New Records of Fish Parasitic Isopods (Crustacea: Isopoda) from the Gulf of Thailand

Simple Summary Parasitic isopods were reported found from marine fishes from many habitat in the world. In Thailand, there is not much study on this parasitic group. This work has compiled all published parasitic isopods documents in Thailand from year 1950 to present include collecting samples from the Gulf of Thailand during the period 2006–2019. New host records were found from four species of parasitic isopods (Cymothoa eremita, Smenispa irregularis, Nerocila sundaica, Norileca triangulata) and two species of parasitic isopods (Argathona macronema, Norileca triangulata) were found first time in the central Indo-Pacific region. Abstract From a total of 4140 marine fishes examined, eight species of parasitic isopods were reported from marine fishes in the Gulf of Thailand. These isopods were identified in two families, Corallanidae (Argathona macronema and Argathona rhinoceros) and Cymothoidae (Cymothoa eremita, Cymothoa elegans, Smenispa irregularis, Nerocila sundaica, Norileca indica and Norileca triangulata). Most of these parasitic isopods were found in the buccal cavity of their fish hosts with one host recorded as follows: C. eremita was found from Nemipterus hexodon, C. elegans was found from Scatophagus argus, N. sundaica was found from Saurida tumbil. The majority of the isopod specimens recorded in this study was S. irregularis, which was found in the buccal cavities of five host fish, Pampus argentius, Alepes melanoptera, Caranx hippos, Parastromateus niger and Terapon jarbua, with a prevalence of 11.67%, 10.43%, 9.78%, 6.10% and 4.21%, respectively. Argathona rhinoceros was found in the nasal cavity and branchial cavity of Epinephelus coioides, whereas A. macronema and N. triangulata were found on the skin of Epinephelus coioides and Seriolina nigrofasciata, respectively. The highest species diversity was found in E. coioides, which harbored two species of parasitic isopods, A. macronema and A. rhinoceros. Cymothoa eremita, C. elegans, S. irregularis and N. triangulata were recorded for the first time in the Gulf of Thailand. The reported discovery of C. eremita, S. irregularis, N. sundaica and N. triangulata in their fish hosts were new recorded hosts. Moreover, A. macronema and N. triangulata were found for the first time in the central Indo-Pacific region.


Introduction
The order Isopoda belongs to the subphylum Crustacea, phylum Arthropoda, with more than 10,300 species found in the deepest oceans to the montane terrestrial habitats [1]. The order Isopoda comprises free-living forms that inhabit various habitats and parasitize mostly fish. Parasitic isopods are typically marine, and usually inhabit the warmer seas [2]. Isopods tend to be small, from 0.5 to 3.0 cm in length, and have a characteristic dorsoventrally flattened body, without a carapace [3]. Most parasitic isopods are ectoparasites, while, for example, Cryptoniscoidea was reported as an endoparasite of crustacean hosts [4]. The large groups of parasitic isopods in marine fishes are members Table 1. Parasitic isopods with their host recorded in Thailand from the literature cited.

Materials and Methods
A total of 4140 marine fish specimens were collected from commercial catches (by trawl nets) and individual fishermen (by fishing rod and net) in Chonburi province ( (Figure 1) during the period 2006-2019. All fish specimens were dead and were immediately transported in a cool box to the laboratory. Host nomenclature and fish taxonomy are according to FishBase [40]. The skin, nasal cavities, mouth and branchial cavities of each fish were examined. Isopods were removed and keep in 70% ethanol. Mouthparts and appendages of isopods were dissected for identification using the key of [13,15,25,32,[41][42][43][44][45][46][47][48][49]. The taxonomy was updated according to the WoRMS catalogue [50].
Isopod specimens were deposited at the Zoological Museum of Kasetsart University (ZMKU). Prevalence and mean intensity were calculated according to Bush et al. [51]. were dissected for identification using the key of [13,15,25,32,[41][42][43][44][45][46][47][48][49]. The taxonomy was updated according to the WoRMS catalogue [50]. Isopod specimens were deposited at the Zoological Museum of Kasetsart University (ZMKU). Prevalence and mean intensity were calculated according to Bush et al. [51].  Most of the parasitic isopods from this survey were usually found in the buccal cavity of the fish host, except for Argathona rhinoceros, Argathona macronema, Norileca indica and Norileca triangulata. Only A. rhinoceros was found in nasal cavities, but in some specimens, they were attached to the branchial cavities of their fish host. A. macronema and N. triangulata were found on the skin of their fish host, while N. indica was found in the branchial cavities. Smenispa irregularis was found in five buccal cavities of five hosts, which can separated in three families: Carangidae (Caranx hippos, Alepes melanoptera, Parastromateus niger), Stromateidae (Pampus argentius) and Teraponidae (Terapon jarbua). Meanwhile, other isopods were found in a single host. Epinephelus coioides harbored two species of isopods, A. macronema and A. rhinoceros. The prevalence of parasitic isopods was highest in P. argentius, followed by A. melanoptera and C. hippos, with a prevalence of 11.67%, 10.43%, and 9.78%, respectively. All of fish in the high prevalence ranking in this study were infected with S. irregularis.

