Individual hosts are often co-infected with multiple parasite species. Evidence from theoretical and empirical studies supports the idea that co-occurring parasites can impact each other and their hosts via synergistic or antagonistic interactions. The fundamental aim of understanding the consequences of co-infection to hosts and parasites requires an understanding of patterns of species co-occurrence within samples of hosts. We censused parasite assemblages in 755 adult, male fathead minnows collected from 7 lakes/ponds in southern Alberta, Canada between 2018 and 2020. Fifteen species of endoparasites infected fathead minnows, 98% of which were co-infected with between 2 and 9 parasite species (mean species richness: 4.4 ± 1.4). Non-random pairwise associations were detected within the overall parasite community. There were particularly strong, positive associations in the occurrences and intensities of the 2 congeneric larval trematodes Ornithodiplostomum sp. and Ornithodiplostomum ptychocheilus that comprised >96% of the 100 000+ parasites counted in the total sample of minnows. Furthermore, the occurrence of Ornithodiplostomum sp. was a strong predictor of the occurrence of O. ptychocheilus, and vice versa. Positive covariation in the intensities of these 2 dominants likely arises from their shared use of physid snails as first intermediate hosts in these waterbodies. These 2 species represent a predictable and non-random component within the complex assemblage of parasites of fathead minnows in this region.

The assassin snail genus Anentome is widespread in Southeast Asia, and is distributed all over the world via the aquarium trade. One species of genus Anentome, Anentome helena, is known to act as intermediate host of parasitic trematodes. This study investigates the taxonomic diversity of larval trematodes infecting A. helena and Anentome wykoffi in Thailand. Larval trematodes were identified by combining morphological and DNA sequence data (cytochrome c oxidase I and internal transcribed spacer 2). Species delimitation methods were used to explore larval trematode species boundaries. A total of 1107 specimens of Anentome sp. were collected from 25 localities in Thailand. Sixty-two specimens of A. helena (n = 33) and A. wykoffi (n = 29) were infected by zoogonid cercariae, heterophyid metacercariae and echinostome metacercariae, with an overall prevalence of 5.6% (62/1107) and population-level prevalences in the range of 0.0–22.3%. DNA sequence data confirmed that the larval trematodes belong to the families Echinostomatidae, Heterophyidae and Zoogonidae. As such, this is the first report of zoogonid cercariae and heterophyid metacercariae in A. helena, and echinostome metacercariae in A. wykoffi. Moreover, this study provides evidence of tentative species-level differentiation between Thai Echinostoma sp. and Cambodian Echinostoma mekongi, as well as within Echinostoma caproni, Echinostoma trivolvis and Echinostoma revolutum.

Trematodes are the main macroparasites in coastal waters. The most abundant and widespread form of these parasites is metacercaria. Their impact on their host fitness is considered relatively low but metacercarial larvae of some species can have deleterious effects on individuals and/or populations. This review focused on the cockle Cerastoderma edule and four species of the genus Himasthla; a common host–parasite system in marine coastal environments. Our aims were (1) to review literature concerning Himasthla continua, Himasthla elongata, Himasthla interrupta and Himasthla quissetensis in cockles; (2) to provide molecular signatures of these parasites and (3) to analyse infection patterns using a 20-year monthly database of cockle monitoring from Banc d'Arguin (France). Due to identification uncertainties, the analysis of the database was restricted to H. interrupta and H. quissetensis, and it was revealed that these parasites infect cockles of the same size range. The intensity of parasites increased with cockle size/age. During the colder months, the mean parasite intensity of a cockle cohort decreased, while infection occurred in the warmest season. No inter-specific competition between trematode parasites was detected. Furthermore, even if the intensity of H. interrupta or H. quissetensis infection fluctuated in different years, this did not modify the trematode community structure in the cockles. The intensity of infection of both species was also positively correlated with trematode species richness and metacercarial abundance. This study highlighted the possible detrimental role of Himasthla spp. in cockle population dynamics. It also revealed the risks of misidentification, which should be resolved by further molecular approaches.

