Cockle as Second Intermediate Host of Trematode Parasites: Consequences for Sediment Bioturbation and Nutrient Fluxes across the Benthic Interface

Trematode parasites are distributed worldwide and can severely impact host populations. However, their influence on ecosystem functioning through the alteration of host engineering behaviours remains largely unexplored. This study focuses on a common host parasite system in marine coastal environments, i.e., the trematode Himasthla elongata, infecting the edible cockle Cerastoderma edule as second intermediate host. A laboratory experiment was conducted to investigate the indirect effects of metacercarial infection on sediment bioturbation and biogeochemical fluxes at the sediment water interface. Our results revealed that, despite high parasite intensity, the sediment reworking and bioirrigation rates, as well as nutrient fluxes, were not impacted. This finding was unexpected since previous studies showed that metacercarial infection impairs the physiological condition of cockles and induces a mechanical obstruction of their feet, thus altering their burrowing capacity. There are several explanations for such contrasting results. Firstly, the alteration of cockle behavior could arise over a longer time period following parasite infection. Secondly, the modulation of cockle bioturbation by parasites could be more pronounced in older specimens burying deeper. Thirdly, the intensity of the deleterious impacts of metacercariae could strongly vary across parasite species. Lastly, metacercarial infection alters cockle fitness through an interaction with other biotic and abiotic environmental stressors.

Parvatrema duboisi (Digenea: Gymnophallidae) Life Cycle Stages in Manila Clams, Ruditapes philippinarum, from Aphae-do (Island), Shinan-gun, Korea

Life cycle stages, including daughter sporocysts, cercariae, and metacercariae, of Parvatrema duboisi (Dollfus, 1923) Bartoli, 1974 (Digenea: Gymnophallidae) have been found in the Manila clam Ruditapes philippinarum from Aphaedo (Island), Shinan-gun, Jeollanam-do, Korea. The daughter sporocysts were elongated sac-like and 307-570 (av. 395) μm long and 101-213 (av. 157) μm wide. Most of the daughter sporocysts contained 15-20 furcocercous cercariae each. The cercariae measured 112-146 (av. 134) μm in total length and 35-46 (av. 40) μm in width, with 69-92 (av. 85) μm long body and 39-54 (av. 49) μm long tail. The metacercariae were 210-250 (av. 231) μm in length and 170-195 (av. 185) μm in width, and characterized by having a large oral sucker, genital pore some distance anterior to the ventral sucker, no ventral pit, and 1 compact or slightly lobed vitellarium, strongly suggesting P. duboisi. The metacercariae were experimentally infected to ICR mice, and adults were recovered at day 7 post-infection. The adult flukes were morphologically similar to the metacercariae except in the presence of up to 20 eggs in the uterus. The daughter sporocysts and metacercariae were molecularly (ITS1-5.8S rDNA-ITS2) analyzed to confirm the species, and the results showed 99.8-99.9% identity with P. duboisi reported from Kyushu, Japan and Gochang, Korea. These results confirmed the presence of various life cycle stages of P. duboisi in the Manila clam, R. philippinarum, playing the role of the first as well as the second intermediate host, on Aphae-do (Island), Shinan-gun, Korea.

Echinostoma mekongi: Discovery of Its Metacercarial Stage in Snails, Filopaludina martensi cambodjensis, in Pursat Province, Cambodia

Echinostoma mekongi was reported as a new species in 2020 based on specimens collected from humans in Kratie and Takeo Province, Cambodia. In the present study, its metacercarial stage has been discovered in Filopaludina martensi cambodjensis snails purchased from a local market nearby the Tonle Sap Lake, Pursat Province, Cambodia. The metacercariae were fed orally to an experimental hamster, and adult flukes were recovered at day 20 post-infection. They were morphologically examined using light and scanning electron microscopes and molecularly analyzed by sequencing of their mitochondrial cox1 and nad1 genes. A total of 115 metacercariae (1-8 per snail) were detected in 60 (60.0%) out of 100 Filopaludina snails examined. The metacercariae were round, 174 µm in average diameter (163-190 µm in range), having a thin cyst wall, a head collar armed with 37 collar spines, and characteristic excretory granules. The adult flukes were elongated, ventrally curved, 7.3 (6.4-8.2)×1.4 (1.1-1.7) mm in size, and equipped with 37 collar spines on the head collar (dorsal spines in 2 alternating rows), being consistent with E. mekongi. In phylogenetic analyses, the adult flukes showed 99.0-100% homology based on cox1 sequences and 98.9-99.7% homology based on nad1 sequences with E. mekongi. The results evidenced that F. martensi cambodjensis snails act as the second intermediate host of E. mekongi, and hamsters can be used as a suitable experimental definitive host. As local people favor to eat undercooked snails, these snails seem to be an important source of human infection with E. mekongi in Cambodia.

A new second intermediate host and phylogenetic relationships based on the ITS2 sequence of Isoparorchis sp. (Digenea: Isoparorchiidae) in Thailand

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.

