Parasitic copepods (Crustacea, Hexanauplia) on fishes from the lagoon flats of Palmyra Atoll, Central Pacific

Abstract We surveyed copepods parasitic on the fishes at Palmyra, a remote atoll in the Central Indo-Pacific faunal region. In total, we collected 849 individual fish, representing 44 species, from the intertidal lagoon flats at Palmyra and recovered 17 parasitic copepod species. The parasitic copepods were: Orbitacolaxwilliamsi on Mulloidichthysflavolineatus; Anuretesserratus on Acanthurusxanthopterus; Caligusconfusus on Carangoidesferdau, Carangoidesorthogrammus, Caranxignobilis, Caranxmelampygus, and Caranxpapuensis; Caliguskapuhili on Chaetodonauriga and Chaetodonlunula; Caliguslaticaudus on Rhinecanthusaculeatus, Pseudobalistesflavimarginatus, M.flavolineatus, Upeneustaeniopterus, Chrysipteraglauca, and Epinephalusmerra; Caligusmutabilis on Lutjanusfulvus and Lutjanusmonostigma; Caligusrandalli on C.ignobilis; Caligus sp. on L.fulvus; Caritusserratus on Chanoschanos; Lepeophtheiruslewisi on A.xanthopterus; Lepeophtheirusuluus on C.ignobilis; Dissonussimilis on Arothronhispidus; Nemesis sp. on Carcharhinusmelanopterus; Hatschekialongiabdominalis on A.hispidus; Hatschekiabicaudata on Chaetodonauriga and Chaetodonlunula; Kroyerialongicauda on C.melanopterus and Lernanthropus sp. on Kyphosuscinerascens. All copepod species reported here have been previously reported from the Indo-Pacific but represent new geographical records for Palmyra, demonstrating large-scale parasite dispersion strategies.


Introduction
Although there have been several surveys of copepods parasitic on Indo-Pacific fishes, including the Great Barrier Reef (Australia), New Caledonia, New Guinea, India, Taiwan and the Hawaiian Islands (Yamaguti 1963, Kabata 1966, Lewis 1968, Pillai 1968, 1985, Cressey and Boyle 1973, Ho and Dojiri 1977, Deets and Dojiri 1990, Ogawa 1991, Ho and Lin 2004, Boxshall and Justine 2005, Tang and Kalman 2005, Palm and Bray 2014, the East Indo-Pacific has received little sampling effort. Lafferty et al. (2008) compared parasite communities, including parasitic copepods, at two coral atolls in the Line Islands chain of the central Pacific (Kiritimati Island and Palmyra Atoll). However, their analysis was limited to broad patterns of richness and abundance of morphospecies, conservatively grouped into broad taxonomic categories. Palm and Bray (2014) listed parasites from Hawaiian fishes, reporting 64 copepod species (13 families) from 298 identified fish species.
Palmyra Atoll is one of the northern Line Islands located in the Indo-Pacific (IP) marine ecoregion (Spalding et al. 2007), 1680 km SSW of Hawaii. It is presently a marine protected area and has not supported regular human settlement since World War II. Palmyra Atoll has a relatively long history with little to no exploitation . All fishing has been prohibited at Palmyra since it became a US National Wildlife Refuge in 2000 (before that, its remoteness kept fishing pressure low).
As part of a larger project assembling food webs at Palmyra Atoll, we have been cataloging the parasites found in the system. This paper is a companion to two others examining different fish parasite taxa (Vidal-Martínez et al. 2012, 2017. We recovered a considerable number of parasitic copepods from 44 fish species. As such, our tabulation adds to the few published species descriptions or host records from the Central Indo-Pacific region (Cressey and Boyle 1973, 1979, Cressey 1977, Ho and Lin 2004, Palm and Bray 2014, with an emphasis on describing diversity of the copepod supracommunity (Bush et al. 1997) at this site. The goal of this study is two-fold. First, we list the copepod species recovered, and note, for each, taxonomic issues and report their prevalence, mean intensity and host species. Second, we then discuss this diversity survey with respect to previously published records for the region.

