Parasite Fauna of the White-Streaked Grouper (Epinephelus ongus) from the Thousand Islands, Java, Indonesia

Fish parasites can cause diseases in humans and lead to commercial losses in fisheries and aquaculture. The objectives of this study were to analyze E. ongus’s parasite fauna regarding food safety and parasite transmission risk between Epinephelus species and test whether E.ongus populations can be distinguished by their parasite community. We studied the metazoan parasite fauna of 30 white-streaked groupers Epinephelus ongus from the Thousand Islands, Java Sea, Indonesia, and compared the parasite community with specimens from Karimunjawa archipelago, Java Sea, from a former study. We used common fish parasitological methods for fish examination and parasite calculations. We found 12 metazoan parasite species, establishing five new host and five new locality records, increasing the known parasite fauna of E. ongus by 21%. No anisakid worms infected E. ongus. All but one (trematode Gyliauchen cf. nahaensis) species have been previously reported from Epinephelus. Parasite abundance of E. ongus differed significantly between the two regions. Due to a certain degree of host specificity to groupers, there is potential risk of parasite transmission from E. ongus into groupers in mariculture or surrounding fishes, which increases (sea) food security related health risks from zoonotic parasites and calls for better monitoring and management plans for E. ongus. The regional separation of the Thousand Islands and Karimunjawa with different food availability and fish ecology causes different parasite abundances, distinguishing two separate E. ongus populations by their parasite fauna.


Introduction
With more than 17, 000 islands and 80, 791 km coastline, Indonesia is the biggest archipelago in the world [1]. Consequently, fisheries and aquaculture play an important role for the national economy [2]. In 2016, behind China, Indonesia ranked second in marine capture production with over 6 million tons [3].
Grouper (Serranidae: Epinephelinae) fisheries is of high economic value for food supply and the live reef fish trade in Indonesia, where they are mainly exported to China, Japan and Singapore [4]. Serranids also contribute to stable livelihoods in developing countries and are important for reef ecosystems as (top) predators [5,6]. A rising demand for serranids increases fishing effort which drives their risk of extinction [7]. It is estimated that 19 grouper species are threatened and especially the live reef food fish trade remains largely unmanaged [7,8]. Further knowledge on the ecology and life history of these commercially important species is needed to predict future implications of increasingly high fishing pressure.
Parasites are one of the most successful life forms on Earth [9]. They can impair their fish hosts and also human consumers when raw or undercooked fish is consumed, causing zoonosis as a disease transmitted from animals to humans [10]. The most well-known marine waterborne disease is anisakiasis [11] caused by nematodes of the family Ansiakidae. Monitoring parasites of food fishes is important to continuously assess sea food health risks. Previous studies have suggested a low risk of anisakiasis in Indonesian waters [12][13][14], but climate change may shift marine boundaries causing different food web compositions in the future [15,16] and there are also other marine helminths with zoonotic potential, e.g., Hysterothylacium or Pseudoterranova [13]. Diverse ecosystems such as Indonesia have huge potential for parasitological research [14]. Parasites provide new insights into fish diet, habitat range and trophic position [17,18] and inform about host ecology due to different infection pathways or accumulation of pollutants [19,20]. Consequently, commercially important and often aquaculture species such as the groupers Epinephelus coioides and E. fuscoguttatus have been subject to a number of disease, parasite and environmental studies [19][20][21][22][23][24][25][26].
Today, fishing pressure is shifting toward smaller species such as E. ongus, which is now targeted as food fish in some regions, e.g., Japan [27,28]. Demand for E. ongus has increased steadily [28,29], also based on landings of E. ongus in Indonesia (Karimunjawa) [28,30]. With only seven previous studies on the parasite fauna of E. ongus in South-East Asia and Australia [28,[31][32][33][34][35][36], we herewith sample this fish species from the Thousand Islands archipelago in close proximity to the heavily polluted Jakarta Bay. Our objectives for this study are to assess the transmission risk of parasites from E. ongus to cogeners and vice versa and to determine parasite-borne health risks of E. ongus to consumers in terms of food safety and to mariculture fish. Additionally, we aim to test whether E.ongus populations can be distinguished by their parasite community. For that we compare our data to a former study where the parasite fauna of white-streaked groupers (similar in size and weight) was analyzed in Karimunjawa, an archipelago off the coast of Java, Indonesia, in 2013 by Neubert et al. [28] and discuss infection patterns, parasite biodiversity and fish ecology data.

