Huffmanela spp. (Nematoda: Trichosomoididae) from Orange-spotted Grouper (Epinephelus coioides, Hamilton, 1822) at Jubail Province, on the Arabian Gulf, Saudi Arabia: A case report

This paper is conducted for identifying the parasite which collected during regular routine inspection for the hygienic condition and health status of sh in 2019 at the central sh market shops at Jubail city, Saudi Arabia. Samples from an orange spotted grouper (Epinephelus coioides) sh showed heavy black streaks between the muscle bers that was considered unmarketable and rejected by the consumer after had been cut and prepared for sealing. The black discolorations were in the form of heavy diffused focal black spots or forming threads of variable sizes embedded in the entire whole-body musculature of the sh. These samples were subjected to parasitological and pathological evaluation. The parasitological examination of the affected muscular tissues revealed Huffmanela spp. eggs infection. The parasitic infestation was identied on the bases of the morphological and ultra-structural characters of the eggs. The histopatholgical investigation of the dark discoloration of the muscular tissues showed severe diffused multifocal granulomatous myositis reactions in the form of chronic inammatory response accompanied with brous connective tissue proliferation around the eggs and larvae together with lymphocytes and macrophages aggregation. Muscular necrosis, edema and evidence of muscular regeneration were also noticed. In conclusion; this case report conrmed the occurrence of Huffmanela spp. infection in the muscles of Epinephelus coioides by using light and scanning electron microscopy. Further molecular assessment is recommended.


Introduction
Epinephelus coioides (Hamour) was considered as the most preferred sh species consumed in the sh markets by 60-72% of Saudis (Burger et al., 2014). Huffmanela spp. (Nematoda: Trichosomoididae, Huffmanelinae) is one of the histozoic nematode parasite infecting marine shes and less frequently freshwater shes (Moravec et al., 1998). Furthermore, they are categorized mainly based only on the morphological feature of their eggs, and life-cycle characters such as host family and targeted tissue (Bullard et al., 2012;Justine and Iwaki, 2014).
Previous studies have shown that Huffmanela infection is one of the most common parasitic infections that adversely affect the health of various sh families, worldwide (Moravec & Garibaldi, 2000;Moravec & Garibaldi, 2003;Moravec et al., 2005;Esteves et al., 2009;Moravec & Justine, 2010;Al-Hasson et al., 2019 andEissa et al., 2020). The site of the lesion preference was species dependent, as previously reported for H. lusitana infecting the muscles of pouting Trisopterus luscus (Ramos et al., 2019), while H. paronai was observed in the skin of Xiphias gladius (Justine, 2004). Also, two species of Huffmanelae were recorded in Japan (H. japonica and H. shikokuensis) and the eggs of these parasites were found in the esh of Upeneus bensasi and Stephanolepis cirrhifer (Moravec et al., 1998). Moreover, Moravec and Campbell, 1991, further recorded the presence of H. schouteni in serosa covering intestine; abdominal cavity and liver from ying shes Hirundichthys a nis and Cypselurus cyanopterus.
The aim of the current study was to identify the causative agent of black muscular tissue discoloration and describing the associated pathological lesions in this sh species.

Study area
The shes; orange spotted grouper (Epinephelus coioides);were caught and examined from water of the Arabian Gulf at the eastern region of the Arabian Gulf; at sh landing stations, market shops and sh auctions at Jubail city ('27 ° 57.9'' N and '49° 40' 43.4'' E), Saudi Arabia.

Fish sample
From January 2017 to December 2019, during the routine examination of 850 (280 female and 570 male) orange spotted grouper (Epinephelus coioides); the examined sh were ranged from 45cm -50 cm with average body weight from 2,500 kg to 2,750 kg.;.only one male sh of orange spotted grouper (Epinephelus coioides) showed intermuscular black discoloration spots like threads. The fresh muscle specimens as well as its internal organs were examined directly under the microscope and different samples were xed and taken for further parasitological, histopathological and ultrastructural studies.

Parasitological examination
Different parts of xed muscle in 10% neutral buffered formalin had multiple black strikes (the black coloration of grade 2) were admitted to the Parasitology Department; Faculty of Veterinary Medicine; Cairo University. These lesions were examined under dissecting Stereoscope and light microscope; further, the scraped eggs from muscles were cleared in lactophenol for one hour, and then mounted in glycerol jelly . The measurements of eggs (n = 20) were photographed and measured using an Olympus AX70 connected to a Nikon camera. Measurements of the examined eggs (per microscopic eld) were recorded in micrometers (µm) with mean (minimum -maximum ± standard deviation).