The genus Isoparorchis (Family: Isoparorchiidae) contains trematodes infecting the air bladder of freshwater catfishes in Asia and Australia. Isoparorchis spp. rely on freshwater shrimps and fishes as intermediate hosts. There is limited information about parasitic infections in freshwater shrimp in Thailand, and Isoparorchis infection in an intermediate host in this country has never been reported. Thus, this study reports infections in freshwater shrimp (Macrobrachium lanchesteri and Caridina sp.), including overall prevalence, mean intensity, morphological characters and molecular analyses. The parasite specimens were analysed by studying their morphological characters, together with a molecular approach based on internal transcribed spacer 2 (ITS2) sequence data. The overall prevalence and mean intensity of Isoparorchis infections were 92% and 1.89, respectively. The metacercariae were identified as Isoparorchis sp. based on their morphological characters and supported by the comparison with published ITS2 sequences of Isoparorchis species. The phylogenetic studies based on the ITS2 region demonstrated that all of the Isoparorchis sp. specimens in this study are distinct from Isoparorchis species in previous reports. Moreover, we show for the first time that the freshwater shrimp M. lanchesteri serves as a second intermediate host of Isoparorchis sp. and we provide a morphological description and molecular characterization of Isoparorchis sp. metacercariae based on ITS2 sequence data to clarify the status of Isoparorchis sp. in Thailand.

Temperature and intraspecific competition are important factors influencing the growth of all organisms, including parasites. The temperature increase is suggested to stimulate the development of parasites within poikilothermic hosts. However, at high parasite densities, this effect could be diminished, due to stronger intraspecific competition. Our study, for the first time, addressed the joint effects of warming and parasite abundances on parasite growth in poikilothermic hosts. The growth of the common fish parasite larvae (trematode Diplostomum pseudospathaceum) within the rainbow trout at different infection intensities and temperatures (15°C and 18°C) was experimentally investigated. The results showed that temperature was positively correlated with both parasite infection success and growth rates. The growth rates increased much more compared to those in many free-living poikilothermic animals. Atypically for a majority of parasites, D. pseudospathaceum larvae grow faster when abundant (Allee effect). The possible causes for this phenomenon (manipulation cost sharing, etc.) are discussed in this study. Importantly, limited evidence of the interaction between temperature and population density was found. It is likely that temperature did not change the magnitude of the Allee effect but affected its timing. The impact of these effects is supposed to become more pronounced in freshwater ecosystems under current climate changes.

The study of priority effects with respect to coinfections is still in its infancy. Moreover, existing coinfection studies typically focus on infection outcomes associated with exposure to distinct sets of parasite species, despite that functionally and morphologically similar parasite species commonly coexist in nature. Therefore, it is important to understand how interactions between similar parasites influence infection outcomes. Surveys at seven ponds in northwest Pennsylvania found that multiple species of echinostomes commonly co-occur. Using a larval anuran host (Rana pipiens) and the two most commonly identified echinostome species from our field surveys (Echinostoma trivolvis and Echinoparyphium lineage 3), we examined how species composition and timing of exposure affect patterns of infection. When tadpoles were exposed to both parasites simultaneously, infection loads were higher than when exposed to Echinoparyphium alone but similar to being exposed to Echinostoma alone. When tadpoles were sequentially exposed to the parasite species, tadpoles first exposed to Echinoparyphium had 23% lower infection loads than tadpoles first exposed to Echinostoma. These findings demonstrate that exposure timing and order, even with similar parasites, can influence coinfection outcomes, and emphasize the importance of using molecular methods to identify parasites for ecological studies.

The trematodes from South American reptiles are poorly known, with only one life cycle completely characterized. We used molecular and morphological methods to characterize diplostomoid metacercariae found in 29 of 86 pointedbelly frogs, Leptodactylus podicipinus (Cope, 1862) collected in a marsh pond in Selvíria, in the central-west region of Brazil. The metacercariae were identified as Heterodiplostomum lanceolatum Dubois, 1936 (Proterodiplostomidae), a rarely reported species that matures in snakes. In phylogenetic analysis of partial sequences from 28S rDNA, H. lanceolatum fell within a polytomy with the proterodiplostomid Crocodilicola pseudostoma (molecular divergence of 4.1%) and other members of the superfamily Diplostomoidea. Our collections provide insights into the ecology of this parasite, in that infected frogs were smaller than uninfected frogs, and metacercariae were more numerous in the abdominal cavity and hindlimb muscles than in abdominal muscles, which suggests directions for future research on the transmission and pathology of this proterodiplostomid.