Sample Collection
From April to June 2018, a total of 30 spearfished Epinephelus ongus were obtained from fishermen in the Thousand Islands archipelago (5°48′07.7′′S 106°30′38.4′′E), north coast of West Java, Indonesia ( Fig. 1 individually frozen in plastic bags, usually for 2-5 days, until examination inside the laboratory. Fish were defrosted in lukewarm water and morphometric data were taken (total length, standard length, total weight and gutted weight). Fluids from inside the plastic bags were examined individually under the stereo microscope (Zeiss, Stemi DV4).

Parasitological Examination
The body cavity was opened and internal organs were removed for subsequent endoparasite examination under the stereo microscope. Fish fillets were examined with translucent light under the stereo microscope. All detected parasites were cleaned from host tissue and collected in 70% ethanol. Selected specimens were transferred into glycerin on microscopic slides following Riemann [37]. Parasites were identified according to keys and original descriptions. After identification, the parasite specimens were deposited at LIPI, Science Centre, Jakarta-Bogor, Indonesia (Accession numbers: 45, 173-181, 542, 1482-1484).

Processing of Parasitological Data
Parasitological parameters prevalence (P), intensity (I), mean intensity (mI) and mean abundance (mA) followed Bush et al. [38]. The Shannon-Wiener diversity index [39] and Pielou index of evenness [40] were used to estimate the diversity of the metazoan parasite fauna, as well as the Berger-Parker index of dominance [41]. These indices were calculated for ecto-and endoparasites pooled, and endoparasites exclusively [22,25,26]. Ecto-to endoparasite ratio was calculated following Rückert et al. [42].

Statistical Analyses
All data, including the raw data by Neubert et al. [28], were analyzed using PRIMER software (version 7.0) to compare the parasite fauna of E. ongus sampled in this study in the Thousand Islands and sampled by Neubert et al. [28] in Karimunjawa. A total of 65 fish were compared and a similarity matrix was calculated by applying a Bray-Curtis similarity measure. The data for the Bray-Curtis index were transformed (fourth root = √√) prior to analysis. Fish without parasites were omitted from the analysis. A multivariant scaling plot (MDS) was used to display the relation between samples based on similarity matrices. The analysis of similarities (ANOSIM) was used to calculate the level of significance between both data sets. The differences between or within the data groups were given by the R value that ranged from − 1 to 1. An R value of 0 indicated no differences. A value of − 1 meant that the identical data point was located outside of the group, whereas with an R value of + 1 they were located within the group. The significance level was stated to p < 0.05. To identify which parasite taxa were responsible for the differences between the examined groups, SIMPER was applied. Additionally, a one-way ANOVA with Student's T test (SigmaPlot Version 11.0) was conducted to determine statistical differences in size of the fish between Karimunjawa [28] and the Thousand Islands. Ectoparasites represented the most predominant parasite group in E. ongus. The isopods Alcirona sp. and Gnathiidae indet. had the highest prevalences of 50.0% and 43.0%. Albeit a lower prevalence (23.3%), the Monogenea Pseudorhabdosynochus sp. had the highest mean abundance (5.4) and mean intensity (23.0) ( Table 2). Fish which served as a host for this monogenean were highly infested (up to 83 worms per fish, Table 2).

