Introduction
Digenetic trematodes (Trematoda: Digenea) form a large and diverse group of endoparasitic flatworms, commonly known as flukes, that generally parasitize various vertebrates during the adult stage [1]. Several digenean species have significant socio-economic and public health impacts, causing pathogenesis, bacterial invasion, inflammation, and severe morbidities and mortalities in fishes, livestock, and humans [1–7]. For instance, co-infections of Cyathocotylidae and motile aeromonads have been linked to the massive mortality of farmed Nile tilapia (Oreochromis niloticus) [5]. The ruminant blood flukes Schistosoma spindale and S. indicum (Schistosomatidae), which are widespread across South Asia, including Thailand, can cause visceral schistosomiasis, resulting in reduced milk yield and increased mortality in sheep and cattle [7]. Additionally, an outbreak of cercarial dermatitis caused by ruminant and avian schistosomes has been reported among rice farmers in South Thailand [6]. Notably, infections of the liver fluke Opisthorchis viverrini sensu lato in humans, which can cause and induce severe complications and mortalities due to bile duct cancer, have been well-documented in the Mekong River countries, i.e., Thailand (primarily the northeastern region), Laos, Myanmar, Cambodia, and Vietnam [8].
The life cycles of digenetic trematodes are complicated, typically involving at least 2 hosts [1]. Vertebrates usually serve as definitive hosts where sexual reproduction occurs, while invertebrates, especially snails, act as intermediate hosts where asexual reproduction occurs. Briefly, digenean eggs are released into aquatic environments via the excreta of the definitive hosts. These eggs are either ingested by snail hosts or hatch into miracidia, which penetrate snail tissues and transform into sporocysts, within which several embryos develop asexually into rediae. Subsequently, the rediae produce numerous cercariae as the final product of the asexual phase. The cercariae then escape the snail hosts, swim to find compatible second intermediate hosts, and encyst as metacercariae. The metacercariae mature into adults after the ingestion of these second intermediate hosts by definitive hosts. Asexual reproduction within snail hosts possibly amplifies cercarial transmission to subsequent hosts during their life cycles, highlighting the vital role of snails in ecosystem dynamics. Furthermore, prevalence data of cercarial infections in snails serve as a valuable indicator of digenetic trematode transmissions from definitive hosts into ecosystems and is useful for predicting the epidemiological status of the targeted parasites [8,9].
In Thailand, investigations on cercarial infections in 1–20 freshwater snail species/subspecies encompassing 1–10 families have been conducted in various scales of administrative areas [6,9–15]. The studies reported 6 to 19 morphologically distinct types across 4 to 9 morphotypes belonging to approximately 19 digenean families [6,9–14,16]. A study by Anucherngchai et al. [9], which focused on central Thailand, reported 9 cercarial morphotypes belonging to 5 digenean families in 8 out of 14 examined freshwater snail species/subspecies in the Chao Phraya Basin. Chontananarth et al. [11] also reported 7 cercarial morphotypes belonging to 7 digenean families in 14 freshwater snail species/subspecies from the Nakhon Nayok Province. The species Melanoides tuberculata (Thiaridae) was identified as the most susceptible to cercarial infection. However, Wiroonpan et al. [14], who comprehensively investigated cercarial infections in freshwater snails in Bangkok Metropolis, reported 8 cercarial morphotypes belonging to 10 digenean families in 12 infected snail species/subspecies; the Bithynia siamensis siamensis (Bithyniidae) was the most susceptible to cercarial infection.
The Bangkok Metropolitan Region (BMR) is a flat alluvial plain in central Thailand that comprises the Bangkok Metropolis, Nonthaburi, Nakhon Pathom, Samut Sakhon, Samut Prakan, and Pathum Thani Provinces. Despite its modest size compared to the rest of the country, BMR is epidemiologically significant because it is the most densely populated area in Thailand with a substantial migrant workforce [17]. In addition, this region features a vast array of water resources, including an extensive canal network catering to diverse land uses, such as agriculture, fisheries, livestock husbandry, and residential areas [18,19]. The BMR canal networks also presumably offer a diverse range of foods, habitats, and routes for the existence and distribution of digeneans and their hosts [20,21]. Moreover, most areas of the BMR canal networks are a public source of water where the country’s population can engage in activities such as swimming and fishing.
Despite the epidemiological significance of cercarial infections in molluscan intermediate hosts, there is a lack of comprehensive studies on trematode cercariae infections in freshwater snails from BMR. Therefore, this study aimed to investigate the infection status and species diversity of cercariae in freshwater snails from canal networks in the BMR in Thailand.