Distribution of cysts holding Ascocotyle (Phagicola) longa metacercariae in tissues and organs of mugilid

Ascocotyle (Phagicola) longa is an etiological agent of human phagicolosis. Mugilids are the second intermediate host, the first being Heleobia australis, and mugilids predatory birds and mammals are its definitive hosts. The occurrence of cysts holding A. longa metacercariae is described in mugilids with a prevalence of up to 100%. The wide geographical distribution of A. longa and its intermediate hosts coupled with the rise in the consumption of raw or poorly cooked fish may elevate the risk of human infection. Therefore, in this study, we aimed to verify the distribution pattern of cysts holding A. longa in mugilids. The tissue and organ samples of these fish were processed in a domestic blender and examined under a stereoscopic microscope to identify the cysts holding the digenetic metacercariae. Of the 24 (100%) fish samples that were analyzed, 12 of Mugil curema and 12 of Mugil liza possessed cysts holding A. longa metacercariae. Digenetic cysts were identified to be present in the gills, heart, stomach, liver, intestines, mesentery, and muscular tissues collected from M. curema and M. liza. Conclusively, in M. curema, the cysts holding A. longa metacercariae were found to be distributed randomly throughout the fish body in almost every tissue and organ that was examined.

Morphology and Molecular Identification of Echinostoma revolutum and Echinostoma macrorchis in Freshwater Snails and Experimental Hamsters in Upper Northern Thailand

Echinostome metacercariae were investigated in freshwater snails from 26 districts in 7 provinces of upper northern Thailand. The species identification was carried out based on the morphologies of the metacercariae and adult flukes harvested from experimental hamsters, and on nucleotide sequences of internal transcribed spacer 2 (ITS2) and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1) genes. Twenty-four out of 26 districts were found to be infected with echinostome metacercariae in freshwater snails with the prevalence of 40.4%. The metacercariae were found in all 6 species of snails, including Filopaludina martensi martensi (21.9%), Filopaludina doliaris (50.8%), F. sumatrensis polygramma (61.3%), Bithynia siamensis siamensis (14.5%), Bithynia pulchella (38.0%), and Anenthome helena (4.9%). The echinostome metacercariae found in these snails were identified as Echinostoma revolutum (37-collar-spined) and Echinostoma macrorchis (45-collar-spined) morphologically and molecularly. The 2-week-old adult flukes of E. revolutum revealed unique features of the cirrus sac extending to middle of the ventral sucker and smooth testes. E. macrorchis adults revealed the cirrus sac close to the right lateral margin of the ventral sucker and 2 large and elliptical testes with slight indentations and pointed posterior end of the posterior testis. The ITS2 and nad1 sequences confirmed the species identification of E. revolutum, and the sequences of E. macrorchis have been deposited for the first time in Gen-Bank. The presence of the life cycle of E. macrorchis is a new record in Thailand and the snail F. doliaris as their second intermediate host seems to be new among the literature.

Parasite-modified behaviour in non-trophic transmission: trematode parasitism increases the attraction between snail intermediate hosts

Many parasites with complex life cycles cause host behavioural changes that increase the likelihood of transmission to the next host. Parasite modification is often found in trophic transmission, but its influence on non-trophic transmission is unclear. In trematodes, transmission from the first to second intermediate host is non-trophic, suggesting that free-swimming larvae (cercariae) emerging in closer proximity to the next host would have higher transmission success. We performed a series of behavioural experiments with echinostome trematodes and their snail hosts to determine if potential second hosts (ramshorn snail, genus Planorbella Haldeman, 1842) were more attracted to parasitized first hosts (marsh pondsnail, Lymnaea elodes Say, 1821). In a Y maze, a responding snail (Planorbella sp.) was placed in the base and its response to five treatments was assessed: no stimulus, turion duckweed (Lemna turionifera Landolt; a food item), non-parasitized L. elodes, parasitized L. elodes, and finally parasitized versus non-parasitized L. elodes. Snails showed some attraction to uninfected snails, but had a stronger response to infected first host snails. These results indicate that potential second host snails were more attracted to parasitized, heterospecific first host snails over non-parasitized heterospecific snails. This study demonstrates that echinostome trematodes alter snail behaviour by changing navigational choices in uninfected potential hosts through a chemical communication mechanism.

Monorchis parvus and Gymnophallus choledochus: two trematode species infecting cockles as first and second intermediate host

The most deleterious stage of a trematode life cycle occurs in the first intermediate host where the parasite penetrates as a miracidium and asexually multiplicates in sporocysts or rediae. When infection advances, other organs can be occupied with severe effects on host individual health and population dynamics. Existing studies focused on these host/parasite systems are still scarce due to the usual low prevalence in ecosystems. Using cockles (Cerastoderma spp.) and two trematode species (Monorchis parvus and Gymnophallus choledochus) infecting these bivalves as first and second intermediate host, the present work aimed to (1) summarize the most relevant literature and (2) provide new information regarding this host/parasite system, taking advantage of a 21-year monthly database from Banc d'Arguin (France). This long-term monitoring showed that different trematode species display varying host size range preference (6–38 and 31–36 mm for M. parvus and G. choledochus, respectively). The occurrence of coinfection was lower than expected, raising some questions related to parasite interspecific competition. This review improved our understanding of the processes shaping the prevalence and distribution of parasitism. This study highlighted that beyond constant trematode assemblage monitoring, there is a need to identify the main predictors of rediae/sporocysts infection, such as the definitive host dynamics and miracidium infection processes, for future better management of host severe disease and mortality episodes.