Parasite Community
With regard to endoparasites, the nematode Hysterothylacium sp. was the most common taxon with a prevalence 1 3 of 20%, followed by the digeneans Gyliauchen cf. nahaensis and Cainocreadium epinepheli with prevalences of 16% and 10%, respectively. The digenean Ca. epinepheli was the most abundant taxon in the present study (see Table 1). Gyliauchen cf. nahaensis (Table 3; Fig. 2), usually host specific to herbivorous fish (43), is herewith reported for the first time from an Epinephelus species. Two taxa of Philometra were isolated from E. ongus, Philometra epinepheli (from the operculum) and Philometra sp. 1 (subcutaneous inside the mouth). The latter might represent a new species. It differed from already recorded Philometra species from groupers in Indonesia with two liplike structures at the mouth, a distinctively shaped swollen esophagus, a different body length and site of infection. So far, only three Philometra species have been recorded from E. ongus: P. epinepheli, P. cf. lateolabracis and P. ocularis [28]. Philometra sp. 1 differs from P. epinepheli by larger gravid females (280 mm vs. 70-190 mm) and a different infection site (subcutaneous at mouth vs. subcutaneous at operculum) [22,28,44]. Philometra sp. 1 differs from P. lateolabracis in morphology (shorter uterus, ovaries do not reach up to the nerve ring) and in infection site (subcutaneous at mouth vs. gonads) [28,[45][46][47]. Philometra sp.
1 differs from P. ocularis by the lack of four large fleshy anterior papillae, a round mouth and in infection site (subcutaneous at mouth vs. eye cavity) [28,48]. The herewith recorded Philometra sp. 1 shares most morphological characters (ovaries not reaching up to nerve ring, similar body length of 240 mm, comparable swollen esophagus) with Philometra sp. found by Kleinertz [22] in E. coioides and E. areolatus, and by Rückert [21] in E. coioides and E. fuscoguttatus. In Kleinertz [22] Philometra sp. 2 infested the gonads, gills, gill cavity, mesentery, mouth cavity, nose cavity and opercula, whereas in Rückert [21] Philometra sp. 3infested the gills, gill cavity, mouth cavity and nose cavity. In both previous studies, Philometra sp. 2 and 3 were not found subcutaneous at the mouth.
The Berger-Parker index of dominance reached 0.62 when considering the total parasite fauna, but decreased to 0.3 for endoparasites only. The total parasite fauna had a high Shannon-Wiener index of diversity of 1.41. For only endoparasites, the Shannon-Wiener index decreased to 0.54. This trend goes along with a Pielou index of evenness, which was 0.57 including the ectoparasites and decreased to 0.28 for only endoparasites.

