Prevalence and Morphological Identification of Digenean Trematode Species Recovered from Infected Organs of Lizardfish (Saurida undosquamis)

Helminthic parasites of the marine fish (Saurida undosquamis) are investigated. Out of 192 specimens of the Saurida undosquamis were collected from the local market of Misurata city in Libya. The prevalence rate of Trematoda helminths in examined fish Saurida undosquamis, was high. Based on the morphological and morphometric characterization represented different species of Trematoda (Lecithochirium sp., Lecithochirium grandiporum, Aponurus laguncula, Neotorticaecum sp, Allocreadium sp, and Gonocerca sp.) was isolated of the total examined fish (185%, 96.4%) were infected with different species of Trematoda. The density of Trematoda was around 9390, and they were collected from the gills, stomach, and anterior and posterior parts of the intestine in all seasons, but Trematode infection was observed in the liver and muscles, the high-density rate in winter (4199), followed by autumn (3291), with non-significant differences between density rates in all seasons (P > 0.05). This study found a high infection rate with Lecithochirium sp. in the examined Saurida undosquamis. 

Introduction

Parasites are accepted among the most detrimental effects on fish stocks all over the world. They caused retardation in growth, lead to diseases and may even cause the deaths of whole host fish. Digeneans are an important group of helminthic parasites; that usually invade the gastrointestinal tract of marine Piscean hosts (Mishraet al., 2013). Marine fishes are the common host for various species of digenetic trematodes.

The digenea are one of the major taxa of parasitic Platyhelminthes and they are invariably endoparasites. Digeneans are heterogeneous and require more than one host to complete their life cycle. (Paperna & Dzikowski, 2006).

The lizardfish (family Synodontidae) genus Saurida comprises about 20 species found in tropical and subtropical seas. A single species, recorded from the Mediterranean is a Red Sea-Suez Canal migrant (Golani, 1993).

Usually, 80% of marine fish are infected by parasites in Arab countries due to secondary infection by bacteria and other pathogenic microorganisms, (Taha & Ramadan, 2017). Few previous studies have been done on Saurida undosquamis, to diagnose infection by Digenea trematodes. According to previous studies, many parasites affect the internal organs of Saurida undosquamis such as the intestines, muscles, liver, and kidney, and often show symptoms of infection of these parasites (Banerjeeet al., 2017; Eissaet al. 2010). Different species of digenean, have infected all species of Saurida, and are used as their final or intermediate hosts, Eissaet al. (2010).

Cavalleroet al. (2019) recorded a few previous studies in Libya to determine the prevalence of infection rates from different organs of Saurida undosquamis fish. This study was aimed at the determination of the prevalence and identification of some digenean species from different infected organs in lizardfish in Misurata, Libya.

Materials and Methods

Fish sampling: The study included 192 lizardfish Saurida undosquamis ranging in weight (26–235 g) and length (14–32 cm). The current study was conducted from June 2018 to May 2019, and 15 to 20 fish were collected from the local market of Misurata, Libya. They were transferred in an ice box to the laboratory of the Zoology Department, Faculty of Science.

Lizardfishes were examined only for internal parasites. Fish were dissected, and the whole body cavity, muscles, liver, and digestive system were carefully examined at first by the naked eye for the presence of helminths. On the other hand, the organs (gills, liver, stomach, anterior intestines, and posterior intestines) were separated by dissecting scissors and forceps and placed in Petri dishes containing the normal saline. Then, a scalpel and forceps were used to scrape fish contents gently. All Petri dishes were carefully examined for the presence of Trematodes by dissecting and light microscopes. Finally, the Trematodes were isolated in a sterile container containing normal saline, (Hoffman, 1967).

