crocodilians, birds and mammals (Digenea: Schistosomatidae) have long been considered as closely related, but distinct evolutionary lineages. Recent morphological and molecular studies have considered these families as sister taxa within the Schistosomatoidea. Representatives of both families have similar furcocercous cercariae and similar two-host life cycles, but have different definitive hosts, distinct reproductive patterns and different morphologies. Sequences including approximately 1800 bases of the small subunit ribosomal DNA and 1200 bases of the large subunit ribosomal DNA were generated from representatives of eight spirorchiid genera. These sequences were aligned with pre-existing sequences of Schistosomatidae and other representatives of the Diplostomida and analysed for phylogenetic signal using maximum parsimony and Bayesian inference. These analyses revealed that the Spirorchiidae is paraphyletic and that the turtle blood flukes are basal to the highly derived schistosomatids. Three genera of spirorchiids from marine turtles form a sister group to the Schistosomatidae and five genera of spirorchiids from freshwater turtles occupy basal positions in the phylogeny of tetrapod blood flukes. Marine turtles are considered to be derived from freshwater turtles and the results of the current study indicate that the spirorchiid parasites of marine turtles are similarly derived from a freshwater ancestor. The close relationship of the marine spirorchiids to schistosomatids and the basal position of the marine transmitted Austrobilharzia and Ornithobilharzia in the schistosomatid clade suggests that schistosomatids arose after a marine turtle blood fluke ancestor successfully colonised birds.     

Vermetid gastropods as key intermediate hosts for a lineage of marine turtle blood flukes (Digenea: Spirorchiidae), with evidence of transmission at a turtle rookery

Blood flukes of the family Spirorchiidae Stunkard, 1921 are significant pathogens of marine turtles, both in the wild and in captivity. Despite causing considerable disease and mortality, little is known about the life cycles of marine species, with just four reports globally. No complete life cycle has been elucidated for any named species of marine spirorchiid, but the group is reported to use vermetid and fissurellid gastropods, and terebelliform polychaetes as intermediate hosts. Here we report molecular evidence that nine related spirorchiid species infect vermetid gastropods as first intermediate hosts from four localities along the coast of Queensland, Australia. ITS2 rDNA and cox1 mtDNA sequence data generated from vermetid infections provides the first definitive identifications for the intermediate hosts for the four species of Hapalotrema Looss, 1899 and Learedius learedi Price, 1934. Additionally, we provide a new locality report for larval stages of Amphiorchis sp., and evidence of three additional unidentified spirorchiid species in Australian waters. Based on the wealth of infections from vermetids during this study, we conclude that the previous preliminary report of a fissurellid limpet as the intermediate host for L. learedi was likely mistaken. The nine species found infecting vermetids during this study form a strongly supported clade exclusive of species of the other two marine spirorchiid genera for which sequence data are available; Carettacola Manter & Larson, 1950 which falls sister to the vermetid-infecting clade + a small clade of freshwater spirorchiids, and Neospirorchis Price, 1934 which is distantly related to the vermetid-infecting clade. We provide further evidence that spirorchiid transmission can occur in closed system aquaria and show that spirorchiid transmission occurs at both an important turtle rookery (Heron Island, southern Great Barrier Reef, Australia) and foraging ground (Moreton Bay, Australia). We discuss the implications of our findings for the epidemiology of the disease, control in captivity, and the evolution of vermetid exploitation by the Spirorchiidae.     

Elucidation of the first definitively identified life cycle for a marine turtle blood fluke (Trematoda: Spirorchiidae) enables informed control

Blood flukes of the family Spirorchiidae are significant pathogens of both free-ranging and captive marine turtles. Despite a significant proportion of marine turtle mortality being attributable to spirorchiid infections, details of their life cycles remain almost entirely unknown. Here we report on the molecular elucidation of the complete life cycle of a marine spirorchiid, identified as Amphiorchis sp., infecting vermetid gastropods and captive hatched neonate Caretta caretta in the Oceanogràfic Aquarium, in Valencia, Spain. Specimens of a vermetid gastropod, Thylaeodus cf. rugulosus (Monterosato, 1878), collected from the aquarium filtration system housing diseased C. caretta, were infected with sporocysts and cercariae consistent with the family Spirorchiidae. We generated rDNA sequence data [internal transcribed spacer 2 (ITS2) and partial 28S rDNA] from infections from the vermetid which were identical to sequences generated from eggs from the serosa of the intestine of neonate C. caretta, and an adult spirorchiid from the liver of a C. caretta from Florida, USA. Given the reliability of these markers in the delineation of trematode species, we consider all three stages to represent the same species and tentatively identify it as a species of Amphiorchis Price, 1934. The source of infection at the Oceanogràfic Foundation Rehabilitation Centre, Valencia, Spain, is inferred to be an adult C. caretta from the western Mediterranean being rehabilitated in the same facility. Phylogenetic analysis suggests that this Amphiorchis sp. is closely related to other spirorchiids of marine turtles (species of Carettacola Manter & Larson, 1950, Hapalotrema Looss, 1899 and Learedius Price, 1934). We discuss implications of the present findings for the control of spirorchiidiasis in captivity, for the better understanding of epidemiology in wild individuals, and the elucidation of further life cycles.     

