Morphological study and molecular epidemiology of Anisakis larvae in mackerel fish

Objective: To examine and study the morphology, epidemiology, and molecular phylogeny of Anisakis larvae in blue mackerel [Scomber australasicus (Cuvier, 1832)] and Indian mackerel [Rastrelliger kanagurta (Cuvier, 1816)] using light microscope, scanning electron microscope, molecular phylogeny, and species delimitation methods for confirmation and investigation of Anisakis species and their evolutionary relationship. Methods: A total of 90 fish (45 per species) were purchased from a department store in Chiang Mai, Thailand. Anisakis samples were investigated for morphological characteristics using light and scanning electron microscopes. Molecular phylogeny and species delimitation methods based on the cox2 gene were performed. Results: The prevalence, mean intensity (Mean±SEM), and mean abundance of Anisakis larvae (Mean±SEM) in blue mackerel were 77.78%, 6.74±1.320, and 5.24±1.107, respectively, and in Indian mackerel, these values were 13.33%, 2.50±0.764, and 0.33±0.159, respectively. Scanning electron microscopy showed the detail of morphological characteristics and provided the different shapes of mucron and excretory pores in Anisakis larvae congruent with the phylogenetic tree. The species tree was congruent with the phylogenetic tree. Conclusions: The prevalence, mean intensity, and mean abundance of Anisakis larvae were higher in blue mackerel. To the best of our knowledge, this is the first time that Anisakis pegreffii was found in blue mackerel in Thailand. The phylogenetic tree also supported the morphological data of Anisakis larvae. However, species delimitation based on cox2 revealed 1-3 possible cryptic species in this genus. Anisakis spp. contamination of fish products is unpleasant and a health concern considering human infection with larvae (anisakiasis) arises.

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.

Morphological and Molecular Characteristics of Anisakis typica Larvae in Two Species of Threadfin Bream, Nemipterus hexodon and N. japonicus, from the Gulf of Thailand

The third stage larvae (L3) of Anisakis typica were detected in 2 species of threadfin bream, Nemipterus hexodon and N. japonicus, from the Gulf of Thailand, and were morphologically and molecularly characterized. Total 100 threadfin breams, 50 Nemipterus hexodon and 50 N. japonicus, were examined with naked eyes after the opening of abdominal cavity with scissors. Almost all infected larvae remained alive and active even the fish were transported for 1-2 days. Anisakid larvae were exclusively distributed in the body cavity and rarely in the liver. The prevalence of A. typica L3 were 68.0% and 60.0% in N. hexodon and N. japonicus and their infection intensities were 3.5 and 4.2 per fish infected each. Morphological and morphometric analysis were performed by viewing specimens under both a light microscope and a scanning electron microscope. Interestingly, the protruded mucron of Anisakis typica under SEM showed a distinct cylindrical shape that differed from the cone shape of A. simplex. The protruded mucron could be used to identify A. typica L3 larvae in the future. A comparison of the ITS1-5.8S-ITS2 rDNA nucleotide sequences of these species revealed high blast scores with A. typica. Conclusively, it was confirmed that A. typica L3 are prevalent in threadfin breams from the Gulf of Thailand, and their morphological and molecular characters are something different from those of other anisakid larvae, including A. simplex and A. pegreffii.

Morphological and Molecular Identification of Stellantchasmus dermogenysi n. sp. (Digenea: Heterophyidae) in Thailand

We tried a series of morphological and molecular approaches to identify a new species of Stellantchasmus (Digenea: Heterophyidae) originating from the wrestling half-beaked fish, Dermogenys pusillus of Thailand. Adult worm samples of the new species were recovered from hamsters experimentally infected with the metacercariae from D. pusillus in Thailand. Two isolates (Thai and Korean) of Stellantchasmus falcatus were used as comparative control groups. Worm samples of 3 Stellantchasmus groups were morphologically observed and molecularly analyzed with the mitochondrial cytochrome c oxidase 1 gene. The morphological characteristics of S. dermogenysi n. sp. are similar to S. falcatus originating from brackish water fish, but minor difference was noted including the absence of the prepharynx, position of the ovary near the ceca end, smaller body size, and shorter esophageal length. A phylogenetic tree derived from neighbor-joining and maximum-likelihood methods suggests that S. dermogenysi n. sp. is separated from S. falcatus supported by high bootstrap values. The relative divergences persist between these host-specific trematodes, which we suggest should be recognized as 2 distinct species. Comparisons of S. dermogenysi n. sp. with S. falcatus isolated from mullets in Thailand and Korea indicate a genetic divergence of mitochondrial DNA of 19.4% and 21.7%, respectively. By the present study, a new species, Stellantchasmus dermogenysi n. sp. (Digenea: Heterophyidae), is proposed in Thailand based on molecular evidences, in addition to minor morphological differences between S. falcatus and the new species.

Phylogenetic analysis reveals cryptic species diversity within minute intestinal fluke, Stellantchasmus falcatus Onji and Nishio, 1916 (Trematoda, Heterophyidae)

OBJECTIVE@#To examine the taxonomic boundaries in the Stellantchasmus populations in different hosts and reveal the cryptic speciation of the Stellantchasmus in Thailand based on both morphological and molecular approaches.@*METHODS@#Stellantchasmus falcatus (S. falcatus) s.l. was collected from different hosts throughout Thailand. The worms were examined and measured. The nuclear ITS2 gene and the mitochondrial COI gene were used to investigate the S. falcatus s.l. from Dermogenus pusillus and Liza subviridis.@*RESULTS@#Stellantchasmus was one of food-borne trematode that widely distribute throughout Thailand. The comparison of S. falcatus s.l. from Dermogenus pusillus and Liza subviridis indicated a genetic divergence of nuDNA with 3.6% and mtDNA with 19.3%, respectively. Morphological characteristics were fairly different in the ratio of body size, length of prepharynx, and ratio of organ size.@*CONCLUSIONS@#Our results provide initial evidence that S. falcatus s.l. from different hosts tend to be a different species based on both molecular and morphological characters. Cryptic species complexes are generally found among parasites that tend to have large populations and/or rapid evolution. The degree of genetic diversity existing would suggest the practice of targeted regimes to design and minimize these lections of anthelmintic resistance in the future.

