Identification of Larval Eustrongylides (Nematoda: Dioctophymatoidea) sp. from Channa punctata Bloch, 1793 by Morphological and Molecular Techniques

Objective: This study aimed to identify the larval form of Eustrongylides sp. isolated from the visceral organs of Channa punctata (Bloch, 1793) using morphological and molecular methods. Methods: Fishes were collected from fish farms in Nadia and North 24 Paraganas for the collection of nematodes. The visceral organs were dissected and kept in 0.67% normal saline. Nematodes collected from the abdominal regions and visceral organs for light microscopy study were fixed in 70% ethanol. Morphological features were studied by placing the nematodes in lactophenol. The specimens were later preserved in 70% ethanol containing 5% glycerine. Specimens processed for scanning electron microscopy were fixed in 2.5% glutaraldehyde and postfixed in 1% osmium tetraoxide. Proper identification was done by using standard methology. Molecular studies were performed for the 18S rRNA, ITS1, 5.8S rRNA, ITS2 and 28S rRNA gene fragments using polymerase chain reaction amplification, sequencing and phylogenetic analysis. Results: The morphological characteristics of nematodes were described with the help of light and scanning electron microscopy. Additional features not described earlier like dimensions and shape of the cephalic papillae, absence of somatic papillae, presence of caudal papillae, were identified for the first time. Moreover, molecular studies with ITS regions further confirmed the identification of the nematode. Conclusion: Thus the use of morphotaxonomy along with molecular techniques would help in proper identification of Eustrongylides sp infecting edible fish. Studies on the nematode would help toexplore the intermediate as well as paratenic hosts of the parasite. Data in this regard would contribute significantly to the fish database in regard to parasites infesting edible fishes.

Morphological and molecular characterization of Eustrongylides excisus larvae (Nematoda: Dioctophymatidae) in Sander lucioperca (L.) from Northern Turkey.

The genus Eustrongylides Jägerskiöld, 1909 includes parasitic nematodes (Dioctophymatidae) affecting various fish species and piscivorous birds of freshwater ecosystems. Currently, there is little information on the molecular characterization of E. excisus based on nuclear ribosomal internal transcribed spacer (ITS) rDNA regions. However, before the present study, there had been no reports of characterizing the E. excisus using nuclear small subunit ribosomal RNA (SSU rRNA) and mitochondrial cytochrome c oxidase subunit I (COI) genes sequences. In the present study, Eustrongylides spp. larvae were collected from pike-perch Sander lucioperca (L.) in Northern Turkey, and characterized by sequencing of ITS regions, SSU rRNA, and COI markers. Larvae herein morphologically identified as the fourth stage of Eustrongylides spp. were genetically identified as E. excisus based on the ITS sequence analysis. This study is the first record of SSU rRNA and COI sequences for E. excisus in GenBank. This is also a molecular characterization of E. excisus for the first time in Turkey. The ITS, SSU rRNA, and COI sequences of E. excisus can be used to establish the phylogenetic relationships of Eustrongylides species from Turkey and worldwide for further studies.

Eustrongylides sp. (Nematoda, Dioctophymatoidea) parasitizing Hoplias malabaricus (Actinopterygii: Erythrinidae) collected from the state of Rio de Janeiro, Brazil / Eustrongylides sp. (Nematoda, Dioctophymatoidea) parasitando Hoplias malabaricus (Actinopterygii: Erythrinidae) coletados do Estado do Rio de Janeiro, Brasil

Abstract Hoplias malabaricus (Characiformes, Erythrinidae), trahira, is a neotropical freshwater fish of economic and public health significance. A total of 45 specimens of H. malabaricus commercialized in the municipality of Magé, state of Rio de Janeiro, Brazil, were acquired between April 2016 and April 2018 to investigate the presence of nematode larvae. Twenty of the fish were found parasitized by 347 fourth-stage nematode larvae identified taxonomically as Eustrongylides sp. using morphological, morphometric and molecular data. The parasitic indices were: prevalence 44.44%, mean intensity 17.35, mean abundance 7.71, and range of infection 2-40. Infection sites were musculature, mesentery, abdominal cavity, and serosa of intestine, stomach and liver. This is the first report of Eustrongylides sp. larvae parasitizing H. malabaricus in the state of Rio de Janeiro.