Materials and Methods
Ethics statement
Approval for this study (collecting, temporarily nurturing, and investigating trematode cercarial infections in freshwater snails) was obtained from the Kasetsart University Ethics Committee (Approval No. ACKU61-SCI-034).
Collection and nurture of snails
Thirty-five sites of the canal network encompassing all administrative zones/areas in the BMR (Fig. 1; Table 1) were selected for the survey. Random samplings of freshwater snails, carried out through hand-picking and scooping with a conventional wire-mesh scoop and a hand net, were executed by a single collector, allocating 20 min for each site. This approach was based on the count per unit of time sampling method [22]. The samplings were conducted every 3 months from January 2018 through October 2019, covering 2 cycles of the wet (July–October) and dry (November–June) seasons. The collected snails were transported to the laboratory and identified based on the shell and operculum morphology according to taxonomic keys [23,24]. The snails were temporarily nurtured in 2.5-L perforated, transparent plastic boxes containing 2 L of dechlorinated water, with an appropriate snail-to-box ratio maintained throughout the investigation period for cercarial infection.
Investigation of cercarial infection using the shedding and crushing methods
After acclimatization under laboratory conditions for one day, individual snail specimens were placed in 2-oz, 5-oz, or 9-oz transparent plastic cups, each filled to 3-quarters of its capacity with dechlorinated water (sized appropriately for the snails). Each cup was exposed to a light intensity of approximately 3,000 lux from 6:00 to 14:00 o’clock at room temperature (25±2°C) and then observed under a stereomicroscope. This shedding technique was performed on alternate days over 1 week. Subsequently, the crushing technique was employed, wherein the snail body was separated from its shell and operculum and crushed using Petri dishes containing a small volume of dechlorinated water; the samples were then examined under a stereomicroscope.
Morphological identification of the cercariae
The live cercariae detected in each shedding cup and those separated from the crushed snail tissues were transferred onto glass slides using glass Pasteur pipettes and fixed in a relaxed state using 10% neutral buffered formalin. Some cercarial specimens were vitally stained with 0.5% neutral red solution. The specimens were observed and photographed using an Olympus BX51 microscope equipped with an Olympus DP70 camera (Olympus Corporation, Tokyo, Japan). Multiplanar photomicrographs of mature cercariae were employed to identify them based on their morphological traits and movement behaviors according to the identification keys [1] and descriptions [6,12–14,25]. The morphological features were illustrated using Affinity Designer v1.10.6 (Serif Ltd., Nottingham, UK) and the metric traits (only displayed for dichotoma cercaria) were measured using ImageJ v1.53t (National Institutes of Health, Bethesda, MD, USA) based on the guidelines shown in Supplementary Fig. S2.
Statistical analysis
The prevalence of cercarial infection was assessed as described by Bush et al. [26]. The Chi-square test and odds ratios were used to compare the prevalence between the dry and wet seasons using R base functions. In addition, the exact confidence intervals (CIs) of the prevalences in the 2 seasons were calculated as previously described [27] using the epiR package [28]. These statistical evaluations were executed in R for Windows v4.3.2 (R Foundation for Statistical Computing, Vienna, Austria) through RStudio v2023.12.1 Build 402 (Posit PBC, Boston, MA, USA).
Results
General status of cercarial infection in freshwater snails
In a survey of 35 sites across all administrative zones in the BMR canal network, 29,420 freshwater snails were collected from January 2018 to October 2019, spanning two cycles in the wet and dry seasons. The snail specimens belonged to 11 families, 21 genera, and 24 species/subspecies (Supplementary Table S1; Fig. 2). Among them, 1,275 snails belonging to 12 species/subspecies (Supplementary Table S1; Table 2) were infected with cercariae, revealing an overall prevalence of 4.3%.
Prevalence of cercarial infection based on the locality
Variabilities in the prevalence of cercarial infection were evident across the 35 surveyed sites (Table 1). At the provincial level, the highest mean prevalence of cercarial infections in freshwater snails (5.7%; range, 0.0–4.6%) was observed in Pathum Thani, followed by Nakhon Pathom (5.1%; 1.6–8.4%), Nonthaburi (4.2%; 0.2–6.8%), Samut Sakhon (3.1%; 0.0–6.5%), Bangkok (2.5%; 0.0–4.6%), and Samut Prakan (2.2%; 0.0–5.5%). The highest prevalence (12.4%; 133/1,072) was observed at the Ra Haeng Canal in Lat Lum Kaew, Pathum Thani (PT6). In contrast, no infections were recorded at 4 surveyed sites: BK3, SP1, SP3, and SS3 (Table 1).
