Factors determining community structure of metazoan parasites from brown-spotted grouper, Epinephelus chlorostigma (Valenciennes, 1828) in Southern Saudi Arabia

Several factors influence the structure of parasite communities, which are important components of the biodiversity of various ecosystems. Some of these factors may be related to the environment and/or to the host. The present study evaluated the influence of host size and season on the structure of the metazoan parasite community of the brown-spotted grouper, Epinephelus chlorostigma. One hundred thirty-two fish were collected between March 2018 to February 2019 from the Red Sea, Saudi coast, Southern Saudi Arabia. Eight parasite species were recorded: one copepod (Sarcotaces sp.), one isopod (Argathona rhinoceros), two monogeneans (Pseudorhabdosynochus epinepheli and Megalocotyloides epinepheli), three digeneans (Prosorhynchus epinepheli, Helicometrina nimia, and Erilepturus hamati), and one nematode (Cucullanus epinepheli). The overall prevalence was 43.2% Digeneans were the most frequent parasite species and represented (50.55%) followed by monogeneans (45.05%), crustacean (3.42%), and nematode (0.98%) of the total individual parasites collected from 57 infected fish. Parasite community structure and species composition varied significantly among host size. Positive associations were found between infection parameters of parasite species and host size. Our results suggest that parasite infection parameters were affected by host size and season. Further long-term research is required to conclude the factors determining the structuring of the parasite community of E. chlorostigma.


Introduction
In tropical fish, several factors influence the structure of parasite communities, which are important components of the biodiversity of various ecosystems. Some of these factors may be related to the environment and/or to the host (e.g., feeding, behavior, physiology, sex) [4,31,46].
Temporal and spatial structural changes in marine parasite communities have been associated with seasonal and local variations in biotic and abiotic environmental factors. Variations in parasite species composition and infection levels over time can be attributed also to these factors [20,44,45,47]. In the tropics, structural changes in parasite communities have always been associated with host traits such as age, body size, feeding behavior, host density, and vagility. These can promote high colonisation and exposure rates by new species of parasite and have been associated with parasite communities of high diversity and species richness [26,39,42,46,47].
In tropical regions conducted studies on temporal variation in fish populations [15,44,45,47] revealed that temporal variation in species richness and parasite community structure was affected by biotic and abiotic factors, such as water temperature and oxygen levels, availability of intermediate hosts, as well as human impacts. Even though, there is still little evidence that parasite communities vary over time [15,20,25,46].
Temporal structural changes that related to seasonal and/or local variations in several biotic and abiotic environmental factors can be experienced in metazoan parasite communities. Few studies have addressed this issue in tropical regions, where changes in water temperature are less extreme than in temperate regions, so the factors or processes that can generate variations in these parasite communities are as yet unclear [25,39,46].
E. chlorostigma (Valenciennes) (Pisces: Serranidae), is a widely distributed species in the red sea and one of the most important commercial marine fish in Saudi Arabia [1,12,32]. The habitat recorded for E. chlorostigma was a deep-sea fish because the specimen was caught on the outer slope of the barrier reef, but may be encountered in shallow waters [12,14]. Unexpectedly there is no current studies regarding the parasite community of brownspotted grouper, E. chlorostigma (Valenciennes 1828) (Perciformes: Serranidae); studies have been focused solely on taxonomy of parasites and species catalogue [1,2,5,21,22,35]. Therefore, the parasite community dynamics from this fish should be better investigated. In this sense, the present study aimed to evaluate the influence of host size and season on the metazoan parasite community structure of E. chlorostigma.

Study area, sampling of hosts and parasites
This study was carried out in Al Qunfudhah (19° 7′ 35″ N, 41° 4′ 44″ E), in the Tihamah region on the Red Sea coast. It is located at 290 km to the south from the holy city, Mecca, and 340 km away from Jeddah. Its average monthly weather is shown in Table 1. The high rainfall was recorded in Al Qunfudhah during April, May, November, December, and January. The high temperatures were recorded in May, June, July, August, and September (mostly summer months) ( Table 1).
One hundred thirty-two E. chlorostigma were examined for parasitological study from March 2018 to February 2019. Fish were bought from local fishermen operating landing trawlers along the Al Qunfudhah coast. The length ranged from 400 to 850 mm (all samples were mature according to [12]. Samples were kept fresh or were deepfrozen in individual plastic bags at − 10 °C, until further examination in the laboratory. Thirty-three fish were collected per season in one month at the one time (March for spring, July for summer, September for autumn and January for winter).