Resumo Hoplias malabaricus (Characiformes, Erythrinidae), traíra, é um peixe neotropical de água doce que tem significante impacto na economia e saúde pública. De abril de 2016 a abril de 2018, foram adquiridos 45 espécimes de H. malabaricus comercializados no município de Magé, Estado do Rio de Janeiro, Brasil. Os peixes foram necropsiados e filetados para investigação da presença de larvas de nematoides. Vinte dos peixes coletados estavam parasitados por 347 larvas de nematoides, identificadas taxonomicamente como larvas de quarto estágio de Eustrongylides sp. usando-se dados morfológicos, morfométricos e moleculares, apresentando os seguintes valores: prevalência de 44,44%, intensidade média de 17,35, abundância média de 7,71, e amplitude de variação da infecção de 2-40. Os sítios de infecção foram musculatura, mesentério, cavidade abdominal e serosas do intestino, estômago e fígado. Este é o primeiro registro de larvas de Eustrongylides sp. parasitando H. malabaricus no Estado do Rio de Janeiro.

Morphological and Molecular Characterization of Larval and Adult Stages of Eustrongylides excisus (Nematoda: Dioctophymatoidea) with Histopathological Observations.

The genus Eustrongylides includes nematodes known as the etiological agent of the "big red worm disease." The aim of this work was to identify Eustrongylides spp. larvae from fish and adults from great cormorant (Phalacrocorax carbo) sampled at Lake Trasimeno, Italy, by morphological and molecular analysis. Histopathological description of the lesions in birds was also provided. We described adults of Eustrongylides excisus for the first time in Italy, and we also linked larval stages 3 and 4 to adults. The use of molecular tools combined with the traditional taxonomy will help the identification of the species, including species inquirendae. Moreover, molecular analysis can also help to investigate the role of intermediate and paratenic hosts, to deepen the knowledge about geographical distribution of the different Eustrongylides spp. and to define the zoonotic potential of E. excisus, which has not yet been identified as causal agent of human cases.

Eustrongylides sp. (Nematoda, Dioctophymatoidea) parasitizing Hoplias malabaricus (Actinopterygii: Erythrinidae) collected from the state of Rio de Janeiro, Brazil.

Hoplias malabaricus (Characiformes, Erythrinidae), trahira, is a neotropical freshwater fish of economic and public health significance. A total of 45 specimens of H. malabaricus commercialized in the municipality of Magé, state of Rio de Janeiro, Brazil, were acquired between April 2016 and April 2018 to investigate the presence of nematode larvae. Twenty of the fish were found parasitized by 347 fourth-stage nematode larvae identified taxonomically as Eustrongylides sp. using morphological, morphometric and molecular data. The parasitic indices were: prevalence 44.44%, mean intensity 17.35, mean abundance 7.71, and range of infection 2-40. Infection sites were musculature, mesentery, abdominal cavity, and serosa of intestine, stomach and liver. This is the first report of Eustrongylides sp. larvae parasitizing H. malabaricus in the state of Rio de Janeiro.

The role of freshwater fish in the life cycle of Dioctophyme renale in Southern Brazil.

Brazil stands out by the diverse records of Dioctopphyme renale in different hosts; however, there is little information about the life cycle of the nematode in the region. This study aims to report on third-stage larvae infections in fish in southern Brazil. In this context, 324 fish of different species belonging to Characiformes, Cyprinodontiformes and Siluriformes were collected in an urban area of Rio Grande do Sul State, where domestic and wild hosts were reported with the nematode. Of the total fish examined, 25(7.7%) were found to be parasitized by third-stage larvae of D. renale which were found encysted in the stomach serous membrane and in the celoma cavity of Hoplosternum littorale (Siluriformes) with a prevalence of 53.2% (25/47) and mean intensity of infection of 4.4 larvae/host (1 to 13 larvae). The occurrence of larvae in H. littorale indicates the presence of parasitosis in the region; however, the contribution of this fish species as a source of infection for dogs in urban areas must be considered with caution given the difficulties these dogs may face in the capture and predation of the fish to the point of effectively maintaining the urban cycle of D. renale. In addition, the low level of larvae registered in the total sample of fish examined indicates that these hosts are unlikely to play an important role in the transmission of D. renale to domestic animals in the region of the study.