After collection, the Trematodes were washed several times with normal saline to remove any attached mucus. The different species of Trematodes were kept separately in sterile glass bottles containing 70% ethanol and were labeled with all details regarding each sample. The adult worms of Trematode were fixed in warm ethanol (70%). Then, the worms were flattened gently, followed by dehydration, and then stained in acetic acid alum carmine dye for 2–10 minutes. In addition, they were cleared in xylol, to identify the internal structures of the worms, and finally added to the slides using DPX (Rasheed, 1989).

After mounting worms, they were examined by microscope (Buffalo, N.Y. 1420 U.S.A.) at 10X and 40X and drawn by sketch photos to facilitate identification and classification. The Morphometric measurements of Trematode included the measure of oral and ventral suckers, the distance and length between the oral and ventral suckers, and the total length of adults.

The data analysis compared helminths abundance between the different parasite species using one-way ANOVA at a 5% significance level. The relationship between lengths and weights of Lizardfish with parasite density was determined using linear correlation at a 5% level of significance. The comparisons of parasite abundance with seasonal variations were carried out using one-way ANOVA. This test was performed using the SPSS computer software.

Results

From examined fish were infected with digenean trematodes 185 (96.4%). Fig. 1 shows that the stomach had the highest infection (95.8%), followed by gills (59.9%), whereas the anterior and posterior parts of the intestines had the lowest infection rate (6.8%). In contrast, no Trematode infection was observed in the liver and muscles. On the other hand, the stomach demonstrated the highest infection rate in spring (65%), followed in summer (56.7%). Moreover, the gills showed the highest infection in autumn (43.02%), as illustrated in Table I. Based on the statistical analysis, there was a non-significant difference (P > 0.05) among all different infected organs.

Fig. 1. The prevalence rates of trematode infection based on the infected organs.

Table I. Total Prevalence Rate with the Seasonal Variations of Different Organs

Seasons	Stomach	Anteriorintestine	Posteriorintestine	Gills	Total
Winter	57 (54.8%)	6 (5.8%)	7 (6.7%)	34 (32.7%)	104
Spring	44 (64.7%)	2 (2.9%)	3 (4.4%)	19 (27.9%)	68
Summer	38 (56.7%)	3 (4.47%)	1 (1.49%)	25 (37.3%)	67
Autumn	45 (52.3%)	2 (3.5%)	2 (2.3%)	37 (43.02%)	86

According to the density of infection in different infected organs, the stomach had the highest density of infection (92.9%), followed by gills (6.6%), as shown in Fig. 2. In contrast, Table II shows that no infection was observed in the liver and muscles, based on the seasons, winter was reported the highest density (44.7%), followed by autumn (35.04%), while there were similar densities in spring and summer. Moreover, based on the one-way ANOVA, a non-significant difference (P > 0.05) was found between density infections of infected organs.

Fig. 2. The density infection of trematode species based on organs.

Table II. Density Infection Rates in Saurida undosquamis According to Infected Organs

Seasons	Gills	Stomach	Anterior intestine	Posterior intestine	Total density rate
Winter	211	3962	16	10	4199
	5.02%	(94.4%)	(0.38%)	0.24%	44.7%
Spring	51	922	5	3	981
	5.20%	(93.9%)	(0.51%)	0.30%	10.4%
Summer	91	824	3	1	919
	9.90%	(89.7%)	(0.33%)	0.11%	9.78%
Autumn	269	3017	2	3	3291
	8.2%	(91.7%)	0.06%	0.09%	35.04%

According to the morphological and morphometric features of different species of Trematodes collected from different regions of Saurida undosquamis, some species of trematodes were recovered: (Lecithochirium sp., Lecithochirium grandiporum, Aponurus laguncula, Aponurus sp., Neotorticaecum sp., Allocreadium sp, and Gonocerca sp.). Table III shows the prevalence and density rates of trematode species isolated from various infected organs, Lecithochirium sp. was recorded with the highest rates of prevalence (94.3%) and density (94.05%), followed by Lecithochirium grandiporum (44.3% and 5.66%) respectively. Whereas, the lowest rates of prevalence and density were shown in Lecithochirium sp, Aponurus laguncula, Lecithochirium grandiporum, Neotorticaecum sp., Allocreadium sp, and Gonocerca sp.