Prevalence of Centrocestus formosanus Metacercariae in Ornamental Fish from Chiang Mai, Thailand, with Molecular Approach Using ITS2

The prevalence of Centrocestus formosanus metacercariae was investigated in ornamental fish purchased from a pet shop in Chiang Mai, Thailand, including Carassius auratus (goldfish), Cyprinus carpio (Koi), Poecilia latipinna (Sailfin Molly), Danio rerio (Zebrafish), and Puntigrus tetrazona (Tiger barb). The parasite species was identified by the morphology of worms as well as by a molecular approach using ITS2. The results showed that 50 (33.3%) of 150 fish examined were infected with the metacercariae. The highest prevalence was found in C. auratus (83.3%), and the highest intensity was noted in C. carpio (70.8 metacercariae/fish). The most important morphological character was the presence of 32–34 circumoral spines on the oral sucker. The phylogenetic studies using the rRNA ITS2 region revealed that all the specimens of C. formosanus in this study were grouped together with C. formosanus in GenBank database. This is the first report on ornamental fish, C. carpio, P. latipinna, D. rerio, and P. tetrazona, taking the role of second intermediate hosts of C. formosanus in Thailand. Prevention and control of metacercarial infection in ornamental fish is urgently needed.

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

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.

Phylogenetic analysis reveals cryptic species diversity within minute intestinal fluke, Stellantchasmus falcatus Onji and Nishio, 1916 (Trematoda, Heterophyidae)

Objective To examine the taxonomic boundaries in the Stellantchasmus populations in different hosts and reveal the cryptic speciation of the Stellantchasmus in Thailand based on both morphological and molecular approaches. Methods Stellantchasmus falcatus (S. falcatus) s.l. was collected from different hosts throughout Thailand. The worms were examined and measured. The nuclear ITS2 gene and the mitochondrial COI gene were used to investigate the S. falcatus s.l. from Dermogenus pusillus and Liza subviridis. Results Stellantchasmus was one of food-borne trematode that widely distribute throughout Thailand. The comparison of S. falcatus s.l. from Dermogenus pusillus and Liza subviridis indicated a genetic divergence of nuDNA with 3.6% and mtDNA with 19.3%, respectively. Morphological characteristics were fairly different in the ratio of body size, length of prepharynx, and ratio of organ size. Conclusions Our results provide initial evidence that S. falcatus s.l. from different hosts tend to be a different species based on both molecular and morphological characters. Cryptic species complexes are generally found among parasites that tend to have large populations and/or rapid evolution. The degree of genetic diversity existing would suggest the practice of targeted regimes to design and minimize these lections of anthelmintic resistance in the future.

Developmental and Phylogenetic Characteristics of Stellantchasmus falcatus (Trematoda: Heterophyidae) from Thailand

This study aimed to investigate the infection status, worm development, and phylogenetic characteristics of the intestinal trematode, Stellantchasmus falcatus. The metacercariae of S. falcatus were detected only in the half-beak (Dermogenus pusillus) out of the 4 fish species examined. Their prevalence was 90.0%, and the intensity of infection was 919 metacercariae on average. Worms were recovered from 33 (97.1%) of 34 chicks that were experimentally infected with 200 S. falcatus metacercariae each, and the average recovery rate was 43.0%. The body size and inner organs of S. falcatus quickly increased in the experimental chicks over days 1-2 post-infection (PI). In addition, ITS2 sequence data of this parasite were analyzed to examine the phylogenetic relationships with other trematodes using the UPGMA method. The results indicated that the ITS2 sequence data recorded from trematodes in the family Heterophyidae appeared to be monophyletic. This study concluded that D. pusillus serves as a compatible second intermediate host of S. falcatus in Thailand and that S. falcatus can develop rapidly in the experimental chicks. Data collected from this study can help to close the gap in knowledge regarding the epidemiology, biology, and phylogenetic characteristics of S. falcatus in Thailand.

Molecular Markers for Identification of Stellantchasmus falcatus and a Phylogenic Study using the HAT-RAPD Method

Stellantchasmus falcatus is a minute intestinal fluke in the family Heterophyidae. Metacercariae, the infective stage, were reported in a marine fish, mullet Liza subviridis, and a fresh water fish, Dermogenus pusillus, in Thailand. Adults were found in chicks, rats, cats, and humans. Morphological studies were done for comparing Stellantchasmus sp. worms found in 2 different fish hosts; their shapes and organ arrangements were very similar except for the prepharynx length. Therefore, the present study aimed to compare their DNA fingerprints using the HAT-RAPD method for both types of Stellantchasmus and several other related species. Ten arbitrarily selected primers (OPA-04, OPA-09, OPN-02, OPN-03, OPN-09, OPN-12, OPP-11, OPR-15, OPX-13, and OPAD-01) were used. It was found that OPA-09, OPN-03, and OPAD-01 were able to generate S. falcatus specific fragments in mullets which consisted of 200, 760, and 280 bp, respectively. In addition, the results of morphologic, DNA fingerprinting, and phylogenetic analyses strongly suggest that the fresh water and marine specimens of Stellantchamus may be different species.