Two new species of parasitic copepods from the genera Nothobomolochus and Unicolax (Cyclopoida: Bomolochidae) from Australian waters

A 2016 collaborative survey of commercial fish parasites in Moreton Bay, Queensland, Australia led to the discovery of two new species of parasitic copepods belonging to the family Bomolochidae. Females of Nothobomolochus johndaveorum n. sp. were found attached to the gill filaments of Gerres subfasciatus and Gerres oyena. The new species most closely resembles N. leiognathicola and N. quadriceros. All 3 species possess 3 modified setae on the first antennulary segment that are approximately the same length and have a robust seta on the second antennulary segment adjacent to the 3 modified setae giving a superficial appearance of 4 modified setae on the antennule. The new species can be distinguished from these two species in its possession of longer inner setae on the first two endopodal segments of leg 4: the seta on endopodal segment 1 extends past the midline of the distal segment in the new species vs to the proximal margin of the distal segment in the other two species, and the seta on segment 2 extends well beyond the distal margin of the endopod in the new species vs just to the margin in the other two species. Females and males of Unicolax longicrus n. sp. were found in the nasal sinuses of Sillago maculata and Sillago ciliata. The new species differs from 6 of its 7 congeners in having a leg 4 exopod formula of II, I, 4 rather than II, I, 3 or II, I, 5. The new species resembles U. anonymous in this feature, but differs in its possession of a leg 5 that is relatively longer and less wide, and, whereas U. anonymous possesses inner and outer distal spines on leg 5 that are approximately the same length, those of the new species are relatively longer and asymmetrical. Unicolax longicrus n. sp. is unique among its congeners in its possession of a leg 4 with highly elongated endopodal segments 2 and 3, from which its name is derived. In addition to describing the two new species, host and locality reports for all species of Nothobomolochus and Unicolax are reviewed.


INTRODUCTION
As part of a concerted effort to survey parasites of commercial fish of Moreton Bay, Queensland, Australia, a diversity of fish species were examined for parasitic copepods. Two new species of copepods belonging to the family Bomolochidae Claus, 1875 collected
Etymology: This species is named in honor of John Page and Dave Thompson for their generosity in providing us with host specimens.
Leg 1 (Fig. 2F) biramous, with flattened lamellate rami; members of leg pair joined by intercoxal sclerite (Fig. 2G) bearing lingulate process ornamented with elongate spinules on lateral margins. Coxa with inflated plumose seta, with blunt-apex drawn out into elongate tip. Basis ornamented with patch of rounded spinules proximally, outer plumose seta (Figs. 2D and 2F), and inner seta reduced to small rounded tubercle (arrowhead in Fig. 2F). Exopod (Figs. 2D and 2F) 2-segmented; proximal segment armed with large spine on dorsal surface ornamented with lingulate spinules and narrow tip; compound distal segment formed by fusion of segments 2 and 3, bearing 6 plumose setae (only 3 shown in Fig. 2D; plumosity not figured in Figs. 2D and 2F), 1 naked spine and 1 serrated spine on dorsal surface (Fig. 2D). Secondary cuticular thickenings present on ventral surface of compound distal segment, not indicative of original segmental articulations. Endopod 3-segmented; all segments flattened and expanded transversely; first and second segments each with inner plumose seta and ornamented with patch of small spinules on ventral surface and hair-like setules on outer margin; second segment with additional hair-like setules along inner margin; third segment armed with 5 plumose setae and with hair-like setules along inner margin.
Leg 3 (Fig. 3C) biramous, with unornamented intercoxal sclerite. Coxa with inner plumose seta. Basis with outer naked seta on raised base. Exopod 3-segmented; first segment with outer spine and hair-like setules on inner margin; second segment with outer spine and inner seta; third segment with formula: II, I, 5. All outer spines with bilateral spinulation and subterminal flagellum. Terminal spine with spinulation on inner margin only and lacking flagellum. Endopod 3-segmented; first segment with plumose inner seta and hair-like setules along outer margin; second segment with 2 plumose setae and setules along inner and outer margins; third segment with 2 inner setae plus 2 spines bearing fine spinulation bilaterally, and ornamented with hair-like setules along outer margin.