Table III. Prevalence and Density Infection Rates in Saurida undosquamis According to Different Trematode Species

	Lecithochirium sp	Lecithochirium grandiporum	Neotorticaecum sp
Prevalence rate	94.2%	44.3%	3.7%
Density rate	94.05%	5.66%	0.07%
	Aponurus laguncula	Gonocerca sp	Allocreadium sp
Prevalence rate	4.7%	0.52%	0.52%
Density rate	0.13%	0.01%	0.01%

According to the morphological and morphometric features of Lecithochirium sp., collected from different regions of Saurida undosquamis. Recent publications present diverse opinions concerning the classification and taxonomy of trematodes and the following keys were consulted for identification of the present trematodes: Phylum: Platyhelminthes, Class: Trematoda, Order: Azygiida Schell, Suborder: Hemiurata, Family: Hemiuridae, Subfamily: Hemiuroidea (Faust, 1929), Genus: Lecithochirium (Luhe, 1901), Species: Lecithochirium–spp. (Yamaguti, 1958; Lühe, 1901).

According to the morphological characteristics, the body with a small size, smooth tegument, and small oral sucker, with a length of 12.96 μm and width of 14.9 μm. The ventral sucker was bigger than the oral sucker and close to the anterior region, having a length of 36.96 μm and a width of 36.29 μm. The distance between the suckers was 71.10 μm. The intestinal shape was simple, with two testes, symmetrical and spherical. They were located in the front half of the body below the ventral sucker, with a length of 23.84 μm and a width of 21.81 μm. The ovary was elliptical and located in the back half of the body. The uterus was full of eggs extending just before the tail, ranging from 14.21 to 17.64 μm in length and 9.04–10.07 μm in width, as illustrated in Figs. 3A, 3B.

Fig. 3. (A, B): A general view of the adult worm of Lecithochirium spp. from Saurida undosquamis, showing the different structures of the body. O.S, oral sucker; V.S, ventral sucker; PH, Pharynx; O.E, Esophagus; TS, two testes are equal and opposite; O, the ovary has an elliptical shape and is located in the back half of the body; U.T, uterus; I.C, Intestinal Caeca; E, Eggs.

Regarding the Lecithochirium grandiporum, the small body size, with a smooth tegument. The oral sucker was small, with a length of 8.72 μm and a width of 9.23 μm. The ventral sucker was larger than the oral and close to the anterior region (with a length of 20.97 μm), a width of 25.11 μm, and a distance between the suckers was 26.10 μm. The intestines were simple, and the uterus was full of eggs extending just before the tail. The length of the posterior part of the body down from the ventral sucker was 82.07 μm. The eggs ranged between 9.88 and 14.38 μm in length and 8.06–9.41 μm in width, as illustrated in Figs. 4A, 4B.

Fig. 4. (A, B): A general view of the adult worm of Lecithochirium grandiporum from Saurida undosquamis. O.S, oral sucker; V.S, ventral sucker; PH, Pharynx; O.E, Esophagus; U.T, uterus; I.C, Intestinal Caeca; E, Eggs.

According to the morphological and morphometric features of Neotorticaecum sp., collected from the gills, stomach, and anterior intestines of Saurida undosquamis. Recent publications present diverse opinions concerning the classification and taxonomy of trematodes and the following keys were consulted for identification of the present trematodes: Phylum: Platyhelminthes, Class: Trematoda, Subclass: Digenea, Order: Plagiorchiida, Suborder: Hemiurata, Family: Didymozoidae, Subfamily: Didymozoidae ‘juveniles’, Genus: Neotorticaecum, Species: Neotorticaecum sp. (Yamaguti 1958; Kurochkin and Nikolaeva, 1978).

