Monogenea of fishes from the lagoon flats of Palmyra Atoll in the Central Pacific

Abstract A survey of the monogeneans of fishes from the lagoon flats of Palmyra Atoll detected 16 species already reported from the Indo-West Pacific faunal region. A total of 653 individual fish from 44 species were collected from the sand flats bordering the lagoon of the atoll. Eighteen species of fish were infected with monogeneans. The monogenean species recovered were: Benedenia hawaiiensis on Acanthurus xanthopterus, Chaetodon auriga, Chaetodon lunula, Mulloidichthys flavolineatus, Pseudobalistes flavimarginatus and Rhinecanthus aculeatus; Ancyrocephalus ornatus on Arothron hispidus; Euryhaliotrema annulocirrus on Chaetodon auriga and Chaetodon lunula; Euryhaliotrema chrysotaeniae on Lutjanus fulvus; Euryhaliotrema grandis on Chaetodon auriga and Chaetodon lunula; Haliotrema acanthuri on Acanthurus triostegus; Haliotrema aurigae on Chaetodon auriga and Chaetodon lunula; Haliotrema dempsteri on Acanthurus xanthopterus; Haliotrema minutospirale on Mulloidichthys flavolineatus; Haliotrematoides patellacirrus on Lutjanus monostigma; Neohaliotrema bombini on Abudefduf septemfasciatus and Abudefduf sordidus; Acleotrema girellae and Acleotrema parastromatei on Kyphosus cinerascens; Cemocotylella elongata on Caranx ignobilis, Caranx melampygus and Caranx papuensis; Metamicrocotyla macracantha on Crenimugil crenilabris; and Pseudopterinotrema albulae on Albula glossodonta. All these monogenean–host combinations represent new geographical records. The monogenean species composition of the Palmyra Atoll is similar to that of the Hawaiian Islands. However, the number of species recovered was lower compared with other localities within the Indo-West Pacific, perhaps due to the geographical isolation of Palmyra Atoll.


Introduction
Several studies on the parasitic fauna of marine fishes have been conducted at different localities in the Indo-West Pacific, including off the Great Barrier Reef (Australia), New Caledonia, Moorea (French Polynesia), Malaysia, South China, and the Hawaiian Islands (Yamaguti 1965, Young 1968, Plaisance and Kritsky 2004, Lim and Justine 2007, Kritsky et al. 2009, Lim and Gibson 2008, 2010, Rehulkova et al. 2010, Palm and Bray 2014, Mendoza-Franco et al. 2017. Palmyra Atoll is one of the northern Line Islands located in the East Indo-Pacific marine ecoregion (Spalding et al. 2007), 1680 km south-south-west of Hawaii. It is presently a marine protected area and lacks regular human settlement since World War II. The Palmyra Atoll represents a relatively long history with little to no exploitation (Lafferty et al. 2008). 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 a part of a larger research project on the role of infectious agents in the Palmyra Atoll ecosystem, we had the opportunity to undertake a survey of the fish parasites from the lagoon flats of this coral atoll. The goals of the present paper were to report the monogenean species recovered and to establish their zoogeographical affinities with respect to the Indo-West Pacific (IWP) ecoregion.

Methods
Between 13 October and 10 November 2009, and 22 June and 28 July 2010, we captured fishes by seine, spear, and hook and line from the intertidal sand flats bordering the lagoon of Palmyra Atoll (5°53′00″N; 162°05′00″W), U.S.A. Immediately after capture, the fish were separated and anesthetised individually with 0.5 ml L −1 of 2-phenoxyethanol (Sigma, St. Louis, MO, USA) in plastic bags with lagoon water to avoid loss or mixing of monogeneans among fishes 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 with a total magnification of 40×. For each individual host, the skin was examined and the gill arches removed, examined, and the monogeneans obtained were counted, preliminarily identified, and most of them (70-80%) fixed in 4% hot formalin, labelled, and stored in vials for later evaluation. The remaining specimens were flattened and mounted in glycerine ammonium picrate mixture (GAP) to study the morphology of sclerotized structures under a compound microscope (Olympus BX-53, Olympus Corporation, Tokyo, Japan). After evaluation, the specimens that had been fixed with GAP were remounted in Canada balsam (Ergens 1969). Unflattened specimens were cleared with Gray & Wess medium or stained in trichrome and mounted in Canada balsam (for details of these techniques, see Vidal-Martínez et al. 2001). In this manuscript, the male copulatory organ of the monogeneans is denoted below as the MCO. Prevalence and mean intensity concepts were applied following Bush et al. (1997). Specimen of each species were deposited in the United States National Parasite Collection, NMNH Invertebrate Zoology, Smithsonian Museum Support Center, MD, USA (USNM), and the Helminthological Collection of the Laboratory of Parasitology, at Centre for Research and Advanced Studies, National Polytechnic Institute, Mérida, Yucatán, México (CHCM).