Occurrence of Dioctophyme renale larvae (Goeze, 1782) (Nematoda: Enoplida) in a new host from southern Brazil / Novo hospedeiro de larvas de Dioctophyme renale (Goeze, 1782) (Nematoda: Enoplida) no extremo sul do Brasil

Abstract Dioctophymatosis is caused by the giant kidney worm Dioctophyme renale which occurs in dogs, cats, and wild mammals. In Brazil, the disease has been diagnosed in dogs from several states around the country. In the present study, the occurrence of D. renale larvae in snakes from southern of Brazil is reported. Three specimens of Philodryas patagoniensis (Serpentes: Dipsadidae) (common names in Brazil: "parelheira", "papa-pinto") roadkill in the county of Capão do Leão, State of Rio Grande do Sul, southern Brazil, were necropsied. Two third-stage larvae of D. renale were found in the coelomic cavity of P. patagoniensis. This study reveals a new host for D. renale larvae in the southern region of the State of Rio Grande do Sul, Brazil. This particular geographic area of the country has stood out as several cases of D. renale infection have been reported in a number of vertebrates from this region including domestic dogs and cats and wild animals such as carnivores, fish, and freshwater turtles.

Resumo Dioctophyme renale, verme gigante do rim, parasita cães, gatos e mamíferos silvestres, sendo que no Brasil, a dioctofimatose canina vem sendo diagnosticada em diversos estados brasileiros. O estudo tem por objetivo registrar larvas de D. renale parasitando serpente no extremo sul do Brasil. Nesse contexto, foram examinados três espécimes de Philodryas patagoniensis (Serpentes: Dipsadidae) encontradas mortas após atropelamento em uma estrada do município de Capão do Leão, Rio Grande do Sul (RS). Duas larvas de terceiro estágio de D. renale foram encontradas na cavidade celomática de P. patagoniensis, a qual representa um novo hospedeiro para larvas de D. renale na região sul do RS, a qual vem se destacando devido aos diversos registros do parasito em cães e gatos domésticos, bem como animais silvestres (carnívoros, peixes, quelônios).

Occurrence of Dioctophyme renale larvae (Goeze, 1782) (Nematoda: Enoplida) in a new host from southern Brazil.

Dioctophymatosis is caused by the giant kidney worm Dioctophyme renale which occurs in dogs, cats, and wild mammals. In Brazil, the disease has been diagnosed in dogs from several states around the country. In the present study, the occurrence of D. renale larvae in snakes from southern of Brazil is reported. Three specimens of Philodryas patagoniensis (Serpentes: Dipsadidae) (common names in Brazil: "parelheira", "papa-pinto") roadkill in the county of Capão do Leão, State of Rio Grande do Sul, southern Brazil, were necropsied. Two third-stage larvae of D. renale were found in the coelomic cavity of P. patagoniensis. This study reveals a new host for D. renale larvae in the southern region of the State of Rio Grande do Sul, Brazil. This particular geographic area of the country has stood out as several cases of D. renale infection have been reported in a number of vertebrates from this region including domestic dogs and cats and wild animals such as carnivores, fish, and freshwater turtles.