Based on the morphological characteristics, the body with a small size, and an elongated body with a smooth cuticle. The oral sucker was small, with a length of 4.67 μm and a width of 3.79 μm. It was subterminal, followed by a muscular pharynx and a large ventral sucker, with a length of 13.89 μm and a width of 14.86 μm. However, the distance between the suckers was 36.86 μm. The Oesophagus was narrow and bifurcated anterior to the ventral sucker and encircled by a gland, and intestinal augmentation was simple ocular cells. In addition, the caeca chambers posterior to the ventral sucker increased in size as they reached the end of the body. The excretory vesicle was saccular, located posterior to the caeca. The length of the eggs was 16.06 μm, and the width was 9.76 μm. as in Figs. 5A, 5B.

Fig. 5. (A, B): A general view of the adult worm of Neotorticaecum from Saurida undosquamis. O.S, oral sucker; V.S, ventral sucker; O.E, Oesophagus; I.C, Intestinal Caeca.

Regarding Aponurus laguncula., it was collected from the gills, and stomach of Saurida undosquamis, Recent publications present diverse opinions concerning the classification and taxonomy of trematodes and the following keys were consulted for identification of the present trematodes: Phylum: Platyhelminthes, Class: Trematoda, Subclass: Digenea, Order: Plagiorchiida, Suborder: Hemiurata, Family: Lecithasteridae, Subfamily: Lecithasterinae, Genus: Aponurus, Species: Aponurus laguncula (Yamaguti, 1958; Looss, 1907).

According to the morphological and morphometric features demonstrated a small body with a smooth tegument. In addition, the oral sucker was small, with a length of 14.30 μm and a width of 15.58 μm. The ventral sucker was bigger than the oral sucker and close to the anterior region, with a length of 35.56 μm and a width of 41.63 μm, and the distance between the suckers was 42.52 μm. The testicles were above each other and located on the front half of the body, below the ventral sucker, with a length of (12.68 μm) and a width of (13.43 μm). The ovary was elliptical and located in the back half of the body, with a length of (10.82 μm) and a width of (11.07 μm). The uterus was full of eggs extending just before the body end, and the eggs ranged from 19.69 to 25.31 μm in length and 10.13–11.43 μm in width, as illustrated in Figs. 6A, 6B.

Fig. 6. (A, B): A general view of the adult worm of Aponurus laguncula from Saurida undosquamis. O.S, oral sucker; V.S, ventral sucker; PH, Pharynx; O.E, Oesophagus; TS, two testes were above each other; OV, the ovary is elliptical and located in the back half of the body; U.T, uterus.

On the other hand, Gonocerca sp. was collected from the gills, the gills of Saurida undosquamis, Recent publications present diverse opinions concerning the classification and taxonomy of trematodes and the following keys were consulted for identification of the present trematodes: Phylum: Platyhelminthes, Class: Trematoda, Subclass: Digenea, Order: Azygiida, Suborder: Hemiurata, Family: Allocreadiidae, Genus: Gonocerca (Manter, 1925), Species: Gonocerca sp. (Yamaguti, 1958; Manter, 1925).

Based on the described morphological characteristics, the body was small, with a smooth tegument, a small oral sucker, a length of 14.90 μm, and a width of 18.06 μm. The ventral sucker was bigger than the oral sucker and was closed to the posterior region, with a length of (32.14 μm) and a width of 32.66 μm. The distance between the suckers was 75.45 μm. The pharynx surrounds the oesophagus. The intestinal tubes were simple. The total length of the eggs was about (44.29–47.83 μm), the width ranged from 22.60 to 26.77 μm, and the total body length was 147.11 μm, as illustrated in Figs. 7A, 7B.

Fig. 7. (A, B): A general view of the adult worm of Gonocerca sp. from Saurida undosquamis. O.S, oral sucker; V.S, ventral sucker; PH, Pharynx; O.E, oesophagus.