Leg 4 ( Fig. 4) biramous, with broad unornamented intercoxal sclerite. Coxa lacking inner seta. Basis with naked outer seta on slightly raised base. Exopod 3-segmented; first segment with long outer spine bearing elongate subterminal flagellum and ornamented with hair-like setules along inner margin of segment; second segment bearing plumose seta and outer spine ornamented with flagellum and unilateral spinulation; third segment with setal formula: III, 4; all spines with flagellum; proximal spine with unilateral spinulation; distal 2 spines with bilateral spinulation. Endopod 3-segmented; all segments ornamented with row of long hair-like setules along outer margin: first segment bearing plumose seta extending just beyond middle of third segment; second segment with inner plumose seta extending beyond tip of ramus, about to middle of outer distal spine; third segment with 3 bilaterally-spinulate spines distally; inner and outer distal spines each with flagellate tip; inner distal spine 30% longer than outer but only 40% as long as apical spine.
Nothobomolochus johndaveorum n. sp. differs from N. saetiger (Wilson, 1911) Vervoort, 1962 in its possession of caudal rami that are longer than wide. It can be differentiated from N. denticulatus (Bassett-Smith, 1898) Vervoort, 1962, N. digitatus Cressey, 1970, N. gerresi Pillai, 1973, and N. thambus Ho, Do & Kasahara, 1983 in its possession of a robust, modified seta on the second antennulary segment adjacent to the 3 modified setae on the pedestal of the first segment, that gives the appearance of a fourth modified process, rather than a typical unmodified seta. The new species further differs from these 4 species in possessing a much shorter third pedigerous somite rather than a swollen third pedigerous somite that completely, or nearly completely, overlaps the fourth pedigerous somite, concealing it in dorsal view.
The new species closely resembles N. leiognathicola Boxshall, 2014 andN. quadriceros Pillai, 1973 in that all species possess a robust, modified seta on the second antennulary segment adjacent to the 3 modified setae on the first segment so that, superficially, they appear to have 4 modified setae on the antennule. It can be differentiated from N. leiognathicola in its possession of an outer element on the distal endopodal segment of leg 4 that is 3/4 the length of the inner element, rather than 1 2 the length as in N. leiognathicola. The new species also possesses mandibular blades that are less asymmetrical: the shorter blade is 3/4 the length of the longer blade vs less than 1 2 the length in N. leiognathicola. In addition, the inner setae on the first 2 endopodal segments of leg 4 are much longer in the new species: the seta on endopodal segment 1 extends past the midline of the distal segment in the new species vs just past the margin of segment 2 in N. leiognathicola, and the seta on segment 2 extends well beyond the distal margin of the endopod in the new species vs just to the margin in N. leiognathicola. Nothobomolochus johndaveorum n. sp. is most similar to N. quadriceros but differs in the number of rows of spinules along the distal segment of the antenna. Whereas the new species possesses 4 rows of spinules, N. quadriceros as figured by Pillai (1973) possesses 9 rows of spinules. The new species can also be distinguished from N. quadriceros as figured by Pillai (1973) in the lengths of setal elements on leg 4: the outer spine on segment 2 of the exopod is relatively longer in the new species, extending past the midpoint of the third exopodal segment rather than just past the distal margin of the second exopodal segment; likewise the seta of endopodal segment 1 is relatively longer in the new species, extending past the midpoint of the third endopodal segment rather than to the distal margin of the second segment; and the distal endopodal segment of the new species bears an inner setal element that is 30% longer than the outer setal element, rather than approximately the same length.
One other species of Nothobomolochus has been reported parasitizing a species of Gerres. Nothobomolochus gerresi was described from Gerres filamentosus Cuvier by Pillai (1973) from Trivandrum (now Thiruvananthapuram), India. In addition to the characters noted above, the new species can be further distinguished from N. gerresi in its possession of a longer outer spine on the first exopodal segment of leg 4, which extends past the midpoint of the third endopodal segment rather than to the distal margin of the second segment, and its possession of 4, rather than 10, rows of spinules along the distal segment of the antenna.