Pathology and morphometry of Hystrichis acanthocephalicus (Nematoda) from Phimosus infuscatus (Pelecaniformes) in southern Brazil / Patologia e morfometria de Hystrichis acanthocephalicus (Nematoda) em Phimosus infuscatus (Pelecaniformes) no Sul do Brasil

Abstract Species of Hystrichis are parasite nematodes of the digestive tract of aquatic birds in South America, Europe and Asia. In Brazil, Hystrichis acanthocephalicus has been reported in Phimosus infuscatus. There are few data on the morphometry of this species and there are no reports on pathological conditions that it causes. Therefore, the purpose of this study was to report morphometric data from H. acanthocephalicus and describe the pathological effects of this parasite on the Phimosus infuscatus proventriculus. Thirty gastrointestinal tracts of P. infuscatus were examined to search for nematodes and H. acanthocephalicus occurred in 83% of hosts. Were measured the total length and body width of males and females, and of their respective cuticular spines, esophagus, spicules and eggs, and the internal and external diameter of copulatory bursa. Histopathological examination revealed parasitic structures in the proventriculus from the lumen (anterior end) to the outer layers of the organ (intermediate and posterior parts), in which we observed inflammatory reaction with infiltration of heterophils, hemorrhage and hemosiderin. The results of this study of histopathology, morphometry and parasitological indices are the first ones reported to H. acanthocephalicus and should contribute to the identification and recognition in cases of outbreaks in the Neotropical region.

Resumo Espécie de Hystrichis são nematoides parasitos do trato digestório de aves aquáticas, na América do Sul, Europa e Ásia. No Brasil, foi registrado Hystrichis acanthocephalicus, em Phimosus infuscatus. Existem poucos dados sobre a morfometria e não há registros relativos à patologia desta espécie. Portanto, o objetivo deste estudo foi relatar dados morfométricos de H. acanthocephalicus e descrever os efeitos patológicos deste parasito no proventrículo de Phimosus infuscatus. Foram examinados trinta tratos gastrintestinais de P. infuscatus para a pesquisa de nematoides e H. acanthocephalicus ocorreu em 83% dos hospedeiros. Mediu-se o comprimento total e largura do corpo de machos e fêmeas, dos espinhos cuticulares, do esôfago, dos espículos, dos ovos, respectivamente, e a largura interna e externa da bolsa copuladora. Nos exames histopatológicos foram encontradas estruturas parasitárias no proventrículo, desde o lúmen (porção anterior) até as camadas mais externas do órgão (porções intermediária e posterior), nas quais se observou reação inflamatória com infiltrado de heterófilos, presença de hemorragia e deposição de hemossiderina. Os resultados desse estudo sobre histopatologia, morfometria e índices parasitológicos são os primeiros relatados para H. acanthocephalicus e deverão contribuir para a identificação e reconhecimento em casos de surtos na região Neotropical.

Pathology and morphometry of Hystrichis acanthocephalicus (Nematoda) from Phimosus infuscatus (Pelecaniformes) in southern Brazil.

Species of Hystrichis are parasite nematodes of the digestive tract of aquatic birds in South America, Europe and Asia. In Brazil, Hystrichis acanthocephalicus has been reported in Phimosus infuscatus. There are few data on the morphometry of this species and there are no reports on pathological conditions that it causes. Therefore, the purpose of this study was to report morphometric data from H. acanthocephalicus and describe the pathological effects of this parasite on the Phimosus infuscatus proventriculus. Thirty gastrointestinal tracts of P. infuscatus were examined to search for nematodes and H. acanthocephalicus occurred in 83% of hosts. Were measured the total length and body width of males and females, and of their respective cuticular spines, esophagus, spicules and eggs, and the internal and external diameter of copulatory bursa. Histopathological examination revealed parasitic structures in the proventriculus from the lumen (anterior end) to the outer layers of the organ (intermediate and posterior parts), in which we observed inflammatory reaction with infiltration of heterophils, hemorrhage and hemosiderin. The results of this study of histopathology, morphometry and parasitological indices are the first ones reported to H. acanthocephalicus and should contribute to the identification and recognition in cases of outbreaks in the Neotropical region.

Morphological characterization of Eustrongylides sp. larvae (Nematoda, Dioctophymatoidea) parasite of Rhinella marina (Amphibia: Bufonidae) from Eastern Amazonia.

