The copepods associated with the coral Astroides calycularis (Scleractinia, Dendrophyllidae) in the Strait of Gibraltar

This paper describes and provides new records of the copepods hosted by the ahermatypic scleractinian Astroides calycularis (Pallas, 1766). This coral species is endemic to the Mediterranean Sea and protected by the Convention on International Trade in Endangered Species of Wild Flora and Fauna (CITES). The coral colonies were collected at both sides of the Strait of Gibraltar. Two new species, the poecilostomatoid Doridicola helmuti and the siphonostomatoid, Asterocheres astroidicola, are described and compared with their congeners. Furthermore, this paper represents the first record of the genus Doridicola associated with a scleractinian coral, the first time that Acontiophorus scutatus is found associated with Cnidaria, and the first report of an Asterocheres species living on scleractinian corals from the European coasts.


Introduction
Copepoda have been extremely successful in developing associations with Cnidaria and they can be found in association with three classes, the Hydrozoa, the Scyphozoa, and the Anthozoa. In doing so, the copepods have undergone considerable morphological modification and adaptation, although they range from relatively unmodified copepods such as Acanthomolgus Humes and Stock, 1972 (associated with gorgonians) to highly modified forms such as Lamippe Bruzelius, 1858 (soft corals), Magnippe Stock, 1978from gorgonians or Mesoglicola Quidor, 1906.
The Anthozoa have a large number of copepod associates, resulting from: (1) the fact that the larger colonies of the hosts provide more space for copepods; (2) the existence of relatively large gastrovascular cavities offering a protected environment; and (3) the greater opportunity for the evolution of host specificity in these very diverse cnidarians (Humes 1985). Both soft corals, within the octocorals, and scleractinian, or hard corals, among the Hexacorallia, serve as hosts for many more copepods than any other groups (Stock 1987;Humes 1995Humes , 1996Huys and Boxshall 2001). Among scleractinians, copepods are the most abundant in shallow-water reef-building hermatypic corals, which are the most conspicuous animals in tropical waters, while they are much less abundant in ahermatypic corals (Stock 1985).
Of the various orders of symbiotic Copepoda, the Poecilostomatoida contains by far the greatest number of species associated with cnidarians. The remaining orders are much less frequent, and they are represented by relatively small numbers although Siphonostomatoida and Harpacticoida are probably much more frequent associates than presently recorded.
The scleractinian coral Astroides calycularis (Pallas, 1766) is endemic to the southwest Mediterranean Sea and can be found from the Strait of Gibraltar to the Gulf of Naples including the Maltese Islands in European waters, and from the Strait of Gibraltar to Cape Bon (Tunisia) in North Africa (Zibrowius 1980(Zibrowius , 1995. Currently this species is protected by CITES and recently the Spanish Government has included this ahermatypic coral as ''vulnerable'' in the National Catalogue of Endangered Species. In a recent survey on the biology of A. calycularis from the Strait of Gibraltar, a variety of species of copepods belonging not only to the order Poecilostomatoida, but also to Siphonostomatoida, have been found associated with this orange coral. The purpose of this work is to describe some of these copepods.

Materials and methods
The colonies of the host scleractinian were individually collected, each one being isolated in a plastic bag, by scuba diving at both sides of the Strait of Gibraltar and immediately fixed in formaldehyde (8-10%) in seawater. Symbiotic fauna was obtained by pouring the wash water through a 100 mm net. The copepods were finally recovered from the sediment retained and preserved in 70% ethanol.
Selected specimens were dissected in lactic acid and examined as temporary mounts in lactophenol. All figures were drawn with the aid of a camera lucida on a Leica DMLB differential interference microscope. In order to detect minute details, two selected specimens of each species were prepared for scanning electron microscopy (SEM) studies: they were post-fixed in 2.5% glutaraldehyde in 0.2 M cacodylate buffer at pH 7.3 and in 1% OsO 4 in the same buffer and subsequently critical-point dried, mounted on stubs, coated with gold-palladium and observed and photographed using a Philips XL 30 SEM. All appendage segments and setation elements are named and numbered using the terminology introduced by Huys and Boxshall (1991).
Material examined in the present paper is deposited in the Museo Nacional de Ciencias Naturales in Madrid (MNCN) and in the collection of the research team Biodiversidad y Ecología de Invertebrados Marinos of the University of Seville (BEIM).