Leg 3 (Fig. 7A) biramous; intercoxal sclerite with rows of spinules in paired lateral fields and cuticular folds. Coxa with inner plumose seta. Basis with outer naked seta. Exopod 3-segmented; first segment with outer spine, segment ornamented with patch of spinules on outer margin and hair-like setules on inner margin; second segment with outer spine and inner seta; third segment with formula: II, 1, 5. All outer spines with bilateral spinulation and subterminal flagellum; proximal 2 spines with more robust bilateral serrations. Endopod 3-segmented; first and second segments each with plumose inner seta and hair-like setules along outer margin; third segment with 2 inner setae plus 2 spines bearing fine spinulation bilaterally, and ornamented with hair-like setules along outer margin.
Leg 4 (Fig. 7B) biramous, with intercoxal sclerite ornamented with spinules in paired lateral fields. Coxa lacking inner seta. Basis with naked outer seta and ornamented with patch of spinules near base of exopod. Exopod 3-segmented; first segment with outer spine bearing subterminal flagellum, segment ornamented with patch of spinules on outer margin and hair-like setules along inner margin; second segment bearing plumose seta, outer spine with flagellum, and spinules at base of spine; third segment with setal formula: II, 1, 4; all spines with flagellum; spinules present at base of proximal spine; distal spine bearing flange on outer margin. Endopod 3-segmented; all segments ornamented with row of short hair-like setules along outer margin; first segment bearing plumose seta extending to 30% of length of third segment; second segment (Figs. 7B and 7C) elongate, length:width ratio 2.5, bearing large spinules in cluster on outer distal margin and spine-like seta extending just beyond midline of distal segment; seta with marginal serrations on distal half; third segment (Figs. 7B and 7C) highly elongate, length:width ratio 3.8, bearing 3 spines along distal margin with large spinules at bases of spines; inner and outer distal spines of unequal length, outer spine as long as segment and 20% longer than inner; both ornamented with lateral serrations distally; middle spine naked, more than 2x longer than outer distal spine.
Leg 5 (Fig. 6D) 2-segmented: protopodal segment with naked outer seta and small patch of spinules on outer distal margin; exopodal segment with 4 patches of spinules and 4 setal elements; outer 2 spines each with subterminal flagellum; terminal seta and inner spine naked; inner spine approximately 30% longer than outer; terminal seta markedly longer than other elements, just longer than segment, nearly twice as long as inner spine and 2.5x longer than outer spine. Leg 6 ( Fig. 5A) represented by 3 setae near oviduct opening.
Most appendages sexually dimorphic, except antenna, mandible, paragnath, maxillule, and maxilla as in female. Antennule (Fig. 8A) 5-segmented; first segment heavily sclerotized at base; second to fourth segments partially fused, 3 distal segments cylindrical. First segment bearing 5 hirsute setae. Second segment bearing 13 hirsute setae and 2 naked setae along anterior margin, and 2 hirsute setae and 1 naked seta on posterior ventral surface; 2 long and 1 short seta present on dorsal surface. Third segment with 2 hirsute and 2 naked setae. Fourth segment bearing 1 hirsute seta, 1 naked seta, and 1 aesthetasc. Distal segment with setal formula: 7 + 1 ae; setae naked. Maxilliped (Fig. 8E) 3-segmented, comprising long syncoxa bearing hirsute seta; middle segment tapering distally, with 1 long and 1 short hirsute setae on medial margin, ornamented with patch of blunt spinules along inner surface and row of elongate spinules along inner margin distally; distal segment incorporated into claw and bearing 1 long and 1 short naked setae; claw with 2 rows of teeth in distal half of concave margin.
Leg 1 (Fig. 9A) biramous, with flattened lamellate rami; intercoxal sclerite flattened, ornamented with row of spinules along free posterior margin. Coxa with inner plumose seta. Basis ornamented with row of spinules proximally, outer hirsute seta, and short inner seta reduced to rounded tubercle. Exopod 2-segmented; proximal segment with outer spine and hair-like setules along inner margin; spine armed with subterminal flagellum and bilateral spinulation; distal segment bearing 6 plumose setae and 1 spine with subterminal flagellum and bilateral spinulation. Endopod indistinctly 3-segmented; first segment with inner plumose seta and hair-like setules on outer margin; second segment partially fused to third, with inner plumose seta and hair-like setules on outer margins; third segment armed with 5 plumose setae and 1 spine with bilateral spinulation; terminal seta shorter than adjacent 4 setae.