The Allocreadium sp. were collected from the stomach of Saurida undosquamis, Recent publications present diverse opinions concerning the classification and taxonomy of trematodes and the following keys were consulted for identification of the present trematodes: Phylum: Platyhelminthes, Class: Trematoda, Order: Plagiorchiida, Family: Allocreadiidae, Genus: Allocreadium, Species: Allocreadium sp. (Yamaguti, 1958; Looss, 1907).

According to the morphological and morphometric features demonstrated, the body was small, with a smooth tegument and a large oral sucker, with a length of 20.73 μm and a width of 19.21 μm. The oral sucker was larger than the ventral sucker and located in the middle of the body, with a length of 6.91 μm and a width of 8.02 μm. The distance between the suckers was 34.55 μm. The testicles were equal and opposite, located in the lower part of the body, with a length of 17.43 μm and a width of 16.04 μm. The ovary was elliptical and located in the body’s back half and above the testicle, with a length of 5.80 μm and a width of 6.84 μm. In addition, the total body length was 122.33 μm, as illustrated in Fig. 8.

Fig. 8. A general view of the adult worm of Allocreadium sp. (new species), from Saurida undosquamis.

Discussion

During this study, we encountered great difficulty in distinguishing between types of parasitic worms based on morphological and standard characteristics only, especially with the presence of high infection, as well as the lack of studies in Libya that can be referred to and relied upon in the classification of types of worms isolated from Saurida undosquamis from Misurata, Libya.

The present study provides the endo helminthic parasitic infestation in 192 Saurida undosquamis fish from June 2018 to May 2019, reported a trematodes Species (Lecithochirium sp., Lecithochirium grandiporum, Aponurus laguncula, Aponurus sp., Neotorticaecum sp., Allocreadium sp, and Gonocerca sp.). They demonstrated no pathognomonic clinical signs and were in good health except in the liver and muscles. However, all the fish examined were infected with trematodes in all seasons (100%). Few previous studies illustrated the prevalence of infection rates of trematodes species from different organs of Saurida undosquamis. In this study, about 96.4% of examined fishes were infected with trematodes species, which were collected from different organs such as gills, stomach, anterior and posterior intestine, suggesting that the distribution of this species within the fish tissue was related to the availability of nutrients. Meanwhile, it was higher than what El-Atabanyet al. (2019) obtained around (34.3%). In contrast, it was similar to the finding of Abd-Elghany (2017), about 44.8% of the Zagazig fish market, which was obtained from the Gulf of Suez. There were some isolated trematodes species from specimens of Saurida undosquamis with few numbers compared to Lecithochirium sp, this might be attributed to differences in host fish species and geographic area. Moreover, showed a 4.7% infection rate of Aponurus laguncula., lower than that was shown in a study conducted by Heba and Abdel-Mawla (2019) in Egypt. This might be attributed to the difference in the place where the fish were obtained. In contrast, Abd-Elghany (2017) reported a lower infection rate (2.4%). The morphology of recovered Neotorticaecum in the present study was identical to those described by Pozdnyakov and Gibson (2008) and Felizardoet al. (2011). In addition, Abdul-Salamet al. (1990) found larval didymozoid from the muscles of the marine fish Nemipterus peromli from the Arabian Gulf. Meanwhile, One fish was infected with Gonocerca sp. collected from the gills. This result was similar to a study by Maheret al. (2019), conducted in Hurghada in the Gulf of Suez, Red Sea, Egypt, which found four Brushtooth lizardfish infected with Gonocerca, but in the stomach, not gills as in the current study. Only one fish was infected with Allocreadium sp. which was isolated from the stomach also, Vainutiset al. (2023) found this species from Russia. This species was recorded for the first time in Saurida undosquamis from Misurata, Libya.

Conclusion

The Conclusion of this study, these findings revealed the highest infection with all Species of Lecithochirium spp. in Saurida undosquamis.