Prevalence and species diversity of cercariae in infected snails
Among the 12 field-infected freshwater snail species/subspecies identified in this study, M. tuberculata exhibited the highest prevalence of cercarial infection at 9.9% (62/624), while the lowest prevalence was observed in Tarebia granifera at 0.2% (7/4,591) (Table 2). Despite its widespread occurrence across 27 sites, M. tuberculata hosted a limited cercariae diversity with only 4 morphologically distinct types across 3 morphotypes. The snail species was moderately frequent within the BMR canal network (detected in 13 sites per sampling period), with a nearly equal ratio of sites with and without cercarial infections (13:14). On the other hand, the B. siamensis siamensis detected in 31 sites, showed the second-highest prevalence rate of infection at 8.5% (743/8,741); additionally, it hosted the greatest diversity of cercariae with 15 morphologically distinct types identified across 11 morphotypes, accounting for up to 40.5% of the total number of distinct cercarial types (Table 2; Supplementary Table S1; Figs. 3, 4). B. siamensis siamensis also demonstrated a high occurrence frequency, averaging 23 sites per sampling period (range, 20–27 sites). Notably, B. siamensis siamensis exhibited a significantly higher proportion of infected sites (23) than uninfected sites (8), accounting for 74.2% of the total number of sites where it was detected.
A total of 37 morphologically distinct types across 15 morphotypes of cercariae (Table 3; Figs. 3, 4) were detected from all the snail specimens. Fifteen morphotypes of cercariae were classified up to the family level, encompassing a range of 15–19 families, as listed in Table 3. Among the 15 cercarial morphotypes, armatae xiphidiocercariae (Figs. 3R–V, 4W–Y) was most prevalent (infection rate, 1.8%; 526/29,420), exhibited the greatest diversity (8 morphologically distinct types) within a morphotype of cercariae (Table 3), and encompassed the broadest snail host spectrum (7 species/subspecies: B. siamensis siamensis, Wattebledia siamensis, Radix rubiginosa, M. tuberculata, T. granifera, Filopaludina martensi martensi, and F. sumatrensis polygramma) (Table 2; Supplementary Table S1). The mutabile cercariae (Fig. 3A, B) and virgulate xiphidiocercariae (Fig. 3P) morphotypes were the second and third most prevalent at 0.9% (258/29,420) and 0.8% (228/29,420), respectively, (Table 3). The combination of these 3 morphotypes represented 77.7% of the overall prevalence of cercarial infection in freshwater snails from the canal network in the BMR. In contrast, 5 cercarial morphotypes, i.e., clinostomatoid cercariae (Fig. 3F, G), lophocercous-apharyngeate cercariae (Fig. 3H, I), ubiquita xiphidiocercaria (Fig. 3Q), megalurous cercaria (Fig. 4f), and pleurolophocercous cercariae (Fig. 4g, h), displayed minimal occurrences, each with a prevalence of less than 0.03% (Table 3). Additionally, they displayed a low cercarial diversity (1–2 morphologically distinct types) within morphotypes (Table 3) and a limited host snail spectrum (range, 1–2 species/subspecies; Table 2).
Gymnophallid fluke cercariae found in freshwater snails
Dichotoma cercaria (Fig. 3E; Supplementary Fig. S2), a cercarial morphotype of marine trematodes belonging to the Gymnophallidae family, was discovered from 4 freshwater snail taxa, i.e., W. siamensis, B. siamensis siamensis, F. martensi martensi, and F. sumatrensis polygramma, with a prevalence of 0.1% (1/792), 0.1% (12/8,841), 0.4% (15/3,504), and 0.3% (12/4,792), respectively (Supplementary Table S1). The prevalence of this cercarial morphotype across all snail samples was 0.1% (40/29,420), with a higher prevalence during the wet season (0.2%; 38/16,473) than in the dry season (0.02%; 2/12,947). Dichotoma cercaria was found to be distributed in nine sites, with the highest prevalence observed in NB4 (1.5%; 18/1,191) and the lowest in NP5 and NB6 (0.1%; 1/923) (Supplementary Table S2; Supplementary Fig. S1).