Monogeneans of fishes from the Palmyra lagoon flats
During this study, 653 individual fish belonging to 44 species were collected ( Table  1). The 16 species of monogeneans infected 18 fish species (Table 2). Those hosts with the most monogenean species were Chaetodon auriga Forsskål and Chaetodon lunula (Lacépède) with four species, and Kyphosus cinerascens (Forsskål), Acanthurus xanthopterus Valenciennes and Mulloidichthys flavolineatus (Lacépède) with two species each. All other fish species harboured one monogenean species. Twenty-six additional fish species were examined, but no monogeneans were found (Table 2).
Chaetodon bellamaris (= C. wiebeli) Kaup from the South China Sea (Zhang et al. 2003), Chaetodon modestus Temminck and Schlegel from the South China Sea (Zhang et al. 2003 Remarks. This species was originally described by Yamaguti (1968) as Haliotrema annulocirrus and transferred to the genus Euryhaliotrematoides, as E. annulocirrus, by Plaisance and Kritsky (2004). Recently, Kritsky (2012) Young (1968) as Haliotrema chrysotaeniae and transferred to the genus Euryhaliotrema by Kritsky and Boeger (2002) as E. chrysotaeniae. Euryhaliotrema chrysotaeniae has a delicate MCO with more than three rings and an elongate meandering vaginal canal. New geographical record for Palmyra Atoll.  Remarks. Euryhaliotrema grandis was described by Mizelle and Kritsky (1969) as Parahaliotrema grandis and subsequently transferred to Haliotrema by Paperna (1972) as Haliotrema grandis. Plaisance and Kritsky (2004) erected the genus Euryhaliotrematoides and transferred several species of Haliotrema to this genus, including Euryhaliotrematoides grandis. Recently, Kritsky (2012) proposed the synonymy of Euryhaliotrematoides with Euryhaliotrema. As a consequence, Euryhaliotrematoides grandis was transferred to Euryhaliotrema as Euryhaliotrema grandis. Euryhaliotrema grandis presents a delicate MCO, comprising approximately two rings. Accessory piece variable, serving as a guide for the distal portion of the MCO and is articulated to the base of MCO. Anchors lacking hinged bases. Ventral anchor with flattened base supporting a short deep root, moderately long superficial root, short shaft and point extending to the level of the tip of the superficial root. Dorsal anchor with a short deep root, elongate superficial root, broad base, short shaft, point extending to the level of the superficial root. Ventral bar V-shaped, with an anteromedial concavity having a straight anterior margin and a posterior rounded expansion. Dorsal bar, rod shaped, straight. New geographical record for Palmyra Atoll. Yamaguti, 1968 Type host. Acanthurus sandvicensis (=A. triostegus) (Linnaeus) (Acanthuridae).

Haliotrema acanthuri
Other host and localities. Yamaguti (1968)  Remarks. This species is characterized by the morphology of its copulatory complex, which has a bell-shaped base and a short cylindrical shaft, from which arises a proper MCO, and a similar, solid, shorter spike projecting from the genital pore. New geographical record for Palmyra Atoll.
Remarks. Haliotrema dempsteri was originally described as Parahaliotrema dempsteri by Mizelle and Price (1964). Later, Young (1968) recorded it from A. mata, A. dussumieri and A. xanthopterus, redescribing and transferring it to Haliotrema. The most relevant morphological characteristics are: haptor subhexagonal, broader than long; one dorsal and one ventral pair of anchors, similar in size and shape; superficial root of each anchor base longer than the deep root; shafts solid and points without formation of a definite angle; wings low and inconspicuous on dorsal anchor shafts, apparently absent on ventral shafts; copulatory complex composed of an MCO and an elongate accessory piece attached to the proximal portion of the MCO shaft, terminating in a recurved tip; and MCO tubular with relatively large base and an undulate shaft. New geographical record for Palmyra Atoll. Yamaguti, 1968 Type host. Parupeneus cyclostomus (Lacépède) (Mullidae).