Absctract Eustrongylides spp. nematodes have birds as final hosts and uses other vertebrates as intermediate/paratenic host (fish, amphibians and reptiles) and have zoonotic potential. In amphibians, the larvae may be located in the subcutaneous tissues, liver and mesentery, between the muscle fibres, especially in the lower limbs. Rhinella marina, which is widely observed in Brazil, has exhibited complex diversity in its helminth fauna, reflecting the unique habitat of the Amazon biome. For the first time, this study describes the morphological aspects of third-stage larvae of Eustrongylides sp. in Rhinella marina from Santa Cruz do Ararí, Marajó Archipelago, Eastern Amazonia, using light and scanning electron microscopy.

Morphological characterization of Eustrongylides sp. larvae (Nematoda, Dioctophymatoidea) parasite of Rhinella marina (Amphibia: Bufonidae) from Eastern Amazonia / Caracterização morfológica de larva de Eustrongylides sp. (Nematoda, Dioctophymatoidea) parasita de Rhinella marina (Amphibia: Bufonidae) da Amazônia Oriental Brasileira

Absctract Eustrongylides spp. nematodes have birds as final hosts and uses other vertebrates as intermediate/paratenic host (fish, amphibians and reptiles) and have zoonotic potential. In amphibians, the larvae may be located in the subcutaneous tissues, liver and mesentery, between the muscle fibres, especially in the lower limbs. Rhinella marina, which is widely observed in Brazil, has exhibited complex diversity in its helminth fauna, reflecting the unique habitat of the Amazon biome. For the first time, this study describes the morphological aspects of third-stage larvae of Eustrongylides sp. in Rhinella marina from Santa Cruz do Ararí, Marajó Archipelago, Eastern Amazonia, using light and scanning electron microscopy.

Resumo Os nematoides do gênero Eustrongylides tem as aves como seus hospedeiros definitivos e utilizam outros vertebrados como hospedeiros intermediários/paratênicos (peixes, anfíbios, répteis), além de apresentar potencial zoonótico. Em anfíbios, as larvas podem estar localizadas em tecidos subcutâneos, fígado, mesentério e entre fibras musculares de membros inferiores. Rhinella marina, anfíbio o qual é distribuído amplamente no Brasil, apresenta uma complexa diversidade em sua helmintofauna, refletindo o seu hábitat no bioma amazônico. O presente estudo descreve, pela primeira vez, os aspectos morfológicos das larvas de terceiro estágio de Eustrongylides sp. em R. marina de Santa Cruz do Arari, Arquipélago do Marajó, Amazônia Oriental Brasileira, utilizando-se microscopia de luz e microscopia eletrônica de varredura.

Dioctophyme renale (Nematoda: Dioctophymatoidea) in the abdominal cavity of Rattus norvegicus in Japan.

We collected 24 brown rats, Rattus norvegicus, in Kanagawa Prefecture in Japan and found one rat harboring a dioctophymatid nematode. A single male and a female worm were recovered from the abdominal cavity and were identified as Dioctophyme renale based on morphologic features and a BLAST DNA sequence analysis. We describe the morphological features of the adult worms and eggs from this extremely rare case of D. renale infection in a brown rat.

Eustrongylides sp. (Nematoda: Dioctophymatoidea) from the stomach of a Nile crocodile, Crocodylus niloticus Laurenti, 1768, in Botswana.

During a study conducted between 2003 and 2005 on the diet of Nile crocodiles in Botswana, two young adult nematodes, one male and one female, belonging to the genus Eustrongylides Jägerskiöld, 1909 were recovered from the stomach contents of one of these animals. The caudal bursa of the male is present and the ejaculatory duct could be identified, but the spicule could not be seen. The vulva of the female has opened and the anus is situated on a terminal protruberance. Measurements and drawings of these specimens are provided, together with some data on the occurrence and life-cycles of members of the genus Eustrongylides in crocodilians world-wide and in African hosts in particular. Piscivorous birds are the usual final hosts of these nematodes. It is probable that the specimens described herein had developed in a paratenic fish host, and that the latter had been eaten by the crocodile.