Leg 2 (Fig. 9B) biramous; intercoxal sclerite ornamented with paired lateral fields of small spinules with three larger spinules on inner margin of each field. Coxa with inner plumose seta. Basis with outer seta. Exopod 3-segmented; first segment bearing outer spine and spinules in patch on outer margin; distal segments with setal formula I-1; II, 1, 5; all outer spines bearing subterminal flagellum and bilateral spinulation. Endopod 3-segmented; first and second segments each with plumose inner seta and hair-like setules on outer margin; third segment with hair-like setules on outer margin and setal formula II, 3; spines with subterminal flagellum and bilateral spinulation; terminal spine twice as long as proximal, with spinulation only on distal 25%.
Leg 3 (Fig. 9C) biramous; intercoxal sclerite ornamented with cuticular folds and paired lateral fields of spinules on raised expansions. Coxa with plumose inner seta. Basis with outer seta. Exopod 3-segmented; first segment with outer spine and patch of spinules on outer margin; second segment lacking outer spine, armed with inner seta; third segment with formula: II, 1, 5, ornamented with small patches of spinules at base of spines; all spines with bilateral spinulation and subterminal flagellum. Endopod 3-segmented; first and second segments each with plumose inner seta and hair-like setules along outer margin; third segment ornamented with hair-like setules along outer margin and 2 inner setae plus 2 spines bearing subterminal flagella and bilateral spinulation; terminal spine twice as long as proximal spine.
Leg 4 (Fig. 9D) biramous, with intercoxal sclerite ornamented with spinules in paired lateral fields on raised bases. Coxa lacking inner seta. Basis with outer seta. Exopod 3-segmented; first segment with outer spine bearing subterminal flagellum and bilateral spinulation, segment ornamented with patch of spinules on outer margin; second segment lacking outer spine but bearing plumose seta; third segment with setal formula: II, 1, 4; small patches of spinules present at bases of spines and innermost seta; all spines with subterminal flagella; first spine otherwise unornamented; terminal spine with serrated margins; terminal setal element with asymmetrical spinulation on inner and outer margins. Endopod 2-segmented; both segments ornamented with row of long hair-like setules along outer margin; first segment bearing plumose seta; second segment elongate, bearing row of spinules on posterior margin and 3 terminal elements; medial element longest; inner and medial element with bilateral spinulation; outer element shortest, with serrated margins.
Leg 5 (Fig. 8C) 2-segmented; protopodal segment with naked outer seta; exopodal segment bearing 2 terminal setal elements and patch of spinules; spinules becoming more elongate towards outer posterior margin; both setal elements with bilateral spinulation; outer element twice as long as inner. Leg 6 not seen.
The new species is distinguished from U. reductus in its lack of conspicuous dorsolateral aliform expansions of the second pedigerous somite. It further differs in having a leg 4 exopod formula of II, I, 4 rather than II, I, 3. The new species can be differentiated from U. ciliatus, U. collateralis, U. mycterobius, U. longispinus, and U. quadrispinulus by its possession of a leg 4 exopod with the setal formula II, I, 4 rather than II, I, 5. The new species resembles U. anonymous in this feature, but differs in its possession of a leg 5 that is relatively longer and less wide. The setation of the fifth leg also differs, whereas U. anonymous possesses inner and outer distal spines on leg 5 that are approximately the same length, those of the new species are relatively longer and asymmetrical (outer spine approximately 40% the length of terminal seta, inner spine approximately 50% the length of terminal seta). Unicolax longicrus n. sp. can also be differentiated from U. anonymous in its possession of a leg 4 with much more elongate endopodal segments 2 and 3. The new species further differs from U. anonymous, U. collateralis, U. reductus, and, to a lesser degree, U. ciliatus, in its possession of outer spines on the exopodal segments, particularly of legs 3 and 4, with smaller, less robust serrations on their margins.
