Nebalia holothurophila, a New Species of the Leptostraca (Crustacea, Malacostraca) Possibly Commensal with the Sea Cucumber, Apostichopus parvimensis (H. L. Clark, 1913), in Southern California

Leptostracans are small (usually 3 to 12 mm) crustaceans characterized by a large, bivalved carapace that covers nearly all of the thorax and the eight thoracic appendages, a short movable rostrum, and leaf-like appendages. Haney et al. (2007) and Olesen et al. (2014) summarized leptostracan general morphology and ecology. Although they are most often found in and on organic-rich substrates, such as estuarine mudflats and algal mats, they have been reported from a wide range of habitats, including seagrass beds, subtidal detrital mats, marine caves, and even deep-sea hydrothermal vents (Bowman et al. 1985; Haney and Martin 2000; Haney et al. 2007; Olesen et al. 2014). At least one species has been described as living on or in a sponge (Ortiz et al. 2011).

Leptostracans are not a diverse group of crustaceans, with only 10 genera and approximately 60 species described to date. More than half of the species (36) belong to the genus Nebalia Leach, 1814, a genus found nearly worldwide. In waters of southern California, relatively few species have been described, although it was noted by Haney et al. (2007) that additional undescribed species exist along this coast, and there has been confusion over the taxonomic status of Nebalia pugettensis Clark, 1932, originally described from Puget Sound and at one time believed to be widespread (Haney et al. 2007). Only six species have been reported from California waters: Nebalia daytoniVetter, 1996; Nebalia gerkenaeHaney and Martin, 2000; Nebalia kensleyiHaney and Martin, 2005a; Nebalia sp. (described by Haney et al. 2007 as widespread in the Pacific Northwest and previously confused with N. pugettensis); Nebalia hessleri (Martin, Vetter, and Cash-Clark 1996); and Nebalia pugettensis (the identifcation of which was questioned by Martin et al. (1996) (Haney et al. 2007).

Beginning in 2013, the second author, while volunteering at the Birch Aquarium of the Scripps Institution of Oceanography (La Jolla, California), noticed that when live specimens of the warty sea cucumber Apostichopus parvimensis (Clark, 1913) were transferred into a new container, sometimes small crustaceans were expelled from the cucumber’s cloaca. Some of these crustaceans were eventually collected and sent to the senior author; these specimens and observations form the basis of this report.

All specimens were collected by the second author when volunteering at the Birch Aquarium in La Jolla, California. The artificial habitats at the Birch Aquarium that contain the sea cucumbers are best described as outdoor, semi-natural tidepools. These pools are made of concrete, but fresh seawater is flowing through them in pulses, approximately every 60-90 sec. Multicellular algae grow naturally in the pools, though algal biomass is less substantial in the lowest pool (where the leptostracans were found), since it is nibbled away by fishes (zebra perch and/or opaleyes). Other macroinvertebrates in the pools include the urchins Strongylocentrotus purpuratus and Mesocentrotus franciscanus; the anemones Anthropleura xanthogrammica and A. elegantissima; the sea stars, Pisaster giganteus, P. ochraceus, and Patiria miniata; and the California spiny lobster, Panulirus interruptus. The bottom of the enclosure is covered by a mixture of sand and shell fragments mixed with small fragments of algae. In the lower pools where the leptostracans were found, the bottom is well disturbed by the fishes, making the substrate well aerated (and hence not anoxic). Chunks of krill, squid, and fish are added to the pools twice per week, and clumps of feather boa kelp, Egregia menziesii, and/or giant brown kelp, Macrocystis pyrifera, are added for the herbivores. As this is a semi-natural tide pool, there is a wide variety of other small invertebrates crawling among the rocks and algae.

