Infection with Clonorchis sinensis (Cobbold, 1875) Metacercariae in Fish from the East Lake of Wuhan: Freshwater Fish in Urban Lakes May Act as Infection Sources of Liver Fluke

The liver fluke disease caused by Clonorchis sinensis is one of the most serious food-borne parasitic diseases in China. Many freshwater fish and shrimps can be infected with C. sinensis metacercariae as the second intermediate hosts in endemic regions. Owing to the lack of infected humans and the good administration of pet dogs and cats in cities of non-endemic regions, few fish are expected to be infected with C. sinensis metacercariae in urban lakes. To determine the infection of C. sinensis metacercariae in freshwater fish and shrimps in urban lakes, a total of 18 fish species and one shrimp species were investigated in the East Lake of Wuhan City. Metacercariae were isolated by artificial digestive juice and identified using morphology and rDNA-ITS2 sequences. Five species of fish, Pseudorasbora parva, Ctenogobius giurinus, Squalidus argentatus, Hemiculter leuciclus, and Rhodeus spp., were infected with C. sinensis metacercariae. The overall prevalence of C. sinensis was 32.5%. The highest prevalence was found in P. parva with 57.9%, while S. argentatus exhibited the highest mean abundance (13.9). Apart from the C. sinensis metacercariae, four species of other trematode metacercariae were also identified across twelve fish species in total. Owing to the consumption of undercooked fish and feeding cats with small fish caught by anglers, there is a potential risk that the small fish infected with C. sinensis metacercariae may act as an infection source to spread liver fluke. Given the complete life cycle of C. sinensis, stray cats and rats were inferred to act as the important final hosts of C. sinensis in urban lakes in non-endemic areas.


Introduction
Clonorchis sinensis (Cobbold, 1875), also known as the oriental liver fluke, is one of the most important fish-borne zoonotic trematodes (FZT).Based on a national survey conducted in 2003 in China, 15 million people were estimated to be infected with C. sinensis in East Asia, of whom more than 12 million were in China [1], and the remainder were distributed across South Korea, Vietnam, and Russia [1][2][3].In China, the major endemic areas were concentrated in four provinces: Guangxi and Guangdong in the south and Heilongjiang and Jilin in the northeast [4].The consumption of raw or undercooked freshwater fish was considered to be responsible for the high prevalence in the endemic regions of East Asia [5].
There are three hosts in the life cycle of C. sinensis: freshwater snails as the first intermediate host, freshwater fish and occasionally shrimps as the second intermediate host, and human or carnivorous mammals (cats and dogs) as the definitive host.In the endemic regions, a high prevalence of C. sinensis metacercariae was commonly reported in some fish species in fishponds, lakes, and rivers [6].The overall infection rate of C. sinensis metacercariae was 37.1%, and a higher than 50% prevalence was found in Pseudorasbora parva and Ctenopharyngodon idelluse in Guangdong Province [7], as well as a high prevalence in P. parva (45.3%) and in Misgurnus anguillicaudatus (41.2%) in Guangxi Province [8].In Heilongjiang Province, a 19.9% overall prevalence was found in 3221 examined fish, and P. parva again exhibited the highest prevalence of 42.6% [9].The habit of building toilets and pigsties next to fishponds in endemic areas contributed to the eggs ending up in pond water, which then caused high infection rates of C. sinensis metacercariae in freshwater fish [5].
In non-endemic regions, C. sinensis metacercariae were also detected in fish in fishponds, lakes, and rivers [10,11].Domestic dogs and cats, usually fed raw fish, were considered the key final hosts to discharge feces with eggs of C. sinensis into waters [5,12,13].Nowadays, in non-endemic regions, few people are infected with C. sinensis, and it is prohibited to discharge feces into urban lakes.Dogs and cats kept as pets are also welladministered from a veterinary standpoint.Thus, the prevalence of C. sinensis metacercariae in fish from urban lakes is expected to be low.
East Lake of Wuhan is one of the biggest urban lakes in China, covering an area of 33 square kilometers.The lake is isolated from the Yangtze River and other rivers [14], and more than 39 fish species were found in the lake [15].The discharge of industrial wastewater and domestic sewage into the East Lake is not allowed.In early studies, C. sinensis metacercariae were found in fish collected from the fish market in Wuhan [16,17].During the third survey of parasitic diseases in Hubei Province, conducted from 2014 to 2015, only one case of infection with C. sinensis was detected, in Chongyang City, far from Wuhan City [18].In recent decades, pet cats and dogs are also well-administered around the East Lake.On the basis of these circumstances, we hypothesized that it is unlikely that C. sinensis metacercariae can be detected in freshwater fish and shrimps in the East Lake of Wuhan.To test this hypothesis, the infection of C. sinensis metacercariae was investigated in fish and shrimps collected from the East Lake.

