A new species of Huffmanela (Nematoda: Trichosomoididae) parasitizing Colomesus psittacus (Tetraodontiformes) from Marajó Island, Pará State, Brazil

The puffer fish Colomesus psittacus , is not commercialized on Marajó Island. They are captured as bycacth and discarded dead in the environment in artisanal fisheries that occur in the estuaries of northern Brazil. In this sense, the objective was to identify the parasites present in the gills and to evaluate the histopathological alterations caused by these nematodes of the genus Huffmanela . Fifty-five fish were analyzed, and thirty-five specimens showed the parasite in the gills. Morphological characteristics suggest that it is a new species of the genus Huffmanela, and the histopathological exams showed an edematous inflammation in the secondary lamella and the presence of eggs of this nematode, which is the first record of this parasite in C. psittacus in Brazil.


Introduction
The puffer fish Colomesus psittacus (Bloch & Schneider, 1801) is often found in freshwater demersal environments but can also inhabit estuarine or marine waters. It has a distribution from Venezuela to the center north of Brazil, including the state of Pará (Brazil) (Cervigon et al., 1992;Camargo & Maia, 2008). Their food source consists of mollusks and other small benthic organisms (Szpilman, 2002).
Puffer fish, as they are popularly known, are still usually captured as bycatch and discarded dead in the environment in high densities by artisanal fisheries that occur in estuaries in northern Brazil (Barros et al., 2011;Fonseca & Souza, 2013). The species C. psittacus is also used as bait in fishing for Epinephelus itajara according to Pereira et al. (2020), and for Brachyplatystoma filamentosum and Sciades parkeri around Marajó Island. However, the species is not of interest as a protein source in northern Brazil because of its toxicity (Krumme et al., 2007).
Due to the importance of this species, knowledge of its parasitic ecology is of fundamental importance. Some previous studies with helminths in C. psittacus have been carried out and have identified the presence of Rohdella sp. (Silva et al., 2013); Rohdella amazonica (Giese et al., 2015), Gnathostoma sp. and Cucullanus marajoara (Pinheiro et al., 2017(Pinheiro et al., , 2018. All of these were intestinal parasites.
Thus, this study sought to taxonomically identify specimens of Trichosomoididae nematodes, and the lesions caused by these parasites found in the gills of C. psittacus on Marajó Island, Pará.

Material and Methods
In the year 2021, fifty-five specimens of C. psittacus were acquired in fishing pens located in the municipality of Soure (00º 43' 00" S; 48º 31' 24" W), Marajó Island, State of Pará. The fish in the region are not used for meat consumption or sale in local shops and the specimens were transported to the laboratory cooled, then the gills were separated in Petri dishes containing 0.9% NaCl saline solution and analyzed under a stereomicroscope (Leica ES2) for the search for helminths.
After verifying the presence of nematodes in the gills, cleaned in physiological solution, they were fixed in A.F.A solution (93 parts of 70% ethyl alcohol, 5 parts of formaldehyde and 2 parts of glacial acetic acid) for morphological and morphometric analyses. Thirty males and females were clarified with Lactophenol Aman (0.5%), observed under light microscopy and photographed under a microscope LEICA DM2500 with a camera system LEICA type DFC310 FX with Software Leica Application Suite V4.4. Measurements are presented in micrometers, unless indicated, as an average followed by the range (minimum and maximum values) in parentheses. The taxonomic classification of nematodes was performed according to Moravec & Campbell (1991), Moravec et al. (1998), Moravec & Fajer-Avila (2000) and Moravec (2001).
For scanning electron microscopy (SEM), thirty-three nematodes were fixed in 3% Glutaraldehyde, washed in 0.2M phosphate buffer solution, each wash for one hour, post-fixed in 1% osmium tetroxide, dehydrated in progressive alcohol for one hour each (50%, 70%, 80%, 90%, 100%), and dried at the critical point of CO 2 , metallized with gold-palladium and observed in a scanning electron microscope TESCAN model VEGA 3.