Results and Discussion
All fish samples were usually found to have only one member of each isopod species, except for E. coioides and C. hippos, for which two specimens were found-they had a mean prevalence of 1.10 and 1.11 individuals/fish, respectively ( Table 2).
The details of the isopods found in this survey were described as follows.

Family Corallanidae
Corallanidae are a small family which are free-living or parasitic to aquatic animals, especially fish [12,46]. All two identified isopod species from the family Corallanidae found in this survey belong to genus Argathona. Argathona can be separated from other collanid genera by differences in the mouthpart morphology [12]. Argathona have a maxillule with an exopod terminating in an unguis-like point at the base of which lie one or more recurved hook-like processes, an endopod with a truncate lobe, a mandible with a narrow cutting edge, a large triarticulate palp, and a molar process is present [12]. Twelve species of Argathona are found in tropical and subtropical marine habitats throughout the Indo-Western Pacific at a depth range of 8-267 m. [46]. Fish temporarily hosting genera of Argathona included Epinephelus, Diagramma, Cromileptes, Plectopomus, Variola (Serranidae), Pseudolabras (Labridae), Lutjanus (Lutjanidae), Tetraodon (Tetraodontidae) and Muraena (Muraenidae). This genus was also found as a parasite on turtle, Chelonia [46]. Two species of Argathona, A. macronama amd A. rhinoceros, were found in this survey.

Argathona macronema
(syn. Aega macronema, Alcirona macronema, Argatbona macronema, Corallana macronema) [12]. The specimen of A. macronema has a length of 15.5 mm, a width of 5 mm, and was found on the skin of Epinephelus coioides, although Bruce [12] reported that they occur commonly in the nasal passages of serranids. This specimen is little bit bigger than the specimens from Australia, which have a length of 10-14.4 mm.
Bruce [12] stated that this species is a common, widely spread species. It is distributed throughout the Indo-Western Pacific Ocean, including the Red Sea, Gulf of Aqaba, Gulf of Suez, Java, Celebes, New Guinea, Madagascar, Gilbert Islands, Fiji Islands, Kenya, Australia and New Caledonia [46,53]. Miers [55] reported Corallana macronema, which was the synonym of A. macronema from the Malaysian region. However, Thailand is a newly recorded geographical location for this parasite, and Epinephelus coioides is newly recordedhost.
Argathona rhinoceros is usually found in nasal cavities of Epinephelus coioides, and the specimen's length is 6.5-22.0 mm, a width of 2.5-10.0 mm, except for one sample that was found in a gill chamber (length 22.0 mm, width 10.0 mm). This species may grow to be as large as 26.0 mm, as Bruce [12] reported from Australia. The size of this parasite retreived from nasal cavities varied according to size of the fish host.

Family Cymothoidae
Isopods in this family are ectoparasites on marine, freshwater and brackish water fishes. Most of them occur in shallow water in tropical and subtropical areas [47]. The position of attachment on the host (buccal cavity, gill chamber) is usually genus-or species-specific [47]. The marine species of the order Isopoda are classified into 12 suborders, among which the suborder Cymothoida includes 29 families. Among the families of the suborder Cymothoida, superfamily Cymothooidea, the family Cymothoidae includes 43 genera [56]. Four genera were found in this study-Cymothoa, Smenispa, Nerocila and Norileca.
The genus Cymothoa was found to constitute only six species in the southwestern Indian Ocean. In Australia, it has eleven species, and in the central Indo-Pacific region it has nine species [49]. Cymothoa is mainly characterized by the general body shape, which is strongly vaulted, with widely separated antennae, a cephalon deeply immersed in pereonite 1, pereonite 7 extending past pleonite 1, a wide pleotelson, pleonite 1 as wide as other pleonites, and uropod rami which are shorter than the pleotelson [22,49].
In this survey, two species of Cymothoa were found: C. eremita from Nemipterus hexodon and C. elegans from Scatophagus argus.
C. eremita was found in the buccal cavity of Nemipterus hexodon, with a female length of 21.0 mm and a width of 10.5 mm. In sampling time, many mancas escape from the brood pouch of the mature female and will find a new host. However, many mancas are still attached to the same host. We found them on the outer side of the operculum and also the anterior part of their host fish. Hadfield et al. [49] explained the character of C. eremita, in that they have anterolateral projections which extend to half the length of the cephalon, a truncate anterior margin of the cephalon, a pleon as wide as the pereon, uropods which do not reach the posterior margin of the pleotelson, an ischium on pereopod 7 with a bulbous protrusion and small lateral projections on the posterolateral margins of pereonite 1.