Posthodiplostomum minimum utilizes a three-host life cycle with multiple developmental stages. The metacercarial stage, commonly known as ‘white grub’, infects the visceral organs of many freshwater fishes and was historically considered a host generalist due to its limited morphological variation among a wide range of hosts. In this study, infection data and molecular techniques were used to evaluate the host and tissue specificity of Posthodiplostomum metacercariae in centrarchid fishes. Eleven centrarchid species from three genera were collected from the Illinois portion of the Ohio River drainage and necropsied. Posthodiplostomum infection levels differed significantly by host age, host genera and infection locality. Three Posthodiplostomum spp. were identified by DNA sequencing, two of which were relatively common within centrarchid hosts. Both common species were host specialists at the genus level, with one species restricted to Micropterus hosts and the other preferentially infecting Lepomis. Host specificity is likely dictated by physiological compatibility and deviations from Lepomis host specificity may be related to host hybridization. Posthodiplostomum species also differed in their utilization of host tissues. Neither common species displayed strong genetic structure over the scale of this study, likely due to their utilization of bird definitive hosts.

Paramphistomosis, caused by Calicophoron daubneyi, is an emerging infection of ruminants throughout Western Europe. Despite its prevalence, many questions remain regarding the basic biology of this parasite and how it interacts with its host. Consequently, there is a need to develop methods to study C. daubneyi in vitro to improve our understanding of rumen fluke biology. Towards this, we aimed to identify a suitable protocol for in vitro excystment of C. daubneyi metacercariae. Six methods that have been used to excyst metacercariae from a number of trematode species were tested with C. daubneyi metacercariae. Three of these achieved an average of >50% excystment whilst one method, which included an acid-pepsin treatment, incubation in reducing conditions and an alkaline/bile salt solution to activate the larvae, consistently gave >80% excystment. The latter protocol also showed no detrimental effect on the motility of newly excysted juvenile (NEJ) parasites when observed for up to 24 h in RPMI 1640 medium post-excystment. The successful production of C. daubneyi NEJs in vitro is a significant step forward, and will enable the discovery of infective stage-specific parasite antigens and facilitate drug screening trials, to aid the development of much needed diagnostic and therapeutic options for paramphistomosis.

Temperature is an important factor influencing the biology of organisms and is intrinsically linked to climate change. The establishment of trematodes in target hosts is potentially susceptible to temperature changes effecting parasite infectivity or host susceptibility, and therefore in order to develop predictive frameworks of host–parasite dynamics under climate change large-scale analyses are required. The present study analyses the thermodynamics of the infectivity of larval trematodes including miracidia, cercariae and metacercariae from experimental data contained in the scientific literature using the Arrhenius critical incremental energy of activation (E*), an accurate measure of temperature-driven reaction rates. For miracidia and cercariae, infectivity increases as the temperature rises reaching a plateau over optimal thermal ranges before declining at higher temperatures. In contrast, metacercarial infectivity is at its greatest at low temperatures, declining with increasing temperature.

Although results from field surveys have linked parasites to oxidative stress in their fish hosts, direct evidence involving experimentally infected hosts is lacking. We evaluated the effects of experimental infections with larval trematodes on induction of oxidative stress in fathead minnows, Pimephales promelas. Juvenile fish were exposed in the laboratory to the larvae (cercariae) of 2 species of trematode: Ornithodiplostomum sp. that develops in the liver, and O. ptychocheilus that develops in the brain. For Ornithodiplostomum sp., lipid peroxidation concentration in liver tissue increased 5 days after exposure and remained higher than controls until the end of the experiment at 28 days. For O. ptychocheilus, liver lipid peroxidation concentration was higher than controls at 5 days, but not thereafter. Sustained elevation in lipid peroxidation concentration for the liver trematode may be explained by direct tissue damage caused by developing larvae in the liver, or by an immune response. These experimental results support those from field studies, indicating that the lipid peroxidation assay may be an effective biomonitor for parasite-induced oxidative stress in fish, and that the nature of the oxidative stress response is species and/or tissue specific.

The changes in prevalence and abundance of the three species of metacercariae in the humour of the eyes of perch Perca fluviatilis in Slapton Ley, Devon, have been monitored over a period of 29 years. Earlier studies had revealed that Diplostomum gasterostei was originally the sole occupant of this niche, but Tylodelphys clavata colonised in 1973 and T. podicipina in 1976. A decline in the number of perch with heavy infections of D. gasterostei was significantly negatively correlated with population abundance of T. clavata and a decline in recruitment rate of D. gasterostei coincided with the population increase in T. podicipina over the period 1976–1979. It was suggested that the decline in population size of D. gasterostei was due to inter-specific competition, but this hypothesis could not be tested experimentally. Subsequent investigations, reported here, confirmed the decline when the data set was extended to 1985. A severe decline in the perch population over the winter of 1984/1985 resulted in the disappearance of D. gasterostei and T. podicipina and this was followed by a slow recovery from 1990 onwards. This natural experiment provided an opportunity to test the hypothesis. Only T. clavata survived throughout the perch crash and the population continued at pre-crash levels up to 1999. Its congener T. podicipina did not re-appear until 1994 and was probably a re-introduction: it did not attain pre-crash levels until 1999. It is likely that D. gasterostei survived the crash as it re-appeared in 1991, but was confined to young of the year fish and barely approached pre-crash levels even in 1999. Its continual low levels cannot be explained by changes in the lake or in densities of snail intermediate or bird definitive hosts. New data revealed that the suspensory ligaments of the eye were the preferred site of all three species and that the eye was partitioned out between them. The data from the post-crash period do not refute but rather confirm earlier conclusions that inter-specific competition is responsible for the decline in D. gasterostei and this remains the preferred hypothesis.