Only five specimens have been found to date, four of which were found in containers (plastic “transfer tubs” approximately 25 × 35 × 15 cm) containing only live specimens of the sea cucumber and one of which (the smallest) was taken from algae rather than from one of the tubs. The leptostracans were noticed after water was expelled from the cloaca of the sea cucumbers as a reaction to their placement in a new container. Notably, the leptostracans were not seen being expelled; rather, they were noticed in the tub later, after the sea cucumber had been removed. All specimens were initially stored in isopropanol or commercial (Everclear) alcohol (75.5% ethanol) and shipped to the Natural History Museum of Los Angeles County (NHMLAC), where two specimens were prepared for SEM by cleaning for 10–20 sec in a Branson 1200 ultrasonic cleaner in a weak solution of Branson GP jewelry soap and distilled water. These two specimens were then dehydrated with 100% ethanol. Specimens were placed in solutions of pure ethanol and distilled water in the ratios 2:1, 1:1, 1:2, and finally into 100% ethanol (20 min per treatment). Once dehydrated, specimens were transferred through a series of ethanol and hexamethyldisilazane (HMDS) solutions in the ratios 2:1, 1:1, 1:2 and finally into 100% HMDS (20 minutes per treatment). Specimens then were transferred from the final 100% HMDS to fresh HMDS and allowed to evaporate overnight; they were then mounted on carbon conductive tabs and coated with gold-palladium using an Emitech K550x sputter coater (Quorum Technologies, LTD, Kent, UK) and imaged using a Hitachi S-3000N variable pressure SEM (Hitachi, Troy, MI) at the NHMLAC. The remaining specimens were maintained in 70% ethanol for permanent storage.

Drawings of the entire animal were made using a Wild M5APO dissecting microscope with a drawing tube attached; drawings of the limbs and mouthparts were made with a Nikon Labophot compound microscope equipped with a drawing attachment.

Whole animal length 3.33 mm. Carapace (Fig. 1A, B) broad, ovate, gently rounded but with slight angle at anteroventral border, approximately level with base of the antennule, carapace length in lateral aspect 1.9 mm long, height 1.4 mm, and 0.875 mm deep (Fig. 1A).

Rostrum (Figs. 1A; 2A, F) long, length approximately 2.5 × width (length 1.10 mm; width 0.44 mm), acute in lateral view but anteriorly broadly rounded in dorsal or ventral view, slightly downturned, extending to approximately twice the length of the eye. Subrostral keel (Fig. 2A) rectangular.

Eyes (Figs. 1A, B; 2B, F; 5A) bluntly rounded distally, with anteroventral angle more acute than anterodorsal angle. Ocular plate above eye (Fig. 2B) acute, extending to approximately half the length of the eyestalk.

Antennular peduncle (Fig. 1C) composed of three articles, the first longer than the second and third combined, third (Fig. 5C) terminating in two stout, smooth spines (Fig. 5C, arrow); antennular scale (Figs. 2D; 5B, D) broadly rounded distally, with row of 15-18 short setae opposite a row of 10-12 longer setae lining anterior margin. Flagellum with 10 short articles.

Antenna (Figs. 2B, E; 3A) with three articles on peduncle, first unarmed, second with a short distolateral spine, third with 5 to 6 progressively larger spines along the anterior proximal border and several terminal setae; flagellum with 15 articles.

Mandible. Not examined.

Maxillule (Figs. 2B, E; 4B, C) with basal endite wide and extremely short, followed by broadly rounded endite lined with approximately 10 terminally spatulate spines along medial margin; palp bearing approximately 15 distally hooked setae along medial margin.

Maxilla (Fig. 4A) with four basal endites, setation of which is 8, 7, 13-16, and 4, respectively; endopod of two segments, proximal article longer than distal, exopod composed of single article; endopod and exopod bearing long, plumose setae.

Pleonites (Figs. 1A, 5F) with crenulate borders bearing rounded teeth.Pleopod 1 (Fig. 5E) with up to 30 stout, distally serrate spines; pleopod 5 (Fig. 3B) with 4 to 5 long, smooth, terminal spines.

As per the genus Nebalia (see Haney et al. 2007).

Holotype (here designated): ♀ (3.33 mm) undissected and used for full body illustrations: California, San Diego County, La Jolla, Birch, 32.8658°N 117.2507°W, preserved in 70% ethanol, 10 Nov 2014. Collector Jerry Jacobs, LACM:DISCO:28304.