Examination of the general morphology of the dichotoma cercaria revealed an elongated oval body with a forked, longifurcate tail that was shorter than the body. The tail stem was shaped like a large, plump rod, with the tail furcae accounting for approximately 3-quarters of its length. Both the oral and ventral suckers were nearly equal in size, the former being pear-shaped and large. The ventral sucker, positioned in the third region of the body from the anterior end, appeared large, muscular, and highly noticeable, with a wide opening that accounted for about 3-quarters of its size. The prepharynx was present, and the pharynx was large and distinctly muscular, featuring a prominent medial slit bordered by distinct ridges on both sides. The esophagus and intestinal caeca were short and located in the middle of the body. The penetration glands were large and spherical, comprising one pair, each flanking the pharynx and the esophagus, with their large tubes entering on both sides of the base of the oral sucker. The excretory bladder was notably large, U-shaped, and connected to the proximal end of the tail. The excretory duct appeared in the middle region of the tail stem and bifurcated at the tail furcae. The excretory pores were prominent and situated at the tip of the tail furcae. The 23 metric morphological traits of the dichotoma cercaria are presented in Supplementary Table S3 and Supplementary Fig. S2.
Seasonal variation of cercarial prevalence
The total prevalence of cercariae in freshwater snails from the BMR canal network was notably higher during the wet season (2.4%; 305/12,947; exact CIs=5.5–6.3%) compared to that in the dry season (5.9%; 970/16,473; exact CIs=2.3–3.0%). This difference was statistically significant, as confirmed by a Chi-square value (χ2) of 218.21 (degrees of freedom [df], 1; P-value<0.001). Moreover, the calculated odds ratio was 2.59 (95% CI, 2.28–2.96), indicating a 2.6 times higher risk of infection during the wet season compared to the dry season. Overall, higher prevalences of almost all morphotypes of cercariae were generally detected in the wet season (Table 3), with a mean prevalence of 0.4% (range, 0.00–2.4%), while a mean prevalence of 0.2% (range, 0.0–1.0%) was detected in the dry season. Two exceptions were noted: pleurolophocercous cercariae and parapleurolophocercous cercariae displayed slightly higher prevalences in the dry season, and ubiquita cercaria and clinostomatoid cercariae were found exclusively in 2 individual snail samples during the dry season.
Discussion
In the present study, the overall prevalence of cercarial infections in freshwater snails from the BMR canal network was 4.3%, a finding slightly divergent from previous investigations in central Thailand; a lower overall prevalence of 3.3% for cercarial infection in freshwater snails from natural ponds and canals in Bangkok [14]. This variation in infection rates could be partially attributed to the unceasing decrease in suitable habitats for potential intermediate and definitive hosts of certain digenean trematodes [29–31] in Bangkok compared to those in the broader BMR. In contrast, earlier studies [9,11] documented overall prevalences of 4.7% and 5.9% for cercarial infection in freshwater snails from Nakhon Nayok and the Chao Phraya Basin, respectively, regions characterized by lower urbanization levels compared to the BMR [32].
Melanoides tuberculata showed the highest cercarial infection rate (9.9%) among the 24 snail species/subspecies examined within the BMR canal network. This observation is consistent with a previous report [10], wherein a cercarial infection rate of 5.5% was observed in M. tuberculata from 11 freshwater snail species in various aquatic environments in the Mae Lao Agricultural Basin at Chiang Rai, Thailand. A previous investigation [12] also documented a higher infection rate of 18.8% in M. tuberculata across Thailand, suggesting a pronounced vulnerability of this snail species to larval trematode infections.
Despite the highest cercarial infection rates in M. tuberculata in the current and prior studies, our results indicate that the B. siamensis siamensis snail may represent a greater potential risk to public health, livestock husbandries, and aquacultures. This assessment was based on 5 key factors exhibited by B. siamensis siamensis: (1) a marginally lower infection rate of 8.5% compared to 9.9% in M. tuberculata; (2) a more extensive geographic presence, detected in 31 sites as opposed to 27 for M. tuberculata; (3) a generally higher occurrence frequency, with B. siamensis siamensis detected in an average of 23 sites per sampling period (range, 20–27 sites) compared to an average of 13 sites for M. tuberculata (range, 7–19 sites); (4) a higher ratio of infected to uninfected sites (23:8) compared to M. tuberculata (13:14); and (5) a markedly greater diversity of hosted trematode cercariae, harboring 15 morphologically distinct types across 11 morphotypes, in contrast to M. tuberculata, which hosted 4 distinct types within 3 morphotypes.