Haliotrema minutospirale
Other host and localities. Yamaguti (1968) recorded this species from gills of P. cyclostomus, P. pleurostigma (Bennett) and P. multifaciatus (Quoy and Gaimard) for Hawai'i. Palm and Bray (2014) , 1 specimen) Remarks. The morphology of the copulatory complex is a relevant characteristic for its identification. Its MCO consists of an anterior, spiral, flanged portion and a posterior, cylindrical portion, enclosed in a sheath of circular muscular fibres. The presence of H. minutospirale on the gills Mulloidichthys flavolineatus from Palmyra Atoll represents both a new host and a new geographical record.
Other host and localities. Previous records (as Haliotrema patellacirrus) on L. lutjanus and L. fulviflamma from South China Sea (Bychowsky and Nagibina 1971). Kritsky et al. (2009)  Remarks. This species can be distinguished from other members in the genus by having an inconspicuously sclerotised MCO, consisting of a simple, curved, short tube with a large initial part and simple, bifid stick-like accessory piece. This species also has V-shaped bars with processes, 'marginal' hooks of different sizes, anchors with a spatulate, recurved and grooved point, and a non-fenestrated haptor. The presence of N. bombini in A. septemfasciatus and A. sordidus off Palmyra Atoll represents both new hosts and geographical records for this species.
Other host and localities. Parastromateus niger from Madagascar (Rakotofiringa et al. 1987 Remarks. This species was originally described as Heteroplectanum parastromatei by Rakotofiringa et al. (1987). However, Domingues and Boeger (2007) considered the genus Heteroplectanum as a junior synonym of Acleotrema. Therefore, this species was transferred to Acleotrema as A. parastromatei. It has a haptor with two squamodiscs, each consisting of 25 to 27 rows of sclerotised pieces. A. parastromatei in K. cinerasens from off Palmyra Atoll represents both a new host and a new geographical record. Only one host was found parasitised.

Polyopisthocotylea Odhner, 1912
Site infection. Gills. Specimens deposited. CHCM No. 552 (paratypes) (1 slide, 1 specimen). Prevalence and mean intensity. 4,8 and 3±1 (n=42). Remarks: Metamicrocotyla macracantha is characterized by having a haptor separated from the body proper by a peduncle and with 26-67 clamps disposed in 2 symmetrical lateral rows. The shape of haptor varies depending on state of contraction and number of clamps. Clamps of microcotylid type, similar in shape, somewhat variable in size; middle clamps are the largest, and those from anterior and posterior ends are the smallest. Testes rounded, normally 16 to 25 in a zigzag line occupying inter-caecal space. The presence of M. macracantha from the gills of Crenimugil crenilabris off Palmyra Atoll represents both a new host and a new geographical record.
Other host and localities. Yamaguti (1966) recorded P. albulae from A. vulpes off Hawai'i. It has also been found on the same host by Palm and Bray (2014)  Prevalence and mean intensity. 70,8 and 17±18 (n=24). Remarks. Pseudopterinotrema albulae presents an asymmetrical, fan-shaped haptor on a posterior extension of the body proper, with nine pedunculate clamps. The clamps have very distinct features (see Yamaguti, 1968 for a detailed description of each clamp). MCO plug-shaped, with two unequal sclerotised filaments at base; genital pore ventromedial. The presence of P. albulae from A. glossodonta off Palmyra Atoll represents both a new host and a new geographical record.