Cymothoa elegans
C. elegans was found in the buccal cavity of Scatophagus argus. The females found in this study had a length of 14.0-15.5 mm and a width of 5.5 mm. C. elegans is characterized by a sublime body that is more than twice as long as it is wide, with nearly parallel sides and very small eyes. The head is sunken into the chest and is wider than it is long, and has fairly short antennae, which are hidden under the head. The fifth segment is the widest. The seventh thorax is the longest and is very broad. The pleon is short and narrow, and the first segment is not hidden. It includes the telson, which is longer and wider than the leon and has posterior rounded corners. The uropods are short.
C. elegans was reportedly found on only two host fishes, Epinephelus fuscoguttatus and Scatophagus argus. The distribution of this parasite was found in Java, Indonesia and the Texas coast [43,[66][67][68]. Therefore, the Gulf of Thailand was a new location for this parasite.

Nerocila sundaica
(syn. Emphylia ctenopbora, Nerocila (Emphylia) sundaica) [12]. Nerocila sundaica was found in the buccal cavity of Saurida tumbil. Females found in this study had a length of 14.0-15.5 mm and a width of 5.5 mm. Nerocila is a large genus of the family Cymothoidae including at least 65 species living attached to the skin or on the fins of fishes [7]. Nerocila usually have pleonites 1 and 2 with ventrolateral processes, and uropods extending beyond the posterior of pleotelson [24]. Bowman [69] explained the character of N. sundaica that have a narrow head. Basal segments of antennae 1 are inflated, and are close to or in contact with one another medially. Distal segments of pereopods 3, 6 and 7 are armed with strong spines on grasping margins. Pereopods 1, 2, 4 and 5 do not have marginal spines, and the dactyls are strongly developed, with that of pereopod 4 being the largest.

Norileca indica
(syn. Livoneca indica, Livoneca ornata, Lironeca indica) [14]. Norileca indica was found in the branchial cavity of Selar crumenophthalmus. Females found in this study had a length of 2.4-2.9 mm and a width of 1.8-2.2 mm. The body of the parasite twists to one side. The characteristics of genus Norileca include pleonites 1 and 2 without ventrolateral processes, and uropods not extending beyond the posterior of pleotelson [14,24]. Pleonite 5 and pleonite 1 are subequal, while pleonite 5 is narrower than pleonite 1 in N. triangulate. Uropods are two-thirds the length of pleotelson [14,24].
The special characterisitcs of N. triangulata include pleonite 5 being manifestly narrower than pleonite 1, and maxilliped palp article 2 being about as long as article 3 [14]. In our study, we found only one female specimen of N. triangulata attached to the skin of Seriolina nigrofasciata. This female isopod has a length of 15.0 mm and a width of 6.0 mm.
N. triangulata was reported found in Rastrelliger kanagurta, Sardinella gibbosa, Parexocoetus brachypterus. This species is distributed aorung Tanimdao Island, the Philippines, Australia and the southeast coast of India [5,14,17,24,81]. The Gulf of Thailand is a new geographical record for this parasite and Epinephelus coioides is a newly recorded host.

