Pathogens, Vol. 12, Pages 52: Prevalence, Morpho-Histopathological Identification, Clinical Picture, and the Role of Lernanthropus kroyeri to Alleviate the Zinc Toxicity in Moron labrax

1. IntroductionRecently, parasitic infestations have induced serious hazards, including higher mortalities and diseases, to the freshwater fish in Egypt [1,2]. Parasitic copepods are commonly present in wild and cultured marine fish [3]. Lernanthropus is the most common genus of copepods and there are more than 100 species isolated from the gills of different species of marine fish [4,5]. Lernanthropus causes the erosion and necrosis of gill filaments [6] with severe desquamation and necrosis of the secondary lamellae and leukocytic infiltration [7]. At the site of parasite attachment, there is complete superficial tissue erosion with exposure of the primary lamellar cartilage, exposure of the blood vessels, and hemorrhage resulting from the grasping action of the mandibles and the maxillae of the parasite [6].Pollution with heavy metals or toxic pollutants in the aquatic ecosystem is a global problem, with potential concern as it can negatively affect fish with health-inducing physiological, biochemical, molecular, and histopathological alterations [8,9,10]. Fish absorb heavy metals from the surrounding water and accumulate in different tissues in various amounts [11]. The metals can enter the bloodstream of fish and gradually accumulate in their tissues [12,13], particularly in the hepatic tissue, where they reach the consumers through the food chain or are bio-transformed and excreted [14]. Hence, parasites, as well as heavy metals, induce serious damage to the biochemical and physiological processes that in turn induce severe impairments to the health and physiology status of fish [15]. Recent reports have addressed various methods for heavy metal chelation such as natural extracts, probiotics, and nanoparticles [13,16,17]. Fish parasites are considered extra sensitive to pollution with heavy metals, as they not only uptake and accumulate toxicants in their tissues, but also produce a physiological response to them [18]. Parasites can be used either as effective indicators or as accumulation indicators, because of the different ways in which they react to anthropogenic pollution [19,20]. There is a relationship between parasitism and pollution, and the role of parasites as bio-indicators of heavy metals pollution [21]. Previous reports have addressed the ability of some parasites to accumulate heavy metal concentrations, such as Acanthocephalans, Cestodes [22], and parasitic nematodes [23,24].

Therefore, the current investigation was carried out to assess the impacts of L. kroyeri infestation. We addressed the prevalence of the parasite in the different seasons, the clinical signs, and the post-mortem changes. The body surface of L. kroyeri using a light microscope was illustrated, besides the bioaccumulation of heavy metals in the tissues of both L. kroyeri and M. labrax. Furthermore, histopathological alterations on the gills and muscles of infected M. labrax were detected.

2. Materials and Methods 2.1. Research Ethics

The protocol of the current study complies with the guidelines and was carried out according to the UK Animals (Scientific Procedures) Act, 1986, and the associated guidelines of the EU Directive for Animal Experiments. The experimental procedures were approved by the Institutional Aquatic Animal Care and Use Committee (IAACUC), Faculty of Aquatic and Fisheries Sciences, Kafrelsheikh University, Kafrelsheikh, Egypt. Approval Code: IAACUC-KSU-038-2022

2.2. Fish Samples

A total number of 200 sea bass (Moron labrax) fish samples were collected alive or freshly dead from the market of the Ezbet-El Borg area, Damietta Province, Egypt, during the period between March 2019 until February 2020. The collected fish were transported on thick ice polyethylene bags to the laboratory of the Animal Health Research Institute, El-Mansoura Branch, where they were examined immediately.

2.3. Clinical ExaminationThe fish were examined for the detection of any clinical abnormalities and external parasites according to Eissa [25]. 2.4. Parasitological ExaminationExamination of the external surface of the fish body was carried out with naked eyes and a hand lens to detect any abnormalities, the gill opercula were removed using scissors, and the gill filaments were transferred to slides with some normal saline and then covered by a cover slide and examined microscopically [26]. The detected crustacean parasites were carefully collected using a fine brush and special needle, transferred into Petri-dish, and washed several times in distilled water then preserved in 70% ethanol and cleared in lactophenol, and then mounted with polyvol [27]. 2.5. Heavy Metals Analysis

The samples were dried at 60 °C for 48 h. Then, the samples were ground to a fine powder and stored in plastic bags until analysis. One gram of each sample was dry-ashed in a muffle furnace at 450 °C for 5 h, and extracted with 20% hydrochloric acid. The samples were measured by Flame Atomic Absorption Spectrometry FAAS (GBC Avanta E, Victoria, Australia; Ser. No. A5616). All of the equipment used was calibrated and uncertainties were calculated. Internal and external quality assurance systems were applied in the Central Laboratory of Environmental Studies at Kafr-Elsheikh University according to ISO/IEC 17025 (2005). All of the measurements, blanks, triplicate measurements of elements in the extracts, and analysis of certified reference materials for each metal (Merck) were routinely included for quality control.