Search data
A total of 138 nematodes were recovered from the gills of 35 (n=55) specimens of C. psittacus, with a prevalence of 63.6%, mean intensity of infection 3.94, mean abundance of 2.50 and range of infection of from 1 to 45 nematodes adults per host. All specimens collected showed characteristics compatible with the genus Huffmanela but could not be attributed to a known species; therefore, a new species is described here. The morphological and morphometric characteristics of this new species are presented below and in Table 1. Trichinellida Hall, 1916 Trichosomoididae Yorke andMaplestone, 1926 Huffmanela Moravec, 1987 Huffmanela psittacus n. sp.  (Based on light microscopy and scanning electron microscopy: Figures 1 to 4) They are small, filiform nematodes, with a narrow anterior end of the body, obtusely rounded, with distinct lips and cephalic papillae ( Figure 1B). Cuticle with well-marked transverse striations. Two broad lateral bacillary bands that extend along almost the entire length of the body with numerous more pronounced cuticular projections in males. Narrow and short muscular esophagus and simple posterior stichosome composed of a row of 27−42 stichocytes with large cell nuclei; shorter stichocytes at the back of the stichosome usually subdivided into 2−4 transverse rings; in larger specimens, 1−2 light-colored stichocytes alternating with darker (more granular) stichocytes ( Figure 1A; Figure 2C, E). Nerve ring surrounding muscular esophagus in approximately one-third of the posterior area, excretory pore not observed. Small presence of post-esophageal pseudocoelomocytes. Males smaller than females.      Figures 2B, 4B). Short vagina. Eggs oval, light in color, not embryonated, polar plugs of eggs partially developed and not protruding. Eggs and thin-shelled. Eggs arranged in one row in the anterior part of the uterus and in two to three rows posteriorly. Single ovary, extending posteriorly to approximately the level of the end of the intestine. Marked oviduct filled with sperm some distance from the posterior end, terminal anus 51.10 (20-73.

Histopathology
Fresh tissue specimens examined using light microscopy showed multiple rounded to oval dark eggs (at different developmental stages) along with larvae and adults. On histological section, the gill arch, lamellae, and filaments showed numerous mature and larval eggs of the parasite dispersed in the gill mucus. The base of the lamella presents edema that promotes detachment of the epithelium from the secondary lamella. There is also a special granulocytic cell and lymphocytic infiltrate at the base of the gill filament ( Figure 5).  Site of infection: gill arch, gills and gill-mucosa.

Etymology:
The specific name psittacus (genitive) relates to the specific name of the host fish (psittacus).

Discussion
The nematodes of the genus Huffmanela described so far are parasites of fish, with the main sites of infection being the skin, oral cavity, musculature, swim bladder, gills and serosa of the intestines (Bullard et al., 2012;Carballo & Navone 2007;Esteves et al., 2009Esteves et al., , 2016Huffman & Moravec, 1988;Moravec & Garibaldi, 2000;. In the gills, the record of infections by nematodes in fish is by the larval stage of Contracaecum sp. in Oligoplites palometa, O. saliens, and O. saurus, and the adult form of Raphidascaris sp. in Pinguipes brasilianus from Brazil by Luque et al. (2011). There are still few studies about the parasitic helminths of C. psittacus, mainly referring to the gills.
The parasites present in the gill mucosa of C. psittacus, in this study, have similar characteristics to those of the Trichosomoididae family. This family includes a genus Huffmanela with the following species described in fish, based on the eggs of these parasites: H. banningi Moravec, 1987 Moravec & Campbell, 1991 in the swim bladder of Exocoetidae in Italy; H. japonica Moravec et al. 1998 in the musculature of Mullidae in Japan; H. shikokuensis Moravec et al. 1998  According to Moravec (1987Moravec ( , 2001 and Gibbons (2010), they are small and filamentous nematodes, with a stichosome composed of a single row of stichocytes with presence of post-esophageal glands. Relatively long cloaca in the male, spicule and spicule sheath absent. Posterior extremity of male with dorsoventral depression, without membranous bursa, provided with a pair of adcloacal papillae. Terminal female anus, oviparous, with vulva near the end of the esophagus. Non-embryonic eggs in the uterus; eggs laid in host tissues. Egg with larvae have heavily pigmented, dark walls, often noticeably thick; whole egg including polar plugs embedded in a thin transparent envelope usually densely covered by tiny spines. The type species is H. carcharhini (MacCallum, 1925).
Adult specimens recorded in fish are: H. huffmani Moravec, 1987  The nematodes in this study showed morphological and morphometric characteristics compatible with Huffmanela, but the adults differ from those previously recorded.
Identification of Huffmanela species is based on keys created by egg characteristics such as size and shell surface morphology (Moravec & Campbell, 1991;Moravec et al., 1998;Moravec & Fajer-Avila, 2000;Moravec, 2001;Conboy & Speare, 2002). According to Moravec & Campbell (1991), Moravec & Garibaldi (2003) H. schouteni has a thin envelope with bulges; Moravec et al. (1998) H. japonica has a thin and smooth envelope, with the presence of protuberances only in developing eggs, and H. shikokuensis smooth surface of the egg; Justine (2007) H. longa has elongated stichocytes and eggs with longitudinal folds, H. ballista egg has a surface marked by imperceptible longitudinal ridges, and H. ossicola has a characteristic egg envelope and numerous filaments. Justine (2011) observed that in H. plectropomi the surface of the eggs has a thick, continuous layer of filaments, but no envelope around the egg. Justine & Iwaki (2014) observed that the eggs of H. hamo had a smooth surface and did not have an envelope or filaments, and the surface of H. lusitana eggs do not have spines or filamentous structures (Ramos et al., 2019).