External organs of fish (skin, fins, nasal pits, eyes, and buccal cavities) and internal organs (stomach, pyloric caeca, intestines, heart, liver, spleen, gall bladder, and gonads) were thoroughly examined for the presence of ectoparasites and endoparasites. Gill arches, placed in Petri dishes filled with sea water, were separated and examined for the presence of parasites. Internal organs (e.g., stomach, pyloric caeca, intestines, heart, liver, spleen, gall bladder, and gonads) were separated and examined for the presence of parasites. Collected parasites were counted, and preserved in 70% ethanol and then specimens were cleared in lactic acid for further examination and species identification. Parasites were identified according to Gibson et al., [16], Al-Mathal [1], Al-Mathal [2], Justine and Henry [21], Justine et al., [22], Bray and Justine [5], and [35]. Host length (host size) was classified into three groups (class 1, ≤ 500 mm, class 2, < 500 to > 700 mm, and class 3 ≥ 700 mm (all samples were mature according to Craig et al. [12].

Data and statistical analysis
Infection levels for each parasite species were described according to Bush et al. [7]. Parasites infecting several host species were considered generalist parasites while specialist parasites, those infecting only one or two host species [18]. Data were statistically analyzed using SPSS software (Version 22.00). Testing of effects of both individual and interacted factors (host size and season) on species richness of metazoan parasite species was analyzed using the General Linear Interactive Model (GLIM) after normalization of the data by log 10 (x + 1) transformation [13,50]. Possible differences in parasite prevalence among host size categories of fish, as well as season were evaluated using a Chi-square test. Possible differences in abundance and intensity among host size and season were tested by Kruskal Wallis. Correlations between host size categories and both prevalence, abundance, and intensity were tested by using the non-parametric, Spearman's rank correlation coefficients (rs). The diversity of parasite species was measured using the Simpson diversity index.
Species richness was the highest among the digeneans parasite species and represented (50.55%) followed by monogeneans (45.05%), crustacean (3.42%), and nematode (0.98%) of the total individual parasites recorded (819) from 57 infected fish. Out of 132 examined fish, 57 (43.2%) fish were found to be infected. The most prevalent parasite species was Pseudorhabdosynochus epinepheli (33.3%), while the least one was C. epinepheli (6.1%) ( Table 2). The overall mean species richness varies significantly by host size (Fig. 1, χ2 = 107.10, P < 0.001). The large-sized fish group (class 3) has the highest mean species richness (Fig. 1, 3.77). Also, fluctuation in species richness per season was found. The highest species richness was recorded in summer (2.58) and the lowest one in winter (Fig. 2, 0.61). There was no significant difference in species richness per season (χ2 = 4.48, P = 0.17). Analysis  of the data with GLIM revealed that host size played a significant role in determining parasite species richness and that there was no interaction between host size and season (Table 3). A positive correlation was found between species richness and host size (length) (r 2 = 0.65, P < 0.001).
The Simpson diversity index of the parasite community was 0.79. The prevalence, mean abundance, and mean intensity of infection (parasite infection parameters) showed a clear tendency per host size. Generally, the observed trend is that parasite infection parameters increase with the host size (host length) of fish; the larger fish harbouring relatively heavy infections (Table 4). Class 3 fish had the highest levels of infection for prevalence and mean abundance across all parasite species. Mean intensity, however, was only highest for three parasite species in the largest fish. The largest host size <)500 to > 700 mm) has the highest prevalence in  (Table 4). There were significant differences in overall prevalence per host size groups (χ2 = 42.95, P > 0.001) and prevalence of all parasite species except A. rhinoceros (χ2 = 4.65, P = 0.09) ( Table 4). Higher mean abundance was observed in higher host size group (largest host size) in Sarcotaces sp.  Table 4). Mean abundance varied significantly in overall mean abundance (χ2 = 51.81, P > 0.001) and all parasite species except A. rhinoceros (χ2 = 4.97, P = 0.08) per host size groups (Table 4). The largest host size has the highest intensity in Sarcotaces sp. (1 ± 0.00), A. rhinoceros (1.71 ± 0.29), and E. hamate (1.21 ± 0.15) when compared to other class groups ( Table 4). The overall mean intensity of infection varied significantly (χ2 = 18.33, P > 0.001) and there was only one parasite species, Prosorhynchus epinepheli among host size groups (χ2 = 6.54, P = 0.038) ( Table 4). The value of the correlation coefficient also indicated a positive correlation between overall prevalence, mean abundance and mean intensity of infection from one side and host size groups from the other side ( Table 4).