Discovery of intermediate hosts for two species of blood flukes Cardicola orientalis and Cardicola forsteri (Trematoda: Aporocotylidae) infecting Pacific bluefin tuna in Japan

Fish blood flukes (Aporocotylidae) are important pathogens of farmed finfish around the world. Among them, Cardicola spp. infecting farmed tuna are considered to be serious threats to tuna farming and have received tremendous attention. We conducted periodical samplings at a tuna farming site in Japan between January and May, 2015 to determine the life cycle of Cardicola spp. We collected over 4700 terebellid polychaetes from ropes, floats and frames of tuna culture cages and found nearly 400 infected worms. Sporocysts and cercariae found in Nicolea gracilibranchis were genetically identified as Cardicola orientalis by 28S and ITS2 ribosomal DNA sequences. This was the first discovery of the intermediate host for this parasite species. Infection prevalence and the abundance of N. gracilibranchis significantly varied between sampling points and the highest number of infected terebellids were collected from ropes. We also demonstrated morphologically and molecularly that asexual stages found in a single Amphitrite sp. (Terebellidae) and adult worms isolated from farmed juvenile tuna were Cardicola forsteri. This is the first report of C. forsteri in Pacific bluefin tuna (PBT) Thunnus orientalis in Japan. Our results demonstrated that all three species of Cardicola orientalis, C. forsteri and Cardicola opisthorchis exist in Japanese farmed PBTs and that they all use terebellid polychaetes as the intermediate hosts.     

Schistosomatium douthitti: effects of thyroxine

Thyroxine-treated flukes showed increased tetrazolium reductase and cytochrome oxidase activities in histochemical preparations. Single male, single female, and pairs of S. douthitti, as well as cercariae of this species, consumed more oxygen when treated with thyroxine than did controls. The same effect was observed in all other invertebrates examined (Haematoloechus sp., Dugesia sp., Tetrahymena pyriformis, and an unidentified strigeoid species of cercaria).Both male and female schistosomes from hyperthyroid hosts are significantly larger than flukes from normal mice (P < 0.025). This condition prevailed whether the hyperthyroid state was established prior to cercarial penetration or after schistosomular migration through the lungs. S. douthitti adults from surgically thyroidectomized rats were significantly smaller than flukes from sham-operated sibling controls (P < 0.01). An accelerated death rate was also observed in hyperthyroid mice infected with S. douthitti.Female schistosomes from unisexual infections were significantly smaller (P < 0.1) than those from bisexual infections, confirming Short's (1952) report.     

Effects of the herbicide atrazine's metabolites on host snail mortality and production of trematode cercariae

Environmental stressors have the potential to greatly impact the transmission of parasites with complex, multi-host life cycles such as those of trematodes. The commonly used herbicide atrazine has been shown to affect the susceptibility of second intermediate hosts (such as larval amphibians) to trematode infection, as well as the longevity and infectivity of the free-swimming cercariae, but not eggs or the free-swimming miracidia that infect the gastropod first intermediate hosts. However, we do not know if this pesticide influences the survival of infected snails or whether it affects cercariae production within, or emergence from, these hosts. In addition, previous studies of host-parasite dynamics have only examined the parent atrazine compound, not any of the long-lasting metabolites commonly present in water bodies. Here, we report that a concentration of 0.33 μg/L of an atrazine metabolite, desethyl atrazine, increased the mortality of freshwater gastropods ( Stagnicola elodes ) infected with a gymnocephalus type of cercaria but not that of uninfected snails or those harboring a mature or dormant infection of Echinoparyphium sp. In contrast, 2 wk of exposure to desethyl atrazine did not affect the emergence of gymnocephalus cercariae from snails, although a trend for a decrease in the emergence of Echinoparyphium sp. cercariae was observed. We suggest that simultaneous trematode infection and exposure to contaminants may represent a significant combined stress to gastropods, but this is likely parasite species-specific as well as dependent on whether cercariae are being actively produced.     