Ultrastructure studies of eggs using Scanning electron microscope (SEM)
The recovered eggs were prepared by xation in 2.5% glutaraldehyde at 4°C for 24 h. Further, the isolated eggs were dehydrated through passing in a grade series of ethanol; each/ 20 min., dehydrated on lter paper Whitman no.1. Then, whole dryness was occurred by CO2 critical point drier (Autosamdri-815, Germany). Finally, the samples were stuck on the stubs, covered with 20 nm gold in a sputter coater (Spi-Module sputter Coater, UK). All the specimens examined and photographed by JSM 5200, Electron probe Microanalyzer, Jeol, Japan at the Faculty of Agriculture, Cairo University, Egypt; number of examined eggs were 10 which photographed by electron microscope .
The morphological identi cation of the collected eggs was carried out according to the described keys and available literature (Moravec and Campbell, 1991;Justine and Iwaki, 2014). The measurements of the different stages of eggs were taken as the length; width of eggs; the length of polar plug; the thickness of the two covering layers of eggs and the thickness of its convoluted larva.

Histopathogical examination
Tissue specimens from infected muscles were xed in 10% neutral buffered formalin, washed in tap water, dehydrated in ascending concentrations of ethanol, cleared in xylene and embedded in para n then sectioned at 5 µ thicknesses. Hematoxylin and eosin (H&E) stain was performed. All tissue processing procedures were performed according to Heil, 2009.  3.1.2. Light and scanning electron microscopy of Huffmanela spp. eggs All the examined eggs with different stages revealed infection of orange spotted grouper muscles with Huffmanela spp. By inspection of muscle 2 stages of eggs were recorded (stage 1: light brown advanced eggs with larva; stage 2: dark brown advanced eggs with developed larva). All the examined eggs were found to be in advanced stage which had larvae.

Gross lesions
The examination of the affected sh showed multifocal black parasitic materials between the muscle bers. Such materials were greyish white to black and appeared as thread-like and/or spots. The parasitic materials were distributed all over the sh esh either super cially in subcutaneous area or deep inside the muscular tissue and they have been concentrated in the esh around the vertebral column. The examined sh and affected muscles were presented in (Fig. 4A&B). The lesion sometimes appeared as a spotted circumscribed focal lesion especially near the myosepta but in other parts of the muscles, the parasitic materials were aggregated in the form of black thread-like clusters separating the myo bers and surrounded with a delicate layer of brous tissue.

Histopathogical ndings
The examined fresh tissue specimens using the light microscope showed multiple rounded to oval black eggs together with larvae deeply embedded in the muscle bers. Dilated lymph vessels and brous connective tissue proliferation were noticed around the parasites [ Fig. 4C]. Lymphatic dilation, edema and mononuclear in ammatory cells in ltration were common nding in these areas (Fig. 4D). The eggs in some of the examined sections were surrounded by shell-like structure and showing brous connective tissue capsule around the eggs; while the nearby muscles of the host showed myodegeneration (Fig. 4E). In other parts of the affected muscles, the eggs were deeply embedded in the myo bers and surrounded with brous connective tissue while the affected myo bers appeared atrophied (Fig. 4F).
The granulomatous reaction appeared either as early granuloma where many of parasitic eggs and larvae were surrounded with in ammatory cells, edema and thin layer of brous connective tissue (Fig. 5A) or appeared as old granuloma with thick layer of lamellate brous connective tissue (Fig. 5B).
Cells of chronic in ammation mainly macrophages and broblasts with prominent muscular necrosis and myophagia were also noticed (Fig. 5C). The granulomatous reaction in other parts was characteristic and showing central area containing remnants of the black parasitic depresses surrounded with mature brous connective tissue layer with regenerated muscular tissue (Fig. 5D). The regenerated myo brils appeared more easinophilic, rosette shape in cross section and with multiple centrally located nuclei ( Fig. 5E) while the necrotic muscles were surrounded with melanophores (Fig. 5F).