Discussion
In the present study, eight metazoan parasite species (6 helminth species and 2 Crustacea), were recorded from one hundred thirty-two E. chlorostigma. The parasite species were one copepod (Sarcotaces sp.), one isopod (A. rhinoceros), two monogeneans (Pseudorhabdosynochus epinepheli and M. epinepheli), three digeneans (Prosorhynchus epinepheli, H. nimia, and E. hamati), and one nematode (C. epinepheli). The recorded species were previously reported by other studies that focused solely on the taxonomy of parasites [1,2,5,21,22,29,35,43]. Digeneans parasite species numerically dominated the parasite community, representing 50.55% and the most diverse group of parasites followed by monogeneans represented 45.05% of the total individual parasites reported. This is in agreement with Villalba-Vasquez et al. [45], Violante-Gonzalez et al. [47], Violante-Gonzalez et al. [48] and Villalba-Vasquez et al. [46]. The overall mean species richness varies significantly by host size; large-sized fish have the highest mean species richness (3.77). Also, fluctuation in species richness per season was found. The highest species richness was recorded in summer (2.58) and the lowest one in winter (0.61). A positive correlation was found between species richness and host size (length). The current study results are consistent with Poulin and Rohde [37] and Villalba-Vasquez et al. [46].
Ectoparasites (monogeneans, copepods, and isopods) and endoparasites (digeneans, cestodes, acanthocephalans, and nematodes) make up the parasite community in marine fish. Ectoparasites are usually transmitted by contact between hosts, but endoparasites are transmitted via trophic pathways [8,28]. The parasite species richness varies substantially between the different hosts of each of these groups [15,37,42,45,46]. Schooling fish are more likely to have abundant ectoparasites than solitary species [26,36] since a transmission stage (e.g., eggs, larvae) is more likely to contact a host when there is a greater density of hosts. This has been recorded in several species of Caligus copepods [36,46].
The current study results indicated that the prevalence of Sarcotaces sp. was higher (8.33%) when compared to that reported by Osman et al. [35] where the infestation rate was 6.74%.
Two monogenean parasite species were identified namely; Pseudorhabdosynochus epinepheli and M. epinepheli. Al-Mathal [2] identified the same monogenean species from E. chlorostigma and reported that the intensity of M. epinepheli from 1-6 per fish where the intensity of Pseudorhabdosynochus epinepheli was over 100 worms per fish.
H. nimia is first described by Linton (1910) from marine and has been reported from numerous marine fishes along the Pacific and Atlantic coasts of the Americas and other areas such as India and Arabian Gulf, H. nimia is found in fishes belonging to at least 9 families [1,3,34]. Al-Mathal [1] reported that H. nimia is one of six digenetic trematodes infecting the hamour fish (E. chlorostigma) in the Arabian Gulf, Saudi Arabia. In the present study, the infection rate of H. nimia was 32.6% (43/132). González et al. [18] reported that the prevalence of H. nimia in Labrisomus philippii, Paralabrax humeralis Acanthistius pictus was 57.5, 10.2, and 7.3% respectively. The intensity of the parasite species was 10.1, 5.4, and 8.6 in Labrisomus philippii, Paralabrax humeralis, Acanthistius pictus respectively. Roumbedakis et al. [41] reported that H. nimia infection rate was 19.49%,the mean abundance was 1.19 ± 4.02, and the mean intensity was 6.13 ± 7.33.
The infection rate of E. hamati was 19.7%. Al-Mathal [1] reported that E. hamati is one of six digenetic trematodes infecting the hamour fish (E. chlorostigma) in the Arabian Gulf, Saudi Arabia. Also, Maghrabi and Gharabawi [27] described E. hamati from stomach of Epinephelus tauvina from the Jeddah coast (Saudi Arabia, Red Sea). Also, this specimen was described from the stomach of Lutjanus carponotatus by Bray et al. [6].
Host body size was one of the factors that determine the structure of the parasite community of E. chlorostigma. There was a clear tendency in infection parameters (prevalence, mean abundance, and mean intensity) per host size. Generally, the observed trend was that parasite parameters increase with the host size (host length) of fish; the larger fish harbouring heavy infection parameters. The value of the correlation coefficient also indicated a positive correlation between overall prevalence, mean abundance, and mean intensity of infection from one side and host size groups from the other side (Table 4). In marine fish, some studies reported that body size on the main predictor of the presence of a particular species, of total abundance, and species richness [26,44,46]. According to Poulin [38], large host size, can enhance parasite colonization because larger individuals eat more food and hence have had more time to accumulate parasites than smaller ones. There were positive correlations between host body size and the abundance of the helminths Sarcotaces sp., Pseudorhabdosynochus epinepheli, M. epinepheli, P. epinepheli, H. nimia, E. hamate, and C. epinepheli. Osman et al. [35] reported that Sarcotaces sp. infest only old and large fishes. The fish size was reported to be correlated with parasite abundance [37].