Parasitism is increasingly recognized as an important determinant of population dynamics, productivity and community structure in intertidal ecosystems, and yet there is very little known about the effect of parasites on polychaetes, which represent a major component of the benthic fauna. We surveyed 11 polychaete species from a mudflat in Otago Harbour, New Zealand, and found that seven of these were infected by five parasite species: four trematodes and one apicomplexan gregarine. The gregarine found in Spirobranchus cariniferus and a strigeid trematode using Streblosoma toddae as its first intermediate host are both likely to have negative fitness impacts on their hosts. Other trematodes found were at the metacercarial stage and thus use polychaetes as second intermediate hosts. The most common, an opecoelid, infected the polychaetes Heteromastus filiformis and Abarenicola affinis at relatively high abundance. There was no indication of parasite-induced mortality in these two hosts based on the relationship between host size and infection intensity. However, a comparison of intact H. filiformis individuals with those that fragmented during collection revealed a significantly higher number of opecoelid metacercariae per segment in the fragments than in the complete individuals, suggesting that infection may compromise the structural integrity of the polychaetes. These results suggest that there exists a great diversity of both trematodes and host–trematode associations within the polychaete fauna, whose ecological impact remains to be quantified.

The trematode Parorchis acanthus is widely distributed and exhibits a broad host spectrum, with larval stages occurring in marine gastropods and adult flukes parasitizing on various seabird species. Unlike the majority of digeneans, its cercariae do not invade a second intermediate host but encyst on a substrate in the open. In a series of laboratory experiments, we investigated factors influencing cercarial emergence and settlement of P. acanthus. Emergence of cercariae from the gastropod intermediate host Nucella lapillus increased with increasing temperature, whereas cercarial encystment success decreased. Numbers of emerged cercariae were highest in the presence of light and water. However, although in much lower numbers, cercariae were also released from N. lapillus and successfully encysted under relatively dry conditions. Cercariae were found to settle predominantly on filtering blue mussels Mytilus edulis, suggesting that cercarial concentration on the valves is largely facilitated by the bivalve's inhalation current. After being inhaled, cercariae actively emerged from the mussels using their suckers and subsequently encysted on the shells, preferably along the rim of the valves. Barnacle epibionts Semibalanus balanoides were observed to impair cercarial settlement on mussels, by disturbing or preying on cercariae. Whilst settlement success of cercariae on M. edulis increased with increasing mussel density, intensity of encysted metacercariae decreased. Furthermore, cercariae settled more efficiently on small than on large mussels. Our findings suggest that the transmission of P. acanthus cercariae is regulated by a range of abiotic and biotic factors, with filtering activity of mussels playing a key role by enhancing accumulation of cercariae on a substrate suitable for transmission to the bird final host.

The effect of ingestion by Lymnaea auricularia on the viability and infectivity of Fasciola gigantica metacercariae was studied. The cyst wall was unaffected by the snail's digestive processes. Two rabbits, each infected with 50 ingested metacercariae, died at 83 and 87 days post-infection. Eight and 10 immature flukes were recovered from the livers, indicating that the metacercariae had remained infective after passing through the intestine of the snail.