Discussion
Based on the previous published data; there are about 21 species of Huffmanella spp. were recorded worldwide till now; these species were identi ed on the basis of various morphological criteria as the eggs morphology (egg length and width; shape of shell smooth or spiny either contain ridges or laments; with envelop or not; the envelop character, which is spinose or aspinose) according to (Moravec and Fajer-Avila, 2000). Additionally, the identi cation was declared by the habitat of eggs either skin; gills; muscles; swim bladder; within bones; serosa of intestine and liver. Also, identi cation was carried out by speci c host species Accordingly; this study was applied on Orange spotted grouper (Epinephelus coioides, Hamilton, 1822) which was severely rejected in sh market due to sever black discoloration of muscles. By parasitological examination of this rejected sh; the examination revealed the presence of Huffmanela spp eggs. The Huffmanela spp was identi ed according to the morphological keys recorded by H. hamo by Justine and Iwaki, 2014); H. banningi (Moravec, 1987); H. japonica (Moravec et al., 1998); H. lusitana (Ramos et al. 2019) and H. shikokuensis (Moravec et al., 1998)..
Our study revealed infection of muscles of Orange spotted grouper with Huffmanela spp. This identi cation was con rmed by the length and width of the eggs as 60-75x 26-36 µm which nearly resembles to previous study with Moravec et al., 1998 whose recorded Huffmanela spp. egg length as 58-69 x 26-30µm. Egg shell in our study had protuberance as recorded by Moravec et al., 1998. Also; in our study, the eggshell envelop was smooth which revealed by scanning electron microscope; this nding con rmed by Moravec et al., 1998. Unfortunately; till now; there are no published molecular data on this tissue parasite for species con rmation. So; the study was con rmed by several points as egg length; width; shell envelop; either these eggs have spine; protuberance; lament or smooth eggshell.
Our study differentiated from other results in H. huffmani which have spine on eggshell; these spines appear triangular as recorded in ultrastructure of transmission electron microscope by Žd'árská et al., 2001; indicating that it is not H. huffmani. In this study the protuberance appeared not regular as in H. huffmani which appeared regular in shape and distribution. Huffmanela huffmani recorded in swimbladder of freshwater shes; Huffman and Moravec, 1988, while our study recorded in muscles of marine shes [Orange spotted grouper].
In the previous study on H. japonica by Moravec et al., 1998, accumulation of eggs in groups, in muscles of the infected goat sh, Upeneus bensasi (Mullidae : Perciformes) were recorded. They found two stages of eggs (less advanced and advanced eggs) as our study; in our study this is the rst time to describe these eggs with scanning electron microscope which not appeared on the previous results by Moravec et al., 1998. There are 5 species of Huffmanela recorded to infect the muscles of marine water shes H. hamo (Justine and Iwaki, 2014); H. banningi (Moravec, 1987); H. japonica (Moravec et al., 1998); H. lusitana (Ramos et al. 2019) and H. shikokuensis (Moravec et al., 1998). The difference of these species according to the length of the eggs; eggshell and it's enveloped if present. The longest eggs of these species were H. banningi (99-108 x 42-45) with its shell spinose with minute canal on the envelop; and H. hamo; H. lusitana and H. shikokuensis were smooth shell with smooth envelop; while H. hamo and H. lusitana were smooth shell with no envelop.
In gross pathological examination, multifocal black parasitic remnants were noticed in the muscular tissue of the infected sh. This black discoloration is characteristic for Huffmanela in many sh species (Esteves et al., 2009 andEsteves et al., 2016). In histopathological examination, dilation of lymphatics may be attributed to the in ammatory reaction evoked by the host tissue against the eggs and/or larvae of the parasite. The chronic in ammatory response that appeared in the form of granulomatous myositis could be agreeable with the ndings of Ramos et al., 2019 who recorded granulomatous reaction in pouting, Trisopterus luscus infected with Huffmanelaspp.. In our study, regeneration of the necrotic muscular tissue was of common occurrence In teleosts, the regeneration ability of their muscles has been con rmed by many authors (Rowlerson et al., 1997;Hans, 2012 andSaera-Vila et al., 2018).

Conclusion
From this study we could conclude that, the Huffmanela spp is reported in orange spotted grouper (Epinephelus coioides) in Saudi Arabia.
In our study, although the Huffmanela spp was recorded in only one reported case in orange spotted grouper, the severity of infection in the muscle caught our attention for the possibility Epinephelus coioides role in dissemination of this parasite to other sh species including groupers' predators in marine ecosystem as sharks. Figure 1 Developmental stages of Huffmanela spp. eggs from Orange spotted grouper; (A) stage 1: amber-shelled egg with bilayer eggshell observable with outer layer (ol) dark colored and innermost typically translucent with developing larva (dl), scale bar: 10 μm. (B; C) stage 2: fully developed brown-shelled egg with developing larva (dl); note protuberance in outer layer (arrow); scale bar: 10 μm.