Experimental Life History and Biological Characteristics of Fasciola gigantica (Digenea: Fasciolidae)

This study was conducted to investigate the life history, morphology, and maturation of larval stages and adult worms of Fasciola gigantica in experimental mice. Lymnaea auricularia rubiginosa was used as the intermediate host, and Oryza sativa was used for encystment of the metacercariae, while Mus musculus was used as the definitive host for maturation study. Fresh eggs from the gall bladder of water buffaloes fully developed into embryonated ones and hatched out at days 11-12 after incubation at about 29ºC. Free-swimming miracidia rapidly penetrated into the snail host, and gradually developed into the next larval stages; sporocyst, redia, and daughter redia with cercariae. Fully-developed cercariae were separated from the redia and shed from the snails on day 39 post-infection (PI). Free-swimming cercariae were immediately allowed to adhere to rice plants, and capsules were constructed to protect metacercariae on rice plants. Juvenile worms were detected in intestines of mice at days 3 and 6 PI, but they were found in the bile duct from day 9 PI. Juvenile and adult flukes were recovered from 16 mice experimentally infected with metacercariae, with the average recovery rate of 35.8%. Sexually mature adult flukes were recovered from day 42 PI. It could be confirmed that experimentally encysted metacercariae could infect and develop to maturity in the experimental host. The present study reports for the first time the complete life history of F. gigantica by an experimental study in Thailand. The obtained information can be used as a guide for prevention, elimination, and treatment of F. gigantica at environment and in other hosts.     

Do Parasitic Trematode Cercariae Demonstrate a Preference for Susceptible Host Species?

Many parasites are motile and exhibit behavioural preferences for certain host species. Because hosts can vary in their susceptibility to infections, parasites might benefit from preferentially detecting and infecting the most susceptible host, but this mechanistic hypothesis for host-choice has rarely been tested. We evaluated whether cercariae (larval trematode parasites) prefer the most susceptible host species by simultaneously presenting cercariae with four species of tadpole hosts. Cercariae consistently preferred hosts in the following order: Anaxyrus ( = Bufo) terrestris (southern toad), Hyla squirella (squirrel tree frog), Lithobates ( = Rana) sphenocephala (southern leopard frog), and Osteopilus septentrionalis (Cuban tree frog). These host species varied in susceptibility to cercariae in an order similar to their attractiveness with a correlation that approached significance. Host attractiveness to parasites also varied consistently and significantly among individuals within a host species. If heritable, this individual-level host variation would represent the raw material upon which selection could act, which could promote a Red Queen “arms race” between host cues and parasite detection of those cues. If, in general, motile parasites prefer to infect the most susceptible host species, this phenomenon could explain aggregated distributions of parasites among hosts and contribute to parasite transmission rates and the evolution of virulence. Parasite preferences for hosts belie the common assumption of disease models that parasites seek and infect hosts at random.     

MicroRNAs resolve an apparent conflict between annelid systematics and their fossil record

Both the monophyly and inter-relationships of the major annelid groups have remained uncertain, despite intensive research on both morphology and molecular sequences. Morphological cladistic analyses indicate that Annelida is monophyletic and consists of two monophyletic groups, the clitellates and polychaetes, whereas molecular phylogenetic analyses suggest that polychaetes are paraphyletic and that sipunculans are crown-group annelids. Both the monophyly of polychaetes and the placement of sipunculans within annelids are in conflict with the annelid fossil record—the former because Cambrian stem taxa are similar to modern polychaetes in possessing biramous parapodia, suggesting that clitellates are derived from polychaetes; the latter because although fossil sipunculans are known from the Early Cambrian, crown-group annelids do not appear until the latest Cambrian. Here we apply a different data source, the presence versus absence of specific microRNAs—genes that encode approximately 22 nucleotide non-coding regulatory RNAs—to the problem of annelid phylogenetics. We show that annelids are monophyletic with respect to sipunculans, and polychaetes are paraphyletic with respect to the clitellate Lumbricus, conclusions that are consistent with the fossil record. Further, sipunculans resolve as the sister group of the annelids, rooting the annelid tree, and revealing the polarity of the morphological change within this diverse lineage of animals.     

