Molecular Phylogenetics of Centrocestus formosanus (Digenea: Heterophyidae) Originated from Freshwater Fish from Chiang Mai Province, Thailand

Abstract

This study aimed to investigate the morphology and reconstruct the phylogenetic relationships of Centrocestus formosanus originating from 5 species of freshwater fish, i.e., Esomus metallicus, Puntius brevis, Anabas testudineus, Parambassis siamensis, and Carassius auratus, in Chiang Mai province, Thailand. Sequence-related amplified polymorphism (SRAP) and phylogeny based on internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunit 1 (CO1) were performed. The results showed similar morphologies of adult C. formosanus from day 5 after infection in chicks. C. formosanus originated from 4 species of freshwater fish had the same number of circumoral spines on the oral sucker, except for those from C. auratus which revealed 34 circumoral spines. The phylogenetic tree obtained from SRAP profile and the combination of ITS2 and CO1 sequence showed similar results that were correlated with the number of circumoral spines in adult worms. Genetic variability of C. formosanus also occurred in different species of freshwater fish hosts. However, more details of adult worm morphologies and more sensitive genetic markers are needed to confirm the species validity of C. formosanus with 34 circumoral spines originating from C. auratus in the future.

Diagnosis of Centrocestus formosanus Infection in Zebrafish (Danio rerio) in Italy: A Window to a New Globalization-Derived Invasive Microorganism

“…The occurrence of C. formosanus infection in zebrafish in Italy underlines the necessity to focus the attention on this invasive parasite, since this is the second case reported in the Italian peninsula [32]. Although its origin and distribution were initially restricted to Asia [4,[9][10][11][12][13]24,31], C. formosanus has been subsequently reported in other countries, including Europe [16][17][18][19][20][21][22]25,26,29,48]. Actually, to the authors' knowledge, this is the fourth case of C. formosanus infection in freshwater fish imported in Europe [19,32,40], confirming the introduction of this parasite in the European area, as well as the possible underestimation of this infection.…”

“…In particular, given the huge numbers of the ornamental fish industry in Europe (approximately 300 million fish, more than 2500 species, of which 60% are of freshwater origin), the importation of ornamental freshwater fish, from countries where C. formosanus is endemic, plays a crucial role in spreading this parasite [35,36,51]. Indeed, Asian countries are the major traders and most of the suppliers of freshwater fish to Europe are involved with C. formosanus infections [4,5,[10][11][12]35,36,51].…”

“…Concerning the diagnosis of C. formosanus, the number of circumoral spines has been acknowledged as a reliable criterion for species identification; however, morphological characteristics could be similar in larval stages, the accurate count of circumoral spines could be challenging, and their number might vary in different fish species [10,11,17]. Therefore, parasite identification should no longer rely only on standard morphological methods, but molecular analyses should be accepted as one of the most effective and accurate approaches for species confirmation [10,11,32]. For these reasons, we propose a fast, cheap and specific PCR-based method to address C. formosanus infection in zebrafish, starting from small pieces of the hosts' gill tissue and avoiding elaborate collection of metacercariae.…”

“…Fish bodies were dissected [38], gill arches were removed with the aid of a stereomicroscope (Leica, Milano, Italy) and prepared as wet mounts to be examined for the presence of encysted metacercariae [5,10,16,21,25,26,28,30,47]. Encysted metacercariae were examined under a light microscope (Leica, Milano, Italy) ( Figure 1B) to evaluate their morphology and to be identified according to published characteristics [5,[10][11][12][13][14][15][16][17]26]. Live encysted metacercariae were also recorded at 40× using a light microscope (Leica, Milano, Italy) (Supplementary Materials Video S1).…”

“…Within fish-borne trematodes, Centrocestus formosanus is a small heterophyid fluke, described for the first time in Taiwan [9] and widely distributed in Asia [4,5,[10][11][12][13][14][15]. Since the 1950s, several authors have reported this species in new continents, although its occurrence could still be underestimated [16][17][18][19][20][21][22].…”