Material and methods
We collected fish by seine, spear, and hook and line from the intertidal sand flats bordering the lagoon of Palmyra Atoll between October 2009 and July 2012. To avoid loss or mixing of parasites among fishes, immediately after capture, we placed fish in individual plastic bags with lagoon water and transported them to the laboratory facility of the Palmyra Atoll Research Consortium (PARC). We examined only freshly killed fish (and the bag water). Observations were under a stereomicroscope. Skin and fins of each host were carefully examined. The gill arches were removed and examined under a stereomicroscope. The copepods obtained were counted, preliminarily identified, fixed in 95 % EtOH, labelled and stored in vials for later evaluation. Then, in the Laboratory of Aquatic Pathology of CINVESTAV-Mérida, specimens were mounted and cleared with lactophenol to identify species based on morphology using an Olympus BX-53 microscope (Olympus Corporation, Shinjuku, Tokyo, Japan). Prevalence and mean intensity concepts were applied following Bush et al. (1997). Synonyms for each host species and copepod species were obtained from FishBase (Froese and Pauly 2018) and World of Copepods (Walter and Boxshall 2018), respectively. Voucher specimens were deposited in the United States National Parasite Collection, Washington, DC (USNPC), and the Helminthological Collection of the Laboratory of Parasitology, at the Centre for Research and Advanced Studies, National Polytechnic Institute, Mérida, Yucatán, México (CHCM).

Copepods of fishes from Palmyra lagoon flats
During this study, 849 individual fish from 44 species were collected. Fourteen of the 44 fish species examined were parasitized by at least one parasitic copepod species. Caranx ignobilis (Forsskål) was host to three copepod species, the most of any fish. Acanthurus xanthop terus Valenciennes, Carcharhinus melanopterus (Quoy & Gaimard), Chaetodon auriga Forsskål, Chaetodon lunula (Lacépède), Lutjanus fulvus (Forster), Mulloidichthys flavolineatus (Lacépède) and Arothron hispidus (Linnaeus) served as host for two copepod species. All other infected species hosted a single copepod species. Thirty fish species were found free of any copepod parasite (Table  1). Ten of the 17 copepod species recovered belong to the Caligidae family (Table 2).  Other host and localities. Scolopsis taenioptera (as S. dubiosus) from Okinawa, Japan (Cressey and Cressey 1989). Coris batuensis (Bleeker) (Labridae) from Lizard Island, Australia (Muñoz and Cribb 2006). Thamnaconus degeni (Regan) (Monacanthidae) from South Australia (Hayward et al. 2011 O. leptoscari, O. uniunquis, and O. williamsi). This second group is based on the second endopodal segment of leg 2 either no inner seta or having 1 inner seta. Particularly, O. williamsi lacks seta on the second endopodal segment of leg 2, as seen in our specimen and the original description provided by Cressey and Cressey (1989). However, Venmathi-Maran et al. (2014) pointed out that O. williamsi carries 1 inner seta in that segment, but this is likely inaccurate. Orbitacolax williamsi has been found on western Pacific fishes from four families, suggesting that this parasite may have a low host specificity.