Microchirus azevia
Coast atlantic  other species, we can observe that the eggs in our study are smaller, and do not match those of any of the recorded species and using SEM we observed the presence of very marked filaments on the surface of the eggshell. Larval egg.
The adult nematodes recorded so far are few, as shown in Table 1, and very well characterized through scientific illustration and morphological description of the adults (Moravec et al., 2005;Carballo & Navone, 2007;Justine, 2007;Al-Hasson et al., 2019). Using scientific illustrations and SEM Carballo & Navone (2007) satisfactorily characterized the species H. moraveci, a parasite of Atherinopsidae fish. Thus, it was possible to distinguish the species of this study, as in its anterior region at the cephalic end it has an oral opening with a circular shape, and simple lips surrounded by a rosette with twelve papillae. In addition, there are two bands of lateral bacillary bands. Therefore, due to the morphological and morphometric differences of the adults and eggs of the nematodes in our research, we can identify a new species, which we have called Huffmanela psittacus n. sp. a parasite of C. psittacus in Marajó Bay, Pará State, Brazil.
In this study Huffmanela psittacus n. sp. adults and eggs were observed only in the gill arch and gills of the host, which triggered mild lymphocytic infiltration of the epithelium between the secondary lamellae present. In Sebastes spp. Conboy & Speare (2002) observed epithelial hyperplasia, spongiosis and mild lymphocytic infiltration of the epithelium and underlying dermis due to the presence of eggs in the gills. In Trisopterus luscus Esteves et al. (2009) observed in myodegeneration, necrosis, calcification and small granulomas with parasites; fish with hyperinfection showed diffuse histiocytic infiltrate and fibrosis, while fish skin, intestinal serosa and swim bladder showed no abnormalities; Bullard et al. (2012) observed intraepithelial inflammation with eosinophilic granulocytes and hyperplasia as well as lymphofollicular hyperplasia in the dermis of Carcharhinus plumbeus. Eissa et al. (2021) observed hemorrhagic, necrotic, and chronic inflammatory cell aggregations in the tissue surrounding the swim bladder, as well as tunnels of necrotic tissue that contained the eggs; Attia et al. (2021b) observed an intense granulomatous reaction with infiltration of mononuclear inflammatory cells and proliferation of fibrous connective tissue in the histopathology of the muscle fibers of Epinephelus coioides containing the nematodes and eggs. In the histological analysis of the gills of C. psittacus, in this study, numerous eggs were observed between the lamellae, which may have caused hyperplasia of the squamous epithelium of the primary and secondary filaments. Changes in fish gills can have several causes, such as the presence of parasites and/or environmental changes (De Lima et al., 2009;Santos et al., 2014).

Conclusions
Based on the differences mentioned, we can therefore say that the nematodes in this research are a new species, we call named Huffmanela psittacus n. sp. This finding represents the first record of a species of Huffmanela in South America and the first record in the gills of a Tetraodontiformes fish, C. psittacus in the state of Pará. We have also added the description of histopathological changes caused by this parasite, thus contributing to the knowledge of its pathogenicity in C. psittacus on Marajó Island.