The new species is most similar to U. quadrispinulus, the only other species of Unicolax known to parasitize a host species of the genus Sillago. Both species possess four spines on the distal exopodal segment of leg 1. The new species differs from U. quadrispinulus in a number of features. There are numerous differences in leg 4 between the new species and U. quadrispinulus: the distal exopodal segment is II, I, 4 in the new species and II, I, 5 in U. quadrispinulus; the setal element on the second endopodal segment is much shorter and more spine-like in the new species, extending only to the midline of the distal endopodal segment, while in U. quadrispinulus this element is a plumose seta that extends well beyond the end of the ramus and is more than 1.5× the length of distal segment. The new species is unusual among species of Unicolax in possessing elongate endopodal segments in leg 4: in the new species the second endopodal segment of leg 4 has a length:width ratio of 2.5 vs. 1.6 in U. quadrispinulus and the distal segment has a length:width ratio of 3.8 vs. 2.4 in U. quadrispinulus. In addition to the differences in leg 4, the lateral terminal spines of leg 5 in U. quadrispinulus are the same length, whereas in the new species the inner distal spine is 30% longer than the outer distal spine. The spinules on the antenna of the new species are also larger and less densely arrayed relative to those on U. quadrispinulus (see Lin & Ho, 2006: fig. 13B). Furthermore, the terminal spine of the distal endopodal segment of leg 3 is longer than the segment itself in U. quadrispinulus while the terminal spine of the new species is shorter than the segment and more blunt.

Unicolax host associations and biogeography
The hosts and localities of all known species of Unicolax are summarized in Table 4. The 8 species of Unicolax have collectively been reported 83 times. The genus parasitizes the nasal cavity of at least 2 orders of fish. Six of 8 known species of Unicolax parasitize fish of the order Scombriformes. Unicolax quadrispinulus and U. longricrus parasitize species of Sillago (family Sillaginidae, Order incertae sedis in Eupercaria). Three fish families are known to host Unicolax. Five species of Unicolax are known to parasitize the Scombridae, 2 the Sillaginidae, and U. longispinus the Centrolophidae.
Species of Unicolax are known from 9 of the 12 marine realms established by Spalding et al. (2007). Of the 83 reports of Unicolax reviewed, 26 (31%) come from the Central Indo-Pacific realm, followed by 15 (18%) from the Western Indo-Pacific, 10 each (12%) in the Temperate Northern Pacific and Tropical Atlantic, and 9 or fewer in the following 5 realms: Temperate Northern Atlantic (n = 9; 11%), Tropical Eastern Pacific (n = 4; 5%), Eastern Indo-Pacific (n = 3; 4%), Temperate Australasia (n = 3; 4%), and Temperate Southern Africa (n = 1; 1%) . There are currently no reports of Unicolax from the Arctic, Temperate South America, or Southern Ocean realms. Two reports of U. ciliatus by Cressey & Cressey (1980) could not be assigned to a realm. The locality was given only as ''China'' (Cressey & Cressey, 1980 pg. 15), and given that the coast of China spans the Central Indo-Pacific and Temperate Northern Pacific realms, a biogeographic realm could not be unambiguously assigned to these records.

DISCUSSION
Current reports suggest Nothobomolochus has a predominately tropical distribution, with 86 reports from tropical ecoregions and 24 reports from temperate regions. The west coast of North America and the east and west coasts of South America remain largely unexplored for species of Nothobomolochus. Given the diversity of potential hosts there, we suspect many new species remain to be described from beloniform, scombriform, and clupeiform fishes in these waters.
Of the 39 species of Nothobomolochus, 25 have been reported a single time, only from the host from which they were described. By far the most widely reported species of Nothobomolochus is N. gibber, which has been reported 28 times from beloniform fish of the families Belonidae and Hemiramphidae. The global distribution of this species, ranging from the Mediterranean Sea to Ascension Island in the Atlantic and a variety of localities spanning the Indian and Pacific Oceans calls into question whether these specimens are, in fact, conspecific. Because of the limited nature of host reports for species of Nothobomolochus, few negative host data available, and questionable conspecificity of specimens reported from a variety of hosts and geographic regions, a precise measure of the host specificity of the genus remains elusive.