Comparison of Epinephelus ongus from the Thousand Islands and Karimunjawa
A combination of both data sets resulted in a total of 23 parasite taxa (17 in Karimunjawa and 12 in Thousand Islands) for E. ongus from Indonesia, 6 of them recorded for both locations ( Table 2). The MDS plot based on parasite abundances shows that the infection patterns at both localities were distinctively grouped (999 permutations) (Fig. 3). The data support a significant difference between both sampling sites (ANOSIM: R = 0.527, p < 0.05), although some samples of Karimunjawa and the Thousand Islands overlap. The major taxa contributing to the separation of the two localities were the gill monogeneans Pseudorhabdosynochus quadratus and the crustaceans Caligus sp. found in Karimunjawa (SIMPER Analysis).  [32,36], three reporting one parasite species [31,33,35] and two from Indonesia with 17 and eight taxa, respectively [28,34]. Though Stolz [34] focused on ectoparasites only, the latter findings suggest that the parasite load in white-streaked groupers from Indonesia underlies considerable variability in the number of isolated parasite species. Ectoparasites predominated the parasite fauna of E. ongus and the most abundant and prevalent one was the monogenean Pseudorhabdosynochus sp. Parasites of this genus are usually host specific and very common in groupers [49] with P. quadratus having been described from E. ongus from New Caledonia in the past [33]. The high infestation of these diplectanid monogeneans coincides with findings of other studies [28,33,34]. Another generalist was the ectoparasite Alcirona sp. which has already been recorded from, e.g., E. areolatus, E. coioides, E. fuscoguttatus, E. quoyanus and Variola albimarginata. Caligus spp. also infected E. areolatus, E. coioides, E. corallicola, E. quoyanus and V. albimarginata in Indonesia [28,34].
Most of the isolated endoparasites have already been found in other serranids in Indonesian waters, e.g., Ca. epinepheli, Philometra spp., Hysterothylacium sp. or Prosorhynchus sp. [19-22, 25, 26, 34]. With three Philometra species so far isolated from E. ongus, this genus is also present in E. areolatus, E. coioides and E. fuscoguttatus and commonly infects groupers [28,50]. In addition, the record of Philometra sp. that is suggested to represent a new species and awaits description adds to this biodiversity in groupers [21,22,44]. New specimen collections from E.ongus are required to confirm its real identity.
Here, similar to earlier studies of E. ongus, we did not find any anisakid nematodes and only Neubert et al. [28] reported Anisakis typica at a low prevalence of 2.9%. No nematodes infected the musculature, where the possible parasite transmission of zoonotic helminths to the consumer takes place. However, post-mortem migration of helminth larvae into the fillet can occur and Hysterothylacium sp. has caused zoonoses in the past, even though it originally infected the intestinal tract of host fishes [51]. With Hysterothylacium sp. larvae present in the studied white-streaked groupers, parasite-borne health risks should not be underestimated, but further studies are needed to better understandand evaluate the infection patterns of zoonotic parasites of E. ongus, which is a species of rising commercial interest.
With many parasite species having high host specificity to groupers, but a low host specifictiy within epinephelids, there is a risk of parasite transmission between E. ongus and its cogeners. This is problematic in and around mariculture facilities, especially under consideration of increasing mariculture activities throughout the region. Consequently, the recorded parasite taxa in this non-cultivated grouper species might still lead to future economic losses [52,53] in maricultured fish and also impacting wild fish stocks.

Ecological Parameters
The parasite fauna was predominated by the ectoparasite Pseudorhabdosynochus sp., implied by a Berger-Parker index of 0.62 for the total parasite fauna and a lower index of 0.3 for only endoparasites. Evenness and diversity indices were higher in the total parasite fauna compared with only endoparasites. Consequently, the taxa are more evenly distributed even when ectoparasites (including the dominant Pseudorhabdosynochus sp.) are considered. This can be explained from a relatively low amount of isolated endoparasites in E. ongus resulting in a very low Shannon-Wiener index of 0.54, compared to other grouper species (E. coioides up to 1.84, 20, E. fuscoguttatus). Both larger sized species have been sampled in more detail with up to 420 specimens per study e.g., [21,22].
Differences in diet and habitat most likely result in the observed less diverse parasite fauna of E. ongus compared to other species [20,24,26]. Though diet and behavior of adult E. coioides, E. fuscoguttatus and E. ongus are approximately the same [54], the parasite composition is different [23,24,28]. Sampled specimens of E. coioides and E. fuscoguttatus often were juveniles, whereas our E. ongus were adults. Juvenile E. coioides and E. fuscoguttatus prefer more shallow waters and live over muddy bottoms, fine sediments or in seagrass beds [54,55], while adult E. ongus live in deeper coral reef habitats and rocky bottoms, often in caves in up to 60 m depth [27,55]. The different ecology, size and age of juvenile and adult E. coioides and E. fuscoguttatus favor a broad accumulation of parasites, which leads to a diverse and species-rich parasite fauna. Until 2016, 57 different parasite species have been recorded in, e.g., E. coioides [22,26], and even from highly polluted regions around Jakarta Bay up to 31 parasite species were found [20]. This differs from E. ongus, with only 30 recorded parasite species so far. Neubert et al. [28] isolated only 17 species in E. ongus in Karimunjawa, a region where the biodiversity is believed to be particularly high [28,29], suggesting that the observed differences in parasite diversity are real and not caused by biased sampling.
An interesting finding was the digenean Gyliauchen cf. nahaensis in E. ongus (Gyliauchenidae, Fig. 2; Table 2). This genus has been recorded in Indonesia only three times, in Sulawesi and Bali [14,56] in herbivorous reef fish. Gyliauchenid trematodes infect the intestine of their hosts in the Indo-West Pacific [57] and have almost exclusively been recorded from fishes from the herbivorous families Acanthuidae, Chaetodontidae, Pomacanthidae, Scaridae, Siganidae and Zanclidae [43,58]. However, Srivastava [59] described the species G. ozakii from the intestine of the carnivorous predator Harpodon nehereus in the Arabian Sea [60]. Lopez [61] isolated Gyliauchen sp. from the carnivorous grouper Plectropomus leopardus in the Philippines. Here, the finding of G. cf. nahaensis in the gills of carnivorous E. ongus likely depicts an accidental infection, because this genus is very host specific and highly adapted to herbivorous fish [43]. Nevertheless, G. cf. nahaensis is a rare parasite in Indonesian waters and our findings reflect parasitogical research potential in this region.