Discussion
The species composition of monogeneans of the fishes from Palmyra Atoll is similar to that reported from other localities in the Indo-West Pacific and the Caribbean regions. These localities include the Great Barrier Reef (Australia), New Caledonia, Moorea (French Polynesia), South China Sea (Young 1968, Kritsky et al. 2009, Rehulkova et al. 2010 and Cuba (Zhukov 1976). Nine of the 16 species recorded in this study have been reported off Hawai'i (see Yamaguti 1968). This was not surprising, since Hawai'i is the closest location where monogeneans of marine fishes have been frequently examined. Some species recorded herein were previously considered endemic to Hawai'i; for example, Pseudopterinotrema albulae, Benedenia hawaiiensis and Haliotrema minutospirale (Palm and Bray 2014).
The absence of monogeneans from 26 of the 44 fish species examined was striking, even with relatively large sample sizes for some of those species (e.g. Liza vaigiensis n=54, Valamugil engeli n=63). Of those fishes that were infected, 14 of 18 species were parasitised by only one monogenean species. The low diversity of monogeneans found in our study is similar to that reported by Lafferty et al. (2008), who found only three species of monogeneans from five fish species captured on the forereef at Palmyra Atoll (n=11-25 individuals) and one monogenean species from a similar sample at the nearby Kiritimati Island. The species richness of monogeneans at Palmyra Atoll (16 monogenean species in 18 fish species) appears to be low compared with other localities, including the Hawaiian Islands, which are themselves remote and with some groups depauperate. Several host species from which monogeneans were absent in this study, have previous records of monogeneans. For example, Pseudorhabdosynochus cupatus, P. melanesiensis, P. vagampullum and P. coioidesis on Epinephelus merra; or Pseudochauhanea sphyraenae and Vallisiopsis sphyraenae on Sphyraena barracuda; or three Haliotrema species reported on Stegastes nigricans (Yamaguti 1968, Lo et al. 1998, Bu et al. 1999, Justine 2005, Hinsinger and Justine 2006. The most likely hypothesis to account for the paucity of monogenean parasites at Palmyra Atoll is its geographical remoteness and small area. The Line Islands are isolated from other island groups in the Pacific and are also remote from the Austro-Malayan-Philippine region, the presumed centre of origin of Indo-West Pacific fishes and their parasites. Since there are also fewer species than those described from off Hawai´i, which is still further from the presumed centre of origin, we suggest the particularly small area of Palmyra Atoll contributes to the depauperate nature of its monogenean fauna. In fact, the low species richness of fishes from the Line Islands (Gosline 1971) is associated with the absence or scarcity of free-living species at Palmyra Atoll compared to other coral atolls in the Indo-West Pacific region (e.g. Adler 1992).
The presence of fish hosts, but often not their directly transmitted monogenean parasites, is consistent with the hypothesis that including a pelagic larval phase in marine animal life cycles is selectively advantageous because these small, morphologically and physiologically distinctive life history phases are incompatible with most of the parasites of juvenile and adult hosts (Strathmann et al. 2002).
This interspecific comparative study is consistent with the experimental studies (e.g., Grutter et al. 2011Grutter et al. , 2017, that evaluate how parasites are transmitted to fish hosts after settlement from the pelagic region. An additional explanation for the low number of monogenean species off Palmyra Atoll at local scale is related to the habitat from which almost all the fish examined were obtained: the lagoon flats. These flats are shallow and the daily temperature range is between 28.2 and 30.1°C (Koweek et al. 2014), perhaps offering an unsuitable environment for monogenean transmission or survival. For example, the negative effect of water temperature on the longevity and infection success of the oncomiracidia of Neobenedenia sp. infecting barramundi (Lates calcarifer) has been demonstrated (Hirasawa et al. 2010;Brazenor et al. 2013).
In conclusion, the number of species and individuals of monogeneans appear to be low in Palmyra lagoon-flat fishes. Filters acting at both local and biogeographical levels (sensu Holmes 1990, Combes 2001) seem to preclude the presence of a rich monogenean fauna. However, monogeneans were studied almost exclusively on fish from the lagoonal flats. The generally low infection prevalence is consistent with a more limited study of five fish species on coral reefs at Palmyra Atoll (Lafferty et al. 2008). Studies on the diversity of the coral reef fish fauna have found important differences in species number and composition between lagoonal flats, backreef and forereef zones (García-Sais 2010; Zhao et al. 2017). These ecological differences in reef zones could also contribute to differences in monogenean species richness and composition. Consequently, comparative studies of the monogenean fauna in different reef zones are needed to determine whether differences in monogenean diversity mirror differences in fish diversity. Palmyra Atoll has not had a permanent human population since WWII, and all fishing has been prohibited since it became a US National Wildlife Refuge in 2000. Consequently, the patterns and processes governing monogeneans diversity obtained in this relatively pristine environment could shed light on patterns of transmission prior to the removal of top predators by fishing, the situation found elsewhere.