2.6. Histopathological ExaminationTissue specimens were collected from the gills and immediately fixed in 10% neutral buffered formalin solution for at least 24 h, then processed using the conventional paraffin embedding technique. Five-micron sections were prepared and then routinely stained with Hematoxylin and Eosin (H&E) according to Suvarna et al. [28], and then examined microscopically. 4. DiscussionLernanthropus is the most common genus of parasitic copepods. There are more than 100 species described from the gills of different marine fish [5]. The current investigation revealed hemorrhagic areas on the body surface with excessive mucous secretion and a marbling appearance of the gills of infected M. labrax with L. kroyeri. These lesions could be attributed to the attachment of the parasites by their rigid claws, feeding activity, severe irritation caused by parasitic movement, and mucous increase as a defense mechanism from the host to overcome the infection, as reported by Abdel-Mawla et al. [29].The present study recorded the isolation of L. kroyeri from the gills of M. labrax. Likewise, Toksen et al. [30], Henry et al. [31], and Eissa et al. [32] isolated the same parasite from the same host and the same site. Meanwhile, El-Deen et al. [33] and Hassanin [34] isolated L. kroyeri from the gills of other fish species such as Mugil cephalus and Moolgarda seheli.In the current prospective study, the prevalence of L. kroyeri was 81%, concurrent with a previous study by Aneesh et al. [35] that recorded 81.4% infection of Strongylura strongylura by L. kroyeri. Additionally, Toksen [5] reported a higher infection rate (100%) by L. kroyeri in Dicentrarchus labrax. Nevertheless, Manera and Dezfuli [6] obtained a lower infection rate (35%) with L. kroyeri in D. labrax. Our paper reports that L. kroyeri infection was the highest during spring (94%), followed by summer (90%), then autumn (78%), and finally winter (31%). This sequence is nearly in agreement with Eissa [25], who also reported that the infection rate with L. kroyeri reached its maximum rate during spring and summer, while the lowest infection was recorded during autumn. These results were inconsistent with Samak and Said [36], who reported that the infection rates with the same parasite reached their maximum rates in autumn and winter (42.5% and 35%), respectively, while their minimum value was 7.5% in spring. These variances in the total infection and seasonal dynamics could be a result of the difference in fish species and the difference in the locality of fish collection.Zn is an essential heavy metal with a permissible limit in the fish muscle of 40 mg/kg [37] or 100 mg/kg [38]. The toxic effect of zinc on aquatic animals depends on several environmental factors, especially temperature, water hardness, and dissolved oxygen concentration. An acute toxic concentration of zinc kills fish by destroying gill tissue and at a chronic toxic level, it induces stress that results in the death of fish [39]. Certain fish parasites can accumulate heavy metals at concentrations significantly higher than those in host tissues or the environment [40,41,42,43,44]. The data of our study revealed that there was a high concentration of Zn in the collected samples, while the concentrations of Cu, Cd, and Co were under the detection limit. In general, the accumulation of Zn was significantly higher in the non-infested tissue in comparison with the infested tissue samples. It is thought that L. kroyeri can absorb Zn from the fish tissue through its alimentary canal and that it accumulates in the parasite tissue, and this finding was verified by analysis of Zn in the parasite tissue. In the same manner, a recent study by Hassanine and Al-Hasawi [45] reported that acanthocephalan accumulates higher concentrations of heavy metals. Concurrent with another study, Szefer et al. [46] suggested that the bioaccumulation of parasites may reflect the higher ability of the host to clear heavy metals. In addition, Thielen et al. [44], Sures and Siddall [47], and Malek et al. [48] considered the parasites beneficial and that they could act as a heavy metal sanitizer for the host. Gills accumulated a higher Zn value compared with the edible part of its fish host. The low ratio of Zn concentration in the host muscle could be a result of the longer exposure time as metal uptake occurs faster in parasites, as stated by Sures [40].Considering the histopathological findings, we illustrated sections of L. kroyeri were distributed in the gills. Similarly, a recent study by Eissa et al. [7] reported the occurrence of L. kroyeri fragments in the gills of D. labrax. The destruction of the secondary lamellar epithelium, goblet cell metaplasia with hemorrhage, and excess mucous secretion could be induced as a tissue reaction to decrease the irritation against the infestation. Concurrent with previous studies, Abdel-Mawla et al. [29], Lester and Hayward [49], Manera and Dezfuli [6], and Ragias et al. [50] reported extensive hemorrhage due to the feeding activity of this parasite. Lymphocytes and eosinophils were found in the gill filaments and arches, and these outcomes have been previously reported [4,5,6,51,52]. In addition, erosion of the gill raker as well as necrosis of the muscles was seen; likewise, Vinoth et al. [53] reported pale gills induced by copepod parasites due to the loss of the gill raker.

Our investigation concluded that, although L. kroyeri has a negative effect on the infected M. labrax, it also plays an important role in the elimination of heavy metals from the tissue of the infected fish through its ability to accumulate heavy metals in its body, which can be advantageous for the infected hosts, allowing them to tolerate much higher concentrations of certain metals. The present results also confirm that L. kroyeri seems to be a good indicator of environmental pollution.

5. Conclusions

To date, our perspective study represents a premier work to report on the efficacy of L. kroyeri to uptake and accumulate heavy metals (zinc). However, L. kroyeri infests M. labrax with a high prevalence in spring and summer and demonstrates excessive mucous secretion, ulceration, marbling appearance of gills, and various histopathological changes in the gills of the infested fish. By detecting various heavy metals (Zn, Co, Cu, and Cd) in the tissues of L. kroyeri and M. labrax, surprisingly, L. kroyeri was found to uptake the highest concentration of Zn in its tissues. Conclusively, the parasitic infestation is an eco-friendly method to uptake heavy metals, and L. kroyeri can be utilized as a natural antitoxic agent, as well as be considered a bio-indicator of toxicity with heavy metals and to lessen the hazardous impact on the aquatic environment for sustaining aquaculture. Future studies are needed to test the activity of other parasites to chelate heavy metals, as well as studies on various fish species.

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