Smenispa irregularis
(syn. Cymothoa irregularis, Cymothoa paradoxa, Enispa irregularis) [14,82]. S. irregularis was found in five species of fish, Caranx hippos, Caranx malam, Pampus argentius, Parastromateus niger and Terapon jarbua, in this study. Most of them were found in the buccal cavity of their fish host. Females found in this study had a length of 16.0-22.5 mm and a width of 8.0-12.0 mm. Martin et al. [82] stated that the diagnostic characteristics of the genus are the strongly vaulted body, a cephalon embedded in pereonite 1, antennula shorter than the antenna, bases set wide apart, the pereon and pleon being co-linear with the sub-parallel lateral margins, pereopods lacking carina on the bases, and the endopods of pleopods 3-5 having large folds. The size of the ovigerous female in our study seems to be smaller when compared with Martin et al. [82] (20-24 mm).
In this survey, we did not find Livoneca sp., although this genus has been recorded in Thailand for a long time. Smit et al. [85] stated that of the approximately 60 species that had been placed in Livoneca up to 1990, most were relocated to Elthusa and Ichthyoxenus, with only three species now remaining in the genus [14]. From our results, two species of parasitic isopods previously had their genus as Livoneca but changed to another genus-Argathona rhinoceros (syn. Livoneca nasicornis) and Norileca triangulata (syn. Livoneca triangulata).
The size of the parasitic isopods on their host in this study in some specimens was not in same range as the literature cited, such as A. macronema and S. irregularis. This can explained by the study by Leonardos and Trilles [86], who studied host-parasite relationships between parasitic isopod Mothocya epimerica on sand smelt Atherina boyeri and found that parasite size increased with host size. The prevalence of isopod infections from this study was only the guideline for advance research because we randomised the marine fish specimens discontinuously, because the prevalence of parasites varied according to the month, and the sex and size of hosts [87].
Low host specificity for parasitic isopod to their fish hosts was also confirmed. The parasitic isopods in each fish species from the literature cited seem to be different from this study. For example, Caranx hippos from the Caribbean area was found to harbour Cymothoa oestrum inside the mouth [47]. Parastromateus niger was found to harbour Cymothoa eremita [57]. Pampus argenteus was found to harbour Cymothoa eremita [23,49]. For only two hosts, A. rhinoceros was found in the nasal cavities of Epinephelus coioides, which is the same as the report in New Calidonia [53], and Scatophagus argus hosted the parasitic isopod Cymothoa elegans, which is the same as the report in Indonesia [67].
The effects of parasitic isopods on their fish hosts have been explained in many records. However, in most cases, the mean intensity is usually only one isopod per infected fish. In this case, infection in low prevalence in a fish population does not seem to affect the economy. However, in some cases, they can change fish behavior, anatomy, or morphology [88]. The swimming capacity of the fish was also found to be affected by parasitic isopods [89]. For instance, Cymothoa exigua sucks so much blood from its host fish's tongue that the tongue atrophies and is destroyed and the isopod remains attached to the remaining tongue stub and is used by the host as a replacement tongue for food manipulation [90]. Although many parasitic isopods feed on fish blood, there are some exceptions, such as Argathona macronema, which feeds on fish mucus, not blood [8]. The low mean intensity with low prevalence seems to not be a danger for marine aquaculture, but the isopod population can increase their number rapidly if this habitat has a high population of fish hosts, such as a fish culture system. They affect fish growth and production, causing economic losses.
Lester [2] explained the life cycle of an isopod of a gravid female which releases eggs into a brood pouch. The eggs embryonate, hatch and undergo two or more moults to form manca or pullus II stage. In this study, some isopod specimens of S. irregularis were found as many mancae from the brood pouch of a gravid female ( Figure 2). The post-mancal juvenile stages (sometimes referred to as the aegathoid stage) have only six pairs of legs, large compound eyes, and highly setose pleopods for active swimming. The juveniles will attach themselves to a convenient fish host and eventually attach to the preferred host-species [46]. After a short free-swimming period, they are parasitic and need to find a fish to take their first meal within one to two days or they will die. From this survey, we found that a specimen of Nemipterus hexodon was attached with many mancae (Figure 2A,B), and this fish specimen had the isopod Cymothoa eremita in its mouth (Figure 2A,B). This finding seems to correspond with that of Lester [2], as in the tongue biters group such as Ceratothoa spp., the mancae move to the preferred site and remain attached to the fish.
In the cymothoids group, they have a short free-living planktonic phase and then attach to fish hosts. Heavy infestations of parasitic juveniles have the potential to kill small fingerlings. So, in a culture system, prevention may done by eradicate immature forms of isopod when they are planktonic [11].
Other than the data regarding the abundance of parasitic isopods in Thailand as seen in the literature review above, the areas which connect to the Gulf of Thailand that have parasitic isopod reports, such as the Malaysian region [55] and the South China Sea [15], were compared with the results of the parasitic survey in this study to discuss the new geographical record of isopod parasites in this region.
Smit et al. [85] mapped the distribution of the marine cymothoids using Spalding et al. [91], and they found that the highest diversity resides within the tropical regions of the central Indo-Pacific, with 79 marine Cymothoidae isopods. Comparing this with our data, all parasitic isopods that we found in the survey were already recored in the central Indo-Pacific region, except for Norileca triangulata, which was distributed around Tanimdao Island, the Philippines, Australia and southeast coast of India.
Argathona macronema, Cymothoa eremita, Cymothoa elegans and Norileca triangulata were newly recorded in Thailand. According to Spalding et al. [91], Norileca triangulata was found for the first time in the central Indo-Pacific region.