Remarks. The genus
Caligus contains approximately 250 species. According to Ho and Lin (2004), before the establishment of C. confusus, specimens of this species were confused with Caligus productus (as Caligus alalongae) Dana, 1852 and Caligus constrictus Heller, 1865. However, these authors pointed out nine characteristics known only for C. confusus. The morphology of our specimens (♂ and ♀) fits with the description of Ho and Lin (2004). Additionally, based on the examination of the present material and also that from previous surveys in the Eastern Pacific (Morales-Serna et al. 2014, 2015, we suggest that the shape of the first segment of the antenna and sternal furca may be useful in identifying C. confusus. Clearly, C. confusus has high affinity for carangid fish; nonetheless, this parasite can also be found on fish from different families. To date, it is distributed in tropical waters of the Eastern Pacific and Indo-Pacific, with no records for the Atlantic Ocean. Lewis, 1967 Type host. Chaetodon miliaris Quoy & Gaimard (Chaetodontidae).
Remarks. Ho and Lin (2004) indicated that the female of C. laticaudus may be identified by a combination of five characteristics (the corpus of the maxilliped with a large, conical protrusion in the myxal region; the terminal elements on last segment of exopod of leg 1 lack accessory processes; outermost element 1 of the four terminal elements of leg 1 exopod about one third of the length of other three elements which are subequal in length; formula of the 3-segmented exopod of leg 4 as I-0; I-0; III; and the terminal three spines on leg 4 subequal in length). Our results support the view that C. laticaudus infects fishes only from the Indo-West Pacific.
Other host and localities. Centropristis striata (as Centropristes striatus) (Serranidae) from North American waters (Wilson 1905 Remarks. Wilson (1905) observed that the genital complex of C. mutabilis varies according to the age of the individuals as well as the developmental stage of the eggs. Also, this author described C. mutabilis as having a short, 2-segmented abdomen. Later, Cressey and Cressey (1980) redescribed this species based on material collected from scombrid fish. These authors noted an incomplete 2-segmented abdomen and at least two other differences from the type specimens; however, such differences were not considered sufficient to propose a new species. Recently, Morales-Serna et al. (2014, 2015 reported C. mutabilis from different host species in the Eastern Pacific, but a molecular analysis revealed relatively high intraspecific genetic divergence among the C. mutabilis isolates. Our specimens share the morphological characteristics described by Cressey and Cressey (1980). Lewis, 1964 Type host. Acanthurus triostegus (Linnaeus) (Acanthuridae).
Remarks. Lewis (1964a) observed that Caligus randalli is morphologically close to C. constrictus Heller, 1865. According to this author, one of the main differences between both species is the length of the urosome. The urosome of C. randalli is one and a half times the length of the urosome of C. constrictus. In the present study, we noted that C. randalli resembles Caligus aesopus Wilson, 1921. However, the urosome in C. aesopus is shorter than in C. randalli. Hayes et al. (2012) included C. aesopus and another nine species of Caligus (C. chorinemy Kroyer, 1863, C. tenax Heller, 1865, C. spinosurculus Pearse, 1951, C. germoi Pearse, 1951, C. rectus Pearse, 1952, C. confusus, C. cordyla Pillai, 1963, C. zylanica Hameed & Pillai, 1986and C. equulae Ho & Lin, 2003 within a cluster of caligid species sharing the following characteristics in the female: bifid postantennal process; bifid posterior process on the maxillule; heavily ornamented apron of the third leg; an inner rosette of large spinules and prominent rib-like structure with a bifid apex, arising near the border with the intercoxal sclerite of leg 3: a massive and strongly incurved spine on the first exopodal segment of leg 3; and a 3-segmented exopod on leg 4 armed with I,I,III spines. Caligus randalli also shares these characteristics, and after a detailed examination. We confirmed that the morphological characteristic of our specimens fit with the description Lewis (1964a) for C. randalli. This is also supported by records of C. randalli in the Central Pacific.

Current host. Lutjanus fulvus (Lutjanidae).
Site of infection. Gills. Prevalence and mean intensity. 3.8 and 1 (n = 26). Specimens deposited. CHCM No. 570 (voucher) (1 vial, 1 specimen ♂). Remarks. Caligus sp. is morphologically close to Caligus laticaudus, mainly by the shape and armature of cephalothoracic appendages and legs. However, our specimen differs from C. laticaudus in the shape and size of the urosome. Unfortunately, the single specimen of Caligus sp. in our collection is not sufficient for a more detailed taxonomic study.
Other host and localities. Acanthurus olivaceus (Acanthuridae) from Hawaii (Hewitt 1971). Naso hexacanthus (Bleeker), Acanthurus triostegus (Acanthuridae), Myripristis sp., Fistularia petimba Lacepède (Fistulariidae) (Lewis 1964a, 1964b, Palm and Bray 2014 Lewis (1964a). However, Hewitt (1971) stated that there is not a useful character to separate Dentigryps Wilson, 1913 from Lepeophtheirus and, therefore, reassigned species of Dentigryps to Lepeophtheirus. As the name L. bifurcatus was preoccupied by L. bifurcatus Wilson 1905, Hewitt (1971 renamed Lewis' species as L. lewisi. The material of the present study corresponds to a male of L. lewisi. The identification of this species was difficult without female specimens; nonetheless, the morphology of our material fits the description provided by Lewis (1964a) for the male of L. lewisi. In addition, this copepod has been mainly found in acanthurid fish from the Central Pacific as in the present work. Lewis, 1964 Type host. Caranx melampygus (Carangidae).