On the other hand, Villalba-Vasquez et al. [45] showed that small body size was linked with greater structuring of parasite infracommunities Parapsettus panamensis. They also found a negative correlation between host body size and the abundance of the helminths Multitestis inconstans and Anisakis sp., and the copepods Parapetalus sp. and Lernanthropus giganteus. Furthermore, smaller fish had higher abundances of these species. This pattern was attributed to the fact that a stronger infracommunity structure occurs in smaller fish due to the differences in parasite accumulation and infection times, rather than differences in host species.
In this study, we detected seasonal variation dynamics of the parasite community structure and most of the parasite species exhibited significant seasonal variation in infection parameters (i.e., prevalence and mean abundance). Infection prevalence and mean abundance differed significantly per season in overall prevalence and Sarcotaces sp., Pseudorhabdosynochus epinepheli, M. epinepheli, P. epinepheli and H. nimia. This pattern also reported for other parasite species in wild fish populations [20,37,45,46,48]. Previous studies reported a relationship between host abundance and the abundance of digeneans, both were influenced by temperature [37,48]. Poulin and Rohde [37] reported that the overall water temperature was correlated positively with both parasite species richness and abundance [37]. Our results are in agreement with Poulin and Rohde [37]. Moreover, environmental variations, vegetation cover, and seasonal variations relating to the dietary behavior of the host may also influence the seasonal infection patterns of metacercariae. These host fishes, the brown-spotted grouper, (E. chlorostigma) is piscivorous feeding on small fishes and crustaceans (mainly stomatopods and crabs) [19]. The high rainfall was recorded in Al Qunfudhah during April, May, November, December, and January. The high temperatures were recorded in May, June, July, August, and September (mostly summer months) ( Table 1). Maghrabi and Gharabawi [27] noticed that the incidence of infection with digenetic trematodes was seen to be high during spring and autumn seasons, with a slight decrease during summer and winter. They attributed this to the differences in the temperature, extensive feeding of fishes, and the availability of the intermediate hosts of these parasites during such seasons.
Monogenoidean species reflected seasonal changes in parasite populations, the ectoparasites are directly affected by their external environment. The size of monogenoidean population can be significantly affected by seasonal variations in environmental temperature, which directly impacts the reproduction, survival time, and behavior of these ectoparasites, as well as the behavior, density, and infection levels of their hosts. Therefore, some of them prefer a higher water temperature for reproduction, while others prefer a cooler temperature for reproduction [17]. In E. chlorostigma, there was infection by the monogenoideans Pseudorhabdosynochus epinepheli, and M. epinepheli during four seasons, but higher levels occurred during summer season. Therefore, these results indicate that reproduction among these two monogenoideans follow a defined pattern, i.e., with prevalence and abundance throughout the year and specifically with the highest peak of prevalence and abundance during the summer season. Chubb [10] reported seasonal differences between or within different genera of monogenoideans to be related to their direct life cycles. Furthermore, his results demonstrate that monogenodeans have developed their life strategy to take advantage of the dry/rainy season cycle. On the other hand, the temperature t directly influences the reproduction of recorded monogenodeans.
Our results indicated that the prevalence of Sarcotaces sp. was higher in warm seasons, spring and summer with prevalence 21.21 and 12.12% respectively. Our results are consistent with Osman et al. [35] who reported that the prevalence of Sarcotaces sp. was higher in warm seasons, and recorded that prevalence was 17.85% and 3.84% in summer and spring respectively. Cirolanidae isopods (Argathona sp.) are typically marine and usually inhabit the warmer seas [9]. The same trend was recorded by the present study in the prevalence of A. rhinoceros per season.
In contrast, nematode parasite, C. epinepheli, recorded a high prevalence in the rainy season (autumn) and represented a new geographical record. This result is in agreement with Vital et al. [49], and Neves et al. [33], in fish hosts of Astronotus ocellatus and Pygocentrus nattereri Kner, 1858 where they reported that there were higher levels of infection by nematodes during the rainy season (autumn), due to the seasonal dietary composition of these hosts, which includes microcrustaceans that are intermediate hosts of nematodes [11].
In conclusion, the present study is considered the first one for the parasite community of E. chlorostigma. Our results suggest that parasite community structure and species composition may vary per host size and season. Study results also provide relevant "baseline" data for the parasite community of E. chlorostigma and further longterm research is needed to conclude the factors determining the structuring of the parasite community of E. chlorostigma.
Author contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by MMI and TSA. The first draft of the manuscript was written by MMI and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Data availability
The manuscript has no associated data.

Conflict of interest
The authors have no relevant financial or nonfinancial interests to disclose.
Ethical approval No approval of research ethics committees was required to accomplish the goals of this study because experimental work was conducted with an unregulated invertebrate species.
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