Most reports of Unicolax come from host species of the family Scombridae. The report of U. longispinus from Psenopsis anomala (Temminck & Schlegel, 1844) (Centrolophidae), the first species reported from a non-scombrid host, is not particularly surprising given that the Centrolophidae are closely related to the Scombridae, an affinity supported by    Ho (2006) (continued on next page) mulitlocus phylogenetic analyses (Betancur et al., 2017). Both families, in fact, are now recognized as members of the order Scombriformes (Betancur et al., 2017). However, the discovery of U. quadrispinulus and U. longicrus parasitizing species of Sillago is unexpected given the distant phylogenetic relationship of these hosts with the Scombridae (see Betancur et al., 2017 fig. 2). This suggests that, not only are other scombrids and related families (i.e., Amarsipidae, Ariommatidae, Arripidae, Bramidae, Caristiidae, Chiasmodontidae, Gempylidae, Icosteidae, Nomeidae, Pomatomidae, Scombropidae, Scombrolabracidae, Stromateidae, Tetragonuridae, and Trichiuridae) candidate hosts of Unicolax, but also that much of the Eupercaria, the largest series of fishes, containing over 6,000 species in 161 families and at least 17 orders, are potential hosts. The fact that most known hosts of Unicolax are widely harvested, economically important fish species is likely a reflection of sampling bias rather than true host distribution. Both the inaccessible microhabitat (i.e., the nasal sinuses) parasitized by these copepods and their small size (0.6-3 mm) has contributed to the slow discovery of species of Unicolax. Given the phylogenetic diversity of fish species hosting members of Unicolax, we predict that careful observation of the nasal sinuses of marine fish will reveal numerous additional scombriform and eupercarian fish host species of Unicolax, many of which are likely new to science. It is likely that diversity of this genus is substantially higher than current records suggest, and this also likely applies to other copepod genera that predominately inhabit fish nostrils.
Five genera of bomolochids are known to live almost exclusively in the nostrils of teleosts: Acanthocolax Vervoort, 1969, Ceratocolax Vervoort, 1965, Naricolax Ho, Do & Kasahara, 1983, Tegobomolochus Izawa, 1976, and Unicolax. These genera share a number of morphological similarities and are thought to be closely related (Huys et al., 2012). It is interesting to consider the potential functional significance of shared morphological features in this group. For instance, the second leg of Ceratocolax, Naricolax, Unicolax, and Tegobomolochus have flattened endopods, and Huys et al. (2012) proposed this modification may help seal the suction cup formed by the ventral cephalothorax, a process documented in other parasitic copepods (e.g., leg 3 of Caligidae by Kabata & Hewitt (1971)). Similarly, males of Naricolax, Unicolax, and Tegobomolochus possess a flattened leg 1, which may assist in sealing the cephalothoracic suction cup.
Other structures have arisen in a number of nostril inhabiting copepods. A pincer-like structure arising from projections of anterior cephalothorax and dorsal projections of the antennae is present in Acanthocolax, Ceratocolax, and Tegobomolochus (Huys et al., 2012). There may be some evidence of this, albeit to a lesser degree, in species of Unicolax. Vervoort (1965) shows U. anonymous to have a protrusion at the anterior margin of the cephalothorax ( fig. 1) and Cressey & Cressey (1980) illustrated a similar protrusion in U. collateralis (see fig. 9A). Perhaps a pincer-like structure is formed between this anterior protrusion of the cephalothorax and the modified spine-like seta of the antennule of these species, which may provide a functional explanation for the heavily sclerotized fourth seta of the antennule in Unicolax. It would be interesting to explore if these pincer-like structures are used for attachment in the nostril, perhaps to the lamellae of the olfactory rosette. A number of nostril inhabiting copepods have also developed dorsal extensions on their body somites (e.g., Ceratocolax, Tegobomolochus, U. anonymous [see Vervoort, 1965 fig. 1], and U. reductus [Cressey & Cressey, 1980 fig. 23a]). Given that dorsal extensions appear to have arisen multiple times in nostril-inhabiting copepods, it is possible that they have functional significance; for instance, they may reduce shearing forces on the copepods or enable them to wedge themselves in small cavities of the nasal passage.
There is considerable variation in the ornamentation on Unicolax appendages. In particular, ornamentation on the margins of spines on legs 2-4 varies from relatively few, robust serrations in U. anonymous, U. collateralis, and U. reductus, to numerous fine serrations along the margins of the spines in U. ciliatus, U. longicrus, U. longispinus, U. mycterobius, and U. quadrispinulus. The inner seta on leg 4 also varies from a typical plumose seta, as seen in U. quadrispinulus, to a more spine-like element in most other species of Unicolax; this element is highly reduced to a short spine-like element in U. reductus. We recommend researchers pay careful attention to the ornamentation of setal elements in species of Unicolax, as these are likely to be useful taxonomic characters.