Among 11 cercarial morphotypes from B. siamensis siamensis snails, classified into 11–15 digenean families, members of 10 families have been associated with varying degrees of public health, veterinary, and socio-economic concerns. For instance, massive mortalities in farmed Nile tilapia fish were linked to Cyathocotylidae spp. and motile aeromonad co-infections [5]. Aporocotylidae, affecting both wild and farmed marine fish, hold economic importance; however, the economic impact has not been reported in Thailand so far [3]. Certain species of schistosomatidae are critical due to their roles in widespread schistosomiasis, affecting humans and livestock globally, especially in tropical regions [1,4]. Philopthalmidae occasionally infect humans; a rare case of conjunctival philophthalmiasis was reported in Thailand [33]. Opisthorchiidae, especially Opisthorchis viverrini sensu lato, with tobacco-pipe-shaped cercariae morphologically similar to pleurolophocercous cercaria I, are medically significant in Thailand [8]. Heterophyids are capable of causing severe clinical manifestations when they accidentally travel to vital organs [34]. These aspects underscore the significant ecological and epidemiological implications of B. siamensis siamensis within the BMR.
In Thailand, earlier investigations on trematode cercariae in freshwater snails revealed a low to moderate diversity, with recorded instances of 5 to 9 morphotypes and 6 to 9 distinct types across the examined 6 to 20 snail species/subspecies [6,9–11,14,35]. In stark contrast, the current study uncovered an obviously higher diversity of trematode cercariae, documenting 37 morphologically distinct types across 15 morphotypes from 24 freshwater snail species/subspecies. This marked increase plausibly aligns with the findings of Gordy et al. [36], who demonstrated a positive correlation between the diversities of trematode communities and snail species.
Among 15 cercarial morphotypes classified across 15–19 digenean families, the armatae morphotype (Plagiorchiidae/Telorchiidae) recorded the highest infection rate at 1.8%, constituting 40.5% of the total infections and exhibiting the greatest diversity with 8 distinct types, likely due to the broadest host range (7 snail species/subspecies) [36]. The small size of the armatae morphotype may enhance larval concentrations within intermediate hosts, boosting the transmission potential [37]. Additionally, its stylet facilitates host penetration, increasing infection efficacy. Moreover, plagiorchiids, which have a broad definitive host range spanning all vertebrate classes and utilize freshwater snails, fishes, and diverse aquatic insects as second intermediate hosts [1], along with telorchiids, which target amphibians and reptiles as definitive hosts and use snails, bivalves, and tadpoles as second intermediate hosts [1], likely promote interactions among the life cycle stages and their compatible hosts within shared ecosystems, enhancing infection opportunities. Regarding the impact on human health, only 5 Plagiorchis species have been reported as human parasites, and their pathological effects are considered minimal [34].
Interestingly, the current study documented the first instance of a gymnophallid digenean in freshwater, identifying dichotoma cercaria in freshwater F. martensi martensi, F. sumatrensis polygramma, B. siamensis siamensis, and Wattebledia siamensis snails in Thailand. Gymnophallidae, a small marine digenean family, primarily utilizes marine bivalves as first and second intermediate hosts, although some members in the metacercarial stage do not encyst and rarely parasitize marine gastropods, brachiopods, and polychaetes [1,38]. Notably, Gymnophalloides seoi, commonly found in the Palearctic oystercatcher (Haematopus ostralegus), also poses health risks to humans, particularly in endemic coastal areas of South Korea, where consumption of raw or undercooked oysters is linked to gastrointestinal issues ranging from mild to severe symptoms [39].
In the present study, the cercarial infection rate in freshwater snails was significantly higher during the wet season (5.9%) than in the dry season (2.4%), exhibiting a 2.5-fold increase. This pattern was consistent across most cercarial morphotypes and aligns with previous findings by Brockelman et al. [40], who noted that seasonal factors like the water temperature and duration and amount of rainfall significantly influence the interactions between hosts and parasites. A previous study has shown that the rainy and monsoon seasons enhance digenean transmission because increased precipitation can lead to higher fecal contamination with digenean eggs from definitive and reservoir hosts to snail habitats [21].
In conclusion, 37 morphologically distinct types across 15 morphotypes of cercariae were detected in 12 of the 24 examined species/subspecies of freshwater snails from canal networks in the BMR, Thailand. Among all freshwater snails identified, the Bithynia siamensis siamensis subspecies exhibited the most significant epidemiological role in snail-transmitted parasitic diseases within the BMR flowing-water networks. The prevalence of cercariae in freshwater snails varied seasonally. The present study is the first to document the presence of gymnophallid larval fluke (dichotoma cercaria) in freshwater intermediate hosts. Additionally, this study provides qualitative and quantitative morphological descriptions and measurement guidelines for the newly discovered gymnophallid cercaria. Nonetheless, molecular approaches and phylogenetic analyses are essential for precisely identifying this cercaria species in the future.
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