Metacercariae of Plagiorchis species 1 were observed to hatch by an active mechanism in a medium free of enzymes. A hatching opening in the bilayered cyst wall was formed by the combined action of caecal fluid extruded by the activated larva through the mouth opening and an internal pressure due to the tendency of the cyst wall to contract on hatching, resulting in an explosive expulsion of part of the metacercarial body. The cyst wall was apparently pierced at any place where the larva delivered its caecal fluid. After excystation the hatching medium contained high phosphatase and proteinase activities and was able to dissolve the inner walls of empty cyst envelopes. The phosphatase activity assayed on 4-methylumbelliferyl phosphate was optimal at pH 3–5. The proteolytic activity was demonstrable on photographic film, Azocoll, and synthetic chromogenic and fluorogenic peptides. A preference for peptides was found which are also susceptible to plasmin. The proteolytic activity was optimal at pH 3–9 and 40–45°C and, when assayed on Suc-Ala-Phe-Lys-MCA, was only due to thiol proteinase(s) according to inhibitor studies. It is suggested that the proteinase(s) represent the hatching enzyme(s) of the metacercaria, because (a) only proteolytic activity was detectable in the pH range optimal for excystment, (b) the inner cyst wall is stabilized by proteins, and (c) the inner wall is dissolved by other proteinases such as trypsin. Enzyme histochemical investigations of metacercariae showed that, in the caeca, acid phosphatase was present mainly before hatching and non-specific esterase developed after hatching. Proteolytic activity was not localized with the methods used although it was suggested that it derived from the caecal fluid. A possible relationship of the thiol proteinase(s) detected in the hatching medium to haemoglobin-digesting proteinases from the gut of schistosomes is discussed.

Scanning and transmission electron microscopical obervations were made on the tegument of excysted metacercariae and adults of the paramphistome. Zygocotyle lunata (Digenea: Trematoda). In accord with other paramphistomes studied, this species lacks spines and mitochondria in the tegumentary syncytium and associated cytons. The newly excysted metacercarie, which possessed relatively few tegumental papillae, were cylindrical in comparison to adults which were distinctly flat. The adults had large numbers of tegumental papillae in the region of the oral sucker and acetabulum.

For the first time the development of the alimentary tract of Levinseniella brachysoma metacercaria (Trematoda: Microphallidae) obtained experimentally from Gammarus oceanicus has been described. The foregut primordium in 16-day-old metacercariae is represented by a syncytial cylindrical cord, resulting from the fusion of embryonic cells. Non-fused parts of the plasma membranes of adjacent cells are revealed as gap cavities within the cord. Later (30th day post infection) the lumen of the foregut is formed as a result of both partial vacuolization of the cytoplasm and by a broadening of the gap cavities, resulting from a thinning of the cytoplasmic spaces between them. Besides the usual organelles, the foregut of the mature metacercaria (42nd day p.i.) contains dense secretory granules in the apical cytoplasm region and numerous microtubules in basal areas. The cellular gastrodermis is formed later than the foregut syncytium (on day 30 p.i.); its large cells contain well-developed Golgi complexes, RER and mitochondria. A noteable inclusion of the gastrodermal cells of mature metacercariae are spherical granules of moderate electron density measuring up to 3 μm in diameter. On the basis of an analysis of the ultrastructural data the possible functioning of the metacercarial alimentary tract is discussed.

Ultrastructural, histochemical and autoradiographical techniques have been used to investigate the development and function of the tegument of both somal and ecsomal body regions in the hemiurid Lecithochirum furcolabiatum. The terms mesocercaria and metacercaria are here adopted for those stages in the copepod second and fish third intermediate hosts respectively on the basis of morphology and on analogy with the Strigeidae. Mesocercariae were obtained by experimental infection of the harpacticoid copepod Tigriopus brevicornis with the cystophorous cercariae (syn. Cercaria vaullegeardi), whilst metacercariae were removed from the body cavities of naturally-infected rockpool teleosts including Gobius paganellus and Blennius pholis. Observations on the mesocercaria show the origin of the ecsoma from the distal half of the excretory vesicle, which at 21 days post-infection is capable of eversion through the terminal pore. The nucleated microrugous surface layer of the ecsoma at this stage is modified or replaced in the metacercaria by anucleate syncytial tegument similar to that of the adult organ. The metacercarial stage is also associated with the more advanced development of the somal tegument. The two stages differ in metabolic activity, the somal tegument of only the metacercaria resembling that of the adult in acid phosphatase distribution. In the mesocercaria the ecsomal tegument showed the strongest uptake of 3H-tyrosine; absorption of the latter was not detected in the metacercaria. 3H-glucose uptake was restricted to the intestinal caeca of both stages.

Experimental infection of Echinoparyphium recurvatum von Linstow (Digenea: Echinostomatidae) cercariae in the snail second intermediate host Lymnaea peregra Müller shows that metacercarial encystment takes place on the lining of the mantle cavity, pericardial cavity and kidney lumen, with the mantle cavity the most preferred site. All three sites are accessible via the body openings. The metacercariae appear to be more susceptible to encapsulation in the visceral mass than in the cavity of the mantle, pericardium and the lumen of the kidney.