Collection of Fish and Shrimps
Fish and shrimps were collected in multiple batches using a net or fish cage with a fine mesh in East Lake (113 • 41 ′ -115 • 05 ′ E, 29 • 58 ′ -31 • 22 ′ N) from April to July in both 2021 and 2022.The fresh fish and shrimps were kept at 5 • C and examined within two days.

Examination of Encysted Metacercariae
First, species of fish and shrimps were identified, and the total length and weight of each fish were measured.The head, scales, viscera, and bones of each fish were removed, and the remaining fish meat was collected for further examination.The muscle tissue of each fish was minced and mixed with artificial gastric juice (67.4 mL hydrochloric acid, 50 g pepsin, diluted with distilled water to 10 L; ten times the volume of the fish meat).The mixture was incubated for 5-8 h at 37 • C, and the digested fish meat was then filtered with a 40-mesh copper sieve.The fluid was placed into a 500 mL beaker for 20 min, and the supernatant was then replaced with a normal saline solution.This procedure was repeated more than 3 times until the supernatant was clear.Finally, the supernatant was discarded, and the sediment was placed into a glass dish in batches [7].The presence of encysted metacercariae was examined under a stereomicroscope.Metacercariae in each fish were counted and preserved in 90% alcohol for morphological and molecular identification.

Morpholgocial Identification of Metacercariae
Based on the characteristics of suckers and excretory bladders, the metacercariae of C. sinensis were differentiated from other trematode species [19].Other metacercariae were also grouped according to these morphological characteristics.According to the number of collected metacercariae, 1 to 11 metacercariae from each group were chosen for further molecular identification.
Species of C. sinensis and other trematode metacercariae were then identified by blasting against the nucleotide database of GenBank using the obtained sequences.

Statistical Analysis
Owing to the limited fish sample size in one month, all the fish specimens collected at different months and years were put together for analysis of infection.Prevalence (infection rate) was defined as the number of fish infected with metacercariae of C. sinensis or other trematodes divided by the number of fish examined ×100%.Mean abundance was calculated as the total number of metacercariae divided by the number of fish examined [21].

Identification of Clonorchis sinensis Metacercariae
Metacercariae with two equal-sized suckers, O-shaped dark excretory bladders, and brownish pigment granules were identified as C. sinensis (Figure 1A).ITS2 sequences of the 11 sequenced specimens were identical and also exhibited 100% sequence identity with a sequence belonging to C. sinensis available in GenBank (MN128618; Table 1).

Identification of Metacercariae of other Trematode Species
Except for Clonorchis sinensis, four forms of other trematode metacercariae were identified based on the shape of metacercariae, wall thickness, and the outline of the entity inside the wall (Figure 1B-E).ITS sequences of the four forms of metacercariae exhibited less than 98.1% identity (Table 2).On the basis of sequences available in GenBank, we inferred that form 1, form 2 and form 3 may belong to different species in the family Heterophyidae (Opisthorchiida) (Table 1), whereas form 4 exhibited the highest similarity (94.7%) with Holostephanus sp. in the family Cyathocotylidae (Diplostomida).

