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.

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.

Investigation of cryptic diversity and occurrence of echinostome metacercariae infection in Anentome helena (von dem Busch, 1847)

Objective: To investigate the parasitic infection of Anentome helena (A. helena) and determine the validity of species boundaries for A. helena by combining molecular phylogeny and morphological approaches. Methods: A total of 325 individuals of A. helena were collected throughout northern Thailand. Shells were measured and compared by t-test. Radulae were investigated by using light and scanning electron microscope. Two partial mitochondrial DNA sequences of COI and 16S rRNA from 36 specimens of A. helena and related species were used to test the validity of the morphospecies. Phylogenetic trees were constructed using neighbour joining, maximum likelihood and Bayesian inference. Infection of A. helena with trematode larva was examined and observed. Results: Morphological examination of A. helena revealed 2 distinct morphospecies. Genetic divergences supported the separation of the two morphotypes into two distinct groups. Both individual and combined analyses of the two nucleotide fragments revealed two phylogroups that corresponded with shell and radula characteristics. In addition, A. helena was found infected with 37-collar spined echinostome metacercariae. The prevalence and intensity of metacercariae was highest in the San Kamphaeng district, Chiang Mai province, with 7.5% and 1.670±0.577, respectively. Conclusions: These findings suggest that comprehensive taxonomic revision of this unrecognised species complex is needed. This study represents the preliminary step to reveal new data on the recent distribution of trematode infection in A. helena. This information may be useful for developing conservation management of the snail and the practice of targeted regimes to reduce anthelmintic resistance in the future.

Investigation of cryptic diversity and occurrence of echinostome metacercariae infection in Anentome helena (von dem Busch, 1847)

Objective:To investigate the parasitic infection of Anentome helena (A. helena) and determine the validity of species boundaries for A. helena by combining molecular phylogeny and morphological approaches.Methods:A total of 325 individuals of A. helena were collected throughout northern Thailand. Shells were measured and compared by t-test. Radulae were investigated by using light and scanning electron microscope. Two partial mitochondrial DNA sequences of COI and 16S rRNA from 36 specimens of A. helena and related species were used to test the validity of the morphospecies. Phylogenetic trees were constructed using neighbour joining, maximum likelihood and Bayesian inference. Infection of A. helena with trematode larva was examined and observed.Results:Morphological examination of A. helena revealed 2 distinct morphospecies. Genetic divergences supported the separation of the two morphotypes into two distinct groups. Both individual and combined analyses of the two nucleotide fragments revealed two phylogroups that corresponded with shell and radula characteristics. In addition, A. helena was found infected with 37-collar spined echinostome metacercariae. The prevalence and intensity of metacercariae was highest in the San Kamphaeng district, Chiang Mai province, with 7.5% and 1.670±0.577, respectively.Conclusions:These findings suggest that comprehensive taxonomic revision of this unrecognised species complex is needed. This study represents the preliminary step to reveal new data on the recent distribution of trematode infection in A. helena. This information may be useful for developing conservation management of the snail and the practice of targeted regimes to reduce anthelmintic resistance 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.

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.

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.