Paratype material: All specimens have the same locality data as holotype. LACM:DISCO:28305 was dissected for illustration of appendages and retained in 70% ethanol. LACM:DISCO:28306 1 juvenile specimen was fixed in isopropanol of unknown concentration, preserved in 70% ethanol. 2 specimens (LACM:DISCO:28307 and LACM:DISCO:28308) were prepared for SEM and are retained as dried specimens that have been sputter coated in gold-palladium alloy although they have been badly damaged by the drying process.

The species is named in recognition of the possible association with the warty sea cucumber, A. parvimensis.

Of the known species of leptostracans along the southern California coast, the new species is immediately distinguishable from Nebalia daytoni by the truncate eyestalk of N. daytoni (Vetter 1996; Haney et al. 2007). Nebalia hessleri is a large species (averaging more than 6.0 mm body length) and is characterized by having acute (rather than rounded) teeth along the posterior borders of the abdominal somites (Martin et al. 1996), although variation exists both ontogenetically and depending on where along the posterior border of the somites these teeth are examined. Juvenile specimens of N. hessleri have more acute teeth along these borders. The other two described California species, Nebalia gerkenae and Nebalia kensleyi, are distinct geographically, with both reported only north of Monterey Bay. Nebalia gerkenae has been found to date only in Bennet Slough, Monterey Bay, California. Nebalia kensleyi has been reported only north of Monterey Bay (Haney and Martin 2005b). Additionally, there are slight but significant morphological differences between these more northern species and the new species described herein. In N. gerkenae, the teeth of the posterior border of the fourth pleonite taper to a more acute point, rather than being gently rounded as in N. holothurophila n. sp. The dorso-distal border of the first and second articles of the antennal protopod bear a stout recurved spine (see Haney and Martin 2000, fig. 3b), whereas this spine is absent on the first such article of the new species. In N. kensleyi, there are 30 to 37 dentate spines along the exopod of pleopod 1, whereas the new species has 30 or fewer.

The identity, characteristics, and geographic boundaries of the more northern species that traditionally was known as N. pugettensis remain in some doubt until such time that a neotype can be designated and described (Haney et al. 2007; Haney and Martin 2000; Martin et al. 1996). Analyses of nucleotide sequence data from cytochrome oxidase subunit 1 (COI) of leptostracans along the West Coast of North America show that clades from the type locality of N. pugettensis indeed differ from those in waters of southern California (Haney, unpublished).

Warty sea cucumbers, A. parvimensis, are known from the intertidal and to a depth of about 60 m from Monterey Bay to southern Baja California, although they are more common from southern California to Baja California, usually inhabiting low-energy environments and associated with rock or other hard substrate (Hamel and Mercier 2008). They are harvested as part of a small sea cucumber fishery in southern California and Mexico (Chávez et al. 2011). Warty sea cucumbers are epibenthic deposit feeders (Muscat 1982; Yingst 1982). Like many holothuroids, the species when threatened can eject internal organs (i.e., autoeviscerate) and grow new ones. It is not known how or if the leptostracans avoid this phenomenon.

Various crustacean taxa are known to live in association with holothuroideans. For instance, caprellidan amphipods and copepods have been collected from the surface of sea cucumbers (Lindsay and Takeuchi 2008; Mahatma et al. 2008). Other species can be found living within the body of the holothuroid, with associations that range from commensal to parasitic. Records include some portunid crabs of the genus LissocarcinusAdams and White, 1849; as well as xanthoid crabs, copepods, and tanaids (Vandenspiegel et al. 1992; Alvaro et al. 2011; Avdeev 2017). Nineteen species of pea crabs have been recorded from the intestine or respiratory tree of sea cucumbers (de Gier and Becker 2020); these records include the species Opisthopus transversusRathbun, 1894, from within the sea cucumber under study herein, A. parvimensis. Although the possibility exists that the new leptostracan species is free-living, or associated with one of the other species in the artificial tidepools, four of the five specimens were found only when the sea cucumbers were isolated in smaller plastic tubs, indicating the strong possibility that these leptostracans are found in, or on, the sea cucumbers. It is possible that the leptostracans are living within the respiratory tree of Apostichopus. Collectors of warty sea cucumbers in the future are encouraged to isolate the live animals for a short period of time (e.g., 24 hr) to see if additional specimens of the leptostracans can be procured.

We thank the staff of the Birch Aquarium for allowing us to see and accession specimens of the new species.