Infectivity of Diplostomum spp. in Arctic charr: aspects of exposure duration and cercariae morphology

The diplostomid flukes, Diplostomum spp., infect fish and cause cataract opacities in the eye lens. The effect of exposure dose on abundance of Diplostomum spp. eye flukes in fish is known, but the effect of the duration of cercariae exposure has not been studied. However, under natural conditions, the temporal window for a successful cercaria attachment on fish is very short and, consequently, differences in infectivity of eye fluke cercariae, in the short-exposure durations of a few seconds, are probably biologically the most meaningful. We investigated infectivity of Diplostomum spp. cercariae originating from snail hosts in 3 lakes (3 Lymnaea stagnalis populations and 1 Radix balthica population) in 6 exposure times, ranging from 5 sec to 15 min, in young-of-the-year Arctic charr Salvelinus alpinus. In addition, we compared the infectivity to the cross-morphology of the cercariae. In the long-exposure duration, i.e., > or = 5 min, infectivity of Diplostomum spp. did not vary between the snail host species (L. stagnalis and R. balthica) of the same lake or across the L. stagnalis populations of 3 different lakes. However, in the short-exposure duration, i.e., < or = 60 sec, Diplostomum spp. cercariae shed from L. stagnalis had higher infectivity than did cercariae from R. balthica of the same lake. This indicates that that there is an interaction between length of cercariae exposure and origin of Diplostomum spp., and that the duration of exposure may influence the results when fish are experimentally infected. Within a lake, cercariae shed from L. stagnalis were also smaller than cercariae shed from R. balthica.     

Trichobilharzia mergi sp. nov. (Trematoda: Digenea: Schistosomatidae), a visceral schistosome of Mergus serrator (L.) (Aves: Anatidae)

Parasitological investigations on red-breasted mergansers (Mergus serrator L.) in Iceland revealed digenean flukes of the family Schistosomatidae. Adult worms were detected in blood vessels of the large intestine and eggs were deposited in the mucosa and surrounded by granulomatous reactions. Traditional morphological methods showed that the flukes have very slender filiform bodies, males are equipped with a short gynaecophoric canal and both suckers and spatulate ends are present on each sex. Among characteristics of the flukes which render them morphologically distinct from other Trichobilharzia species are: i) males—well developed vesicula seminalis (v.s.) consisting of a short v.s. externa and a significantly longer (approx. 3 times) v.s. interna, unusually well developed genital papilla and localization of the first testis a relatively long distance posterior to the gynaecophoric canal; ii) eggs—small and elongated with slightly rounded poles and a short terminal spine. DNA taxonomic techniques confirmed that a new species had been identified, Trichobilharzia mergi sp. n. The sequence data were deposited in GenBank under the accession numbers JX456151 to JX456172. Comparison of the results with our previously published data on characterization of DNA of cercariae isolated from freshwater lymnaeid snails showed that larval development of T. mergi is associated with Radix balthica L. (=Radix peregra Müller, 1774; = Radix ovata Draparnaud, 1805).     

Worm on worm: Two rare genera of Calamyzinae (Annelida,Chrysopetalidae), with a description of new species

Marine annelids in the subfamily Calamyzinae (family Chrysopetalidae) are either symbiotic or free-living forms that have been mainly reported from deep-sea chemosynthetic environments. Symbiotic calamyzines predominantly live in the mantle cavity of bivalves distributing at hydrothermal vents or methane seeps except for two species inhabiting the epidermis of polychaetes and octopuses. In this study, we describe a new species, Calamyzas crambon sp. nov., from Japan and report a new record of Nautiliniella calyptogenicola from the Mariana Trench. We also provide the phylogenetic position of the two species within Chrysopetalidae based on four gene markers (COI, 16S, 18S, and H3).     

Evidence that a lineage of teleost-infecting blood flukes (Aporocotylidae) infects bivalves as intermediate hosts