Infection with Clonorchis sinensis and Other Trematode Metacercariae in Fish and Shrimps
Among the 815 fish (18 species) examined, 265 fish specimens belonging to five species were found to be infected with C. sinensis.A total of 1622 metacercariae were detected, and the overall prevalence was 32.5%.The highest prevalence (57.9%) was found in P. parva, followed by Ctenogobius giurinus, Rhodeus spp., Squalidus argentatus, and Hemiculter leucisculus.The highest mean abundance (13.9 ± 2.8) was found in S. argentatus.No metacercariae were detected in the 219 shrimps, all of which belonged to the same species Macrobrachium nipponense (Table 3).Table 3. Infection of Clonorchis sinensis and other trematode metacercariae in freshwater fish and shrimp from East Lake of Wuhan, China.N, the number of examined fish; ML, the mean fish body length; P, the prevalence of C. sinensis metacercariae; MA, the mean abundance of C. sinensis metacercariae per fish or in 5 g of fish meat; PT, the prevalence of metacercariae of other trematodes.Twelve species of fish were found to be infected with four other types of trematode metacercariae.Their total number was 672, and their prevalence ranged from 6.7% to 100% (Table 3).
As in previous investigations, the topmouth gudgeon P. parva was infected with C. sinensis metacercariae with a high prevalence and mean abundance.The preferred water layer inhabited by topmouth gudgeon, and its thin skin, were proposed as the primary factors that make this species highly susceptible to C. sinensis [9].Hemiculter leucisculus and Rhodeus spp.were also previously recognized as common second intermediate hosts [8,9], but this is the first record of C. giurinus and S. argentatus as the second intermediate hosts of C. sinensis.
The consumption of raw or undercooked freshwater fish is considered to be the main transmission route of C. sinensis in human infections [1,5].Although small fish are generally not used to make sashimi, small fish fried with flour are commonly consumed in China.If such fish is undercooked, it may be dangerous to eat it.In addition, East Lake is popular with recreational anglers, who often use the caught small fish to feed cats, so the cats may become infected with C. sinensis.It is also possible that humans may become infected via the accidental ingestion of C. sinensis metacercariae on their hands after handling infected fish [24], even if the probability of this event is considered rare.Therefore, there is a potential risk that infected fish in East Lake may act as infection sources, transmitting C. sinensis to the final hosts.
Furthermore, the topmouth gudgeon P. parva was accidentally introduced into Europe and Africa with commercial fish and became an invasive fish in the world [25].Fish species in Rhodeinae were caught and usually bred as ornamental fish in China.Consequently, the introduction and trade of P. parva and Rhodeus spp.also inevitably spread C. sinensis metacercariae worldwide.
Besides C. sinensis metacercariae, other trematode metacercariae were frequently detected in freshwater fish.There were 16 species of zoonotic trematode metacercariae encysted in freshwater fish in Korea, and the FTZ belonged to seven families, such as Opisthorchiidae, Heterophyidae, Echinostomatidae, Clinostomidae, Cyathocotylidae, Cryptogonimidae, and Bucephalidae [19].Metacercariae of Metorchis orientalis (Opisthorchiidae), Haplorchis taichui, H. pumilio, and Centrocestus formosanus (Heterophyidae) were found in P. parva and Rhodeus spp. in China [26].In the present study, three species in Heterophyidae and one species in Cyathocotylidae were identified in twelve fish species using the ITS sequence marker.The high prevalence (6.7-100%) of other trematode metacercariae suggested that more attention should be paid to the infection of other FTZ in freshwater fish.
The finding of C. sinensis metacercariae in freshwater fish suggested that C. sinensis could complete its life cycle in East Lake, i.e., that the first intermediate hosts and the final hosts also populate this urban lake.In China, eight species of freshwater snails were recognized as the first intermediate host for C. sinensis: Alocinma longicornis, Parafossarulus striatulus, P. sinensis (Hydrobiidae), Semisulcospira cancellata (Melaniidae), Bithynia fuchsianus, B. robustus (Bithyniidae), Melanoides tuberculata (Thiaridae), and Assiminea lutea (Assimineidae) [5].Alocinma longicornis, P. striatulus, P. sinensis, and B. fuchsianus were considered to be important hosts in Hubei Province [27].During previous surveys of macrozoobenthos, A. longicornis and P. striatulus were found in East Lake [28].The two snail species may act as the first intermediate hosts of C. sinensis.
In endemic regions, an important transmission route is the release of C. sinensis eggs into the external environment via the feces of infected humans or other definitive hosts.The prevalence of C. sinensis was high in cats and dogs in southern Chinese endemic regions [5].In northern endemic regions, the prevalence was higher in cats than in dogs, and pigs were infected at lower levels [13].In a recent survey of parasitic diseases in Hubei Province, only one case of human infection by C. sinensis was found in Chongyang City, far from Wuhan [18].In addition, pet cats and dogs were well-administrated around East Lake, and the discharge of municipal wastewater including feces into the East Lake was forbidden.However, stray cats were occasionally seen around the lake.As there is no investigation of C. sinensis in other final hosts in Wuhan, we can only speculate that stray cats act as the definitive hosts to release eggs of C. sinensis into the East Lake of Wuhan.
Aside from cats, dogs, and pigs [12], rats (Rattus norvegicus) were also viable definitive hosts of C. sinensis under experimental conditions [7,13,29].Indeed, during the epidemiological investigation of C. sinensis in Hubei Province, C. sinensis eggs were detected in the feces of rats [27].The Asian house rat (Rattus tanezumi) is widely distributed in China and it can be found along river banks [30].It is omnivorous, with meat and fish also included in its diet [31].We frequently observed the feces of rats along the banks of East Lake.On this basis, we hypothesize that rats may also act as a reservoir for C. sinensis in urban lakes in non-endemic areas.

Conclusions
Clonorchis sinensis metacercariae were detected in five fish species from East Lake of Wuhan.Given the consumption of undercooked fried fish and feeding cats with small fish caught by anglers, there is a potential risk of infection for humans.The high dispersal ability of P. parva and Rhodeus spp.bred as ornamental fish may also contribute to the spread of C. sinensis metacercariae around the world.In addition, the finding of C. sinensis metacercariae suggested that this liver fluke can complete the life cycle in the East Lake of Wuhan.We hypothesize that stray cats and rats may act as the final or reservoir hosts of C. sinensis in urban lakes in non-endemic areas.To confirm this hypothesis, domestic cats and dogs, stray cats, and wild rats in this area should be investigated for infection with C. sinensis in the future.

Table 1 .
Newly sequenced ITS2/ITS sequences of Clonorchis sinensis and four forms of other trematode metacercariae and their top similarity hits in GenBank.

Table 1 .
Newly sequenced ITS2/ITS sequences of Clonorchis sinensis and four forms of other trematode metacercariae and their top similarity hits in GenBank.

Table 2 .
Pairwise identity values (%) for ITS rDNA sequences of the four forms of other trematode metacercariae.