Lepeophtheirus uluus
Other host and localities. Caranx melampygus (Carangidae) from Oahu, Hawaii (Lewis 1964b, Palm andBray 2014). Reported as Dentigryps ulua on Caranx ignobilis from Heron Island, Australia (Ho and Dojiri 1977 Remarks. Nemesis is one of 12 genera in the family Eudactylinidae and includes about nine species (Mangena et al. 2014). Nemesis species can be divided into two groups by the relative width of the cephalothorax, free thoracic segments and genital segments (Dippenaar et al. 2008). One group (consisting of most of the species) has a fourth free thoracic segment that is much narrower than the preceding three, whereas the other (consisting of N. lamna only) has all four segments of about the same width (Kabata 1979). The identification and comparison of Nemesis species belonging to the first group is difficult because of morphological variation among individuals and the inconsistencies in the literature (Hewitt 1969, Kabata 1979 Remarks. Of the nine genera included in the Hatschekiidae, the most speciose genus is Hatschekia, with approximately 140 valid species so far. According to Uyeno and Nagasawa (2013), H. longiabdominalis may be separated from other congeners by having a fusiform trunk with posterior lobes, the urosome markedly projecting beyond posterior lobes of the trunk, and unique intercoxal sclerites of legs 1 and 2, which strongly project from the middle of the anterior margin and bear four blunt processes on the posterior margin. We observed all of these characters in our specimens. Kabata, 1991 Type host. Chaetodon aureofasciatus Macleay (Chaetodontidae).

Site of infection. Gills.
Remarks. The genus Lernanthropus includes about 120 species and it is one of the commonest genera of parasitic copepods on marine fishes. In this study, a single female of Lernanthropus sp. was collected. We were unable to proceed with the species identification because of the lack of specimens for dissection, which is necessary to observe appendages of the cephalothorax as well as legs 1 and 2. Even with enough material, the identification of Lernanthropus sp. is quite difficult because many species have not been described with sufficient detail (Koyuncu et al. 2012).

Discussion
The present study is the first detailed survey of the diversity and ecological attributes of the parasitic copepods infecting fishes at Palmyra Atoll. All records we report here are new geographical records. Most copepods (10 of 17) belonged to the family Caligidae. Of these ten caligid species, six were in the genus Caligus and two in the genus Lepeophtheirus. These finding are in agreement with the fact that Caligus copepods are mostly found on warm water fishes, while Lepeophtheirus copepod diversity is low in the tropics (Ho and Lin 2004, Suárez-Morales and Gasca 2012, Morales-Serna et al. 2016. However, as far as we know, specific evolutionary or ecological mechanisms underlying this greater diversification Caligus species in the tropics are not well understood. On the other hand, in experiments carried out by Bravo et al. 2010, they suggest that species of Caligus are more active swimmers than species of Lepeophtheirus, which in turns increase transmission between hosts. Clearly, such swimming ability could be contributing to dispersal of Caligus and host switching. Several copepods species can parasitize multiple fish species (Dojiri and Ho 2013). This is the case of C. mutabilis found on Lutjanus monostigma and L. fulvus in the present study however, this species has been reported in at least 13 families of marine fishes from the Atlantic and Pacific oceans (Morales-Serna et al. 2015).
Consistent with observations of the monogenean fauna of Palmyra Atoll fishes (Vidal-Martínez et al. 2017), parasitic copepod richness at Palmyra Atoll qualitatively appears low relative to other localities in the Indo-Pacific region. Most of the fish species we examined (30 of 44) were not parasitized by copepods, even with large sample sizes for some fish species (e.g. Acanthurus triostegus, n = 50). Several fishes that were unparasitized at Palmyra have copepod records at other sites. For example, Acanthurus triostegus, Gymnothorax pictus, Epinephelus merra and Sphyraena barracuda have been reported as hosts of at least one species of parasitic copepod in other localities of the Indo-Pacific (Boxshall andHuys 2007, Palm andBray 2014). Because ectoparasite species richness, host size and age are positively related (Rhode 1993, Muñoz andCribb 2005), the lack of copepods in some host species could be due to our sampling of only young (Chanos chanos) or small individuals (Sphyraena barracuda). Furthermore, the intertidal habitat sampled at Palmyra differs from the more often sampled fore-reef and reef flat habitats, making a direct comparison among studies difficult. More generally, Palmyra's remoteness may contribute to its depauperate copepod parasite fauna. The Line Islands are far from the Austro-Malayan-Philippine region, the presumed center of origin of Indo-West Pacific (IWP) fishes and their parasites. Because we found fewer copepod species than described from Hawaii, which is still further from the presumed center of origin, we suggest that the remote location of the Line Islands and the particularly small size of Palmyra Atoll also contribute to the depauperate nature of the parasitic copepod fauna.