Description
Female. Body ( Figure 1A) 985 mm long (920-1008 mm) (excluding setae on caudal rami) and 362 mm wide (340-400 mm) (greatest width of cephalothorax) based on six females in lactic acid. First pediger separated from cephalosome by distinct dorsal furrow. Ratio of length to width of prosome 1.7:1. Ratio of length of prosome to that of urosome 1.62:1. Segment bearing leg 5 subquadrate, 57.7692.9 mm. Genital double somite ( Figure 1A, B) 1.23 times longer than wide, 134.66109 mm, with prominent anterodorsal bulge    expanding laterally to egg sac attachment area and overhanging narrow posterior part of somite proper ( Figure 1A, B). Genital areas composed of two parts, the first one rounded with short plumose seta and two spiniform processes. Second part subquadrate bearing a longer plumose seta and a small spiniform process ( Figures 1C, 5B).Three free abdominal somites 60657.7, 43.3648, and 57652.9 mm, respectively ( Figure 1A, B). Posterodorsal border of genital segment and of the two first postgenital segments irregular. Caudal ramus ( Figure 1A, B) 3.4 times longer than wide, measuring 81.7624 mm and bearing six setae in terminal area. Outer lateral seta 38.4 mm, dorsal seta 80 mm, both smooth. Outermost terminal seta 89.7 mm, innermost terminal seta 102. 5 mm, both with lateral setules widely spaced. The two median terminal fringed setae 221 mm (outer), 214 mm (inner) with setules on their outer margin. Egg sac elongate, oval. Dorsal surface of body lacking visible ornamentation, except for minute setules as shown in Figure 1A. Rostrum broadly rounded posteroventrally. Antennule ( Figure 1D) seven-segmented, with armature formula: 4, 13, 6, 3, 4+1 aesthete, 2+1 aesthetasc, and 7+1 aesthetasc. Antenna ( Figure 1E) four-segmented; with formula of armature: 1, 1, 3, and 3+2 subequal claws slightly dentate ( Figure 5C). Claws 2.5 times longer than the last segment. Labrum with two large, divergent posteroventral lobes. Mandible ( Figure 2A) minute, with shallow proximal notch followed by a short terminal lash setulose on its outer side. A small lobe bearing three to four teeth between the proximal notch and the lash. Convex margin with a naked outer scale. Maxillule ( Figure 2B) armed with two terminal subequal setae, the inner plumose, and one spiniform process. Maxilla ( Figure 2C) two-segmented; proximal segment (syncoxa) unarmed; distal segment (basis) ornamented with three typical setae: a tiny outer setula (seta III) at base, naked seta (seta II) on anterior surface, and seta armed with strong spinules (seta I) proximal to the base of the main lash and as long as it.
Terminal lash armed along one side with large strong spinules. Maxilliped ( Figure 2D, E) two-segmented; proximal segment unarmed; distal segment long with just one naked long seta which usually is around the segment, terminating in various barbed pointed processes and carrying a spiniform process with setules at its inner margin.
Legs 1-4 ( Figure 3A-D) biramous, with three-segmented rami except the endopod of leg 4, which is two-segmented. Intercoxal sclerite present in legs 1-4. Formula of spines (in Roman numerals) and setae (in Arabic numerals) as follows: Inner coxal seta plumose in all legs on inner margin of basis of all legs ornamented with setules and basal seta of all legs plumose.
Leg 5 ( Figure 1A, B) long, reaching more than half the length of the double genital segment and armed with small spinules; without proximal inner expansion. Two terminal smooth setae, subequal in length.
Male. Body ( Figure 4A) 800 mm long (760-830 mm) (excluding setae on caudal rami) based on four males in lactic acid, with six-segmented urosome. Genital somite ( Figure 4A, B) slightly wider than long. Four postgenital segments. Caudal ramus 2.3 times longer than wide with the usual six setae in terminal area as described for the female. Rostrum as in female. First antenna similar to that of female but with three aesthetascs at locations indicated by dots in Figure 1D. Second antenna slightly different from female in having spinules on the surface of the first and second segments. Labrum, mandible, maxillule, and maxilla resembling those of female. Maxilliped ( Figure 4C) four-segmented; first segment (syncoxa) large but unarmed; second segment (basis) armed with a row of spinules and two inner setae, one of them inserted in a protuberance; third segment (endopod) smallest and unarmed; terminal claw as long as the preceding segments. The claw with terminal lamellae, one long barbed seta and a smaller naked seta at the basal region. Armature on rami of legs 1-4 are as in female, except for the armature of the endopod of leg 1 ( Figures 4D, 5A) since the second lateral spine of the last segment has an additional spinule at its base. The free segment of leg 5 ( Figure 4A, B) small, 21.768.6 mm, tipped with two subequal setae. Leg 6 ( Figure 4B) composed of two ventral subequal setae located at posterolateral corner of genital somite.

Etymology
The specific name helmuti is after Dr Helmut Zibrowius (Station Marine d'Endoume, Marseille) for his important contribution to our knowledge of the biodiversity and distribution of European scleractinians.

Remarks
Among the 46 species currently recognized in the genus Doridicola, 25 species have the two terminal claws of the antenna subequal in size like D. helmuti (Ho and Kim 2001), although none of them has the claw 2.5 times longer than the fourth segment. Furthermore, among these 25 species, only D. antheliae (Humes and Stock, 1973), associated with an actiniarian from Madagascar, and D. rostripes Humes, 1990, living in an octocoral from the Moluccas, have a naked outer scale of the mandible. However, D. antheliae can be easily separated from the new species by: (1) the shape of the mandible; (2) the armature of the maxillule; (3) the seta I and II of the maxilla; and (4) the armature of the maxilliped and leg 5. The mandible of D. anthelinae has the concave margin deeply indented and, in general Coxa Basis Exopod segments Endopod segments Leg 1 0-1 1-0 I-0; I-1; III,I,4 0-1; 0-1; I,5 Leg 2 0-1 1-0 I-0; I-1; III,I,5 0-1; 0-2; I,II,3 Leg 3 0-1 1-0 I-0; I-1; III,I,5 0-1; 0-2; I,II,2 Leg 4 0-1 1-0 I-0; I-1; II,I,5 0-1; II appearance, is stronger than in the new species. The maxillule has four elements in D. anthelinae and three in D. helmuti. The seta I of the maxilla is short, not reaching the terminal lash, and has spinules on both sides in D. anthelinae, while the new species has a long spined internal seta I. The seta II of this appendage is barbed in D. anthelinae and smooth in D. helmuti. The maxilliped has a very different armature in both species and D. anthelinae has an inner expansion on leg 5 which lacks spinules (Humes and Stock 1973). Doridicola rostripes differs from the new species by its broad