Regional Differences
The herewith analyzed fish specimens from the two archipelagos were similar in size and weight ( Table 2). Size is an important factor for comparability because larger fish often have more endoparasites [26]. The fish from the two locations differed in sex ratio ( Table 2, more males in Karimunjawa, more females in the Thousand Islands), but there is no consistency in the literature how or if sex affects parasite abundance in groupers. Samples were obtained 5 years apart: Karimunjawa in 2013 [28] and Thousand Islands in 2018. While pollution and anthropogenic effects can have an impact on the fish parasite fauna of groupers [20], previous studies in Indonesia have shown that seasonal change and natural inter-annual variability seem to have low impact on the metazoan fish parasite fauna [23,26]. Therefore, we conclude that the samples were comparable and we suggest that the significant differences in the parasite abundances of E. ongus seen in the MDS plot (Fig. 3) are mainly caused by different biotic and abiotic conditions at both sites and are not biased by annual change or host factors.
While the MDS plot shows a clear separation of samples, some fish from Karimunjawa and the Thousand Islands are grouped together. Most likely this is because these samples harbored identical parasite species, as the locations share six common parasite species. This minor overlap in parasite abundance is not very surprising because we sampled the same species in a quite similar habitat-but it is all the more interesting to observe a significant difference in infection patterns in white-streaked groupers based on the sample location. The ectoparasites Pseudorhabdosynochus quadratus and Caligus sp. from Karimunjawa were the main factors for the discrepancies. Some gill parasites are thought to accumulate over time, depending on the age of the fish [28]. However, no significant differences in the total fish length between both sites could be detected (Student's T test: p = 0.719). Despite high monogenean infestation, Karimunjawa is considered a healthy marine ecosystem due to high endoparasite diversity, high diversity indices and low ecto-to endoparasite ratios [28,42]. While both localities are important spawning and nursery grounds for groupers [62,63], Karimunjawa has established management plans, fishing guidelines and protected zones for grouper [29]. Grouper abundance might be higher there which might result in higher ectoparasite transmission and lead to higher ectoparasite infestation in this region.

Conclusion
The present study suggests that the parasite fauna of the small-sized white-streaked grouper has less species diversity compared to its larger sized cogeners. Its parasite community differed between the Thousand Islands and Karimujawa archipelagos shown in a comparison between samples from 2013 by Neubert et al. [28] and 2018. This is due to the differences in habitats and regional differences, leading to different ecological needs of the fishes which implies that the parasite composition of E. ongus can indicate different populations at the two sites. Fishing efforts on E. ongus is steadily increasing and threatens their occurrence throughout the Indonesian archipelago in recent years. This study suggests that the fish parasite community can indicate different origin of groupers and possibly distinct populations, supporting the development of better monitoring and management plans for this species in future. Finding numerous new host and locality records and parasites with a certain affinity to groupers, we suggest potential risks of parasite transmission within different grouper species, for example those cultivated in mariculture facilities or free living groupers. While this poses food safety concerns to consumers of fisheries and aquaculture products in Indonesia, increasing fishing efforts on E. ongus call for more research on this grouper species and better monitoring and management plans are needed.
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