The family Aporocotylidae is recognized as having the widest intermediate host usage in the Digenea. Currently, intermediate host groups are clearly correlated with definitive host groups; all known life cycles of marine teleost-infecting aporocotylids involve polychaetes, those of freshwater teleost-infecting aporocotylids involve gastropods, and those of chondrichthyan-infecting aporocotylids involve bivalves. Here we report the life cycle for a marine elopomorph-infecting species, Elopicola bristowi Orélis-Ribeiro & Bullard in Orélis-Ribeiro, Halanych, Dang, Bakenhaster, Arias & Bullard, 2017, as infecting a bivalve, Anadara trapezia (Deshayes) (Arcidae), as the intermediate host in Moreton Bay, Queensland, Australia. The cercaria of E. bristowi has a prominent finfold, distinct anterior and posterior widenings of the oesophagus, a tail with symmetrical furcae with finfolds, and develops in elongate to oval sporocysts. We also report molecular data for an unmatched aporocotylid cercaria from another bivalve, Megapitaria squalida (G. B. Sowerby I) (Veneridae), from the Gulf of California, Mexico, and six unmatched cercariae from a gastropod, Posticobia brazieri (E. A. Smith) (Tateidae), from freshwater systems of south-east Queensland, Australia. Phylogenetic analyses demonstrate the presence of six strongly-supported lineages within the Aporocotylidae, including one of elopomorph-infecting genera, Elopicola Bullard, 2014 and Paracardicoloides Martin, 1974, now shown to use both gastropods and bivalves as intermediate hosts. Of a likely 14 aporocotylid species reported from bivalves, six are now genetically characterised. The cercarial morphology of these six species demonstrates a clear distinction between those that infect chondrichthyans and those that infect elopomorphs; chondrichthyan-infecting aporocotylids have cercariae with asymmetrical furcae that lack finfolds and develop in spherical sporocysts whereas those of elopomorph-infecting aporocotylids have symmetrical furcae with finfolds and develop in elongate sporocysts. This morphological correlation allows predictions of the host-based lineage to which the unsequenced species belong. The Aporocotylidae is proving exceptional in is propensity for major switches in intermediate host use, with the most parsimonious interpretation of intermediate host distribution implying a minimum of three host switches within the family.     

Buffering role of the intertidal anemone Anthopleura aureoradiata in cercarial transmission from snails to crabs

In nature, parasite transmission from one host to the next takes place within complex biotic communities where non-host organisms can reduce transmission rates, for instance by preying on infective stages. We experimentally investigated the impact of four very different non-host organisms on the transmission of the microphallid trematode Maritrema novaezealandensis from its snail first intermediate host to its crustacean second intermediate host. We show that in laboratory mesocosms, accumulation of parasites in juvenile stalk-eyed mud crabs, Macrophthalmus hirtipes (Ocypodidae), was not reduced in the presence of cockles, Austrovenus stutchburyi, barnacles, Balanus sp., or the algae Enteromorpha spp., three organisms whose feeding mode or general abundance could negatively impact the parasite's infective stages (cercariae). In contrast, the presence of the anemone Anthopleura aureoradiata in the mesocosms caused a more than 4-fold reduction in the number of parasites acquired by crabs when compared to control mesocosms. Observations on fluorescent-dyed cercariae confirmed that they are ingested by anemones. Given the often high densities of anemones on mudflats, they may represent an important regulator of the abundance of M. novaezealandensis, and thus of the impact of this parasite on its hosts. These anemones may decrease cercarial transmission for many other trematode species as well. Our results stress the need for studies of parasite transmission in natural contexts rather than under simplified laboratory conditions.     

Temperature and pCO2 jointly affect the emergence and survival of cercariae from a snail host: implications for future parasitic infections in the Humboldt Current system

Ocean warming and acidification are general consequences of rising atmospheric CO₂ concentrations. In addition to future predictions, highly productive systems such as the Humboldt Current System are characterized by important variations in both temperature and pCO₂ level, but how these physical–chemical ocean changes might influence the transmission and survival of parasites has not been assessed. This study experimentally evaluated the effects of temperature (14, 18 and 25 °C) and the combined effects of temperature (∼15 and 20 °C) and pCO₂ level (∼500 and 1400 microatmospheres (µatm) on the emergence and survival of two species of marine trematodes—Echinostomatidae gen. sp. and Philophthalmidae gen. sp.—both of which infect the intertidal snail Echinolittorina peruviana. Snails were collected from intertidal rocky pools in a year-round upwelling area of the northern Humboldt Current System (23°S). Two experiments assessed parasite emergence and survival by simulating emersion-immersion tidal cycles. To assess parasite survival, 2 h old cercariae (on average) were taken from a pool of infected snails incubated at 20–25 °C, and their mortality was recorded every 6 h until all the cercariae were dead. For both species, a trade-off between high emergence and low survival of cercariae was observed in the high temperature treatment. Species-specific responses to the combination of temperature and pCO₂ levels were also observed: the emergence of Echinostomatidae cercariae was highest at 20 °C regardless of the pCO₂ levels. By contrast, the emergence of Philophthalmidae cercariae was highest at elevated pCO₂ (15 and 20 °C), suggesting that CO₂ may react synergistically with temperature, increasing transmission success of this parasite in coastal ecosystems of the Humboldt Current System where water temperature and pH are expected to decrease. In conclusion, our results suggest that integrating temperature-pCO₂ interactions in parasite studies is essential for understanding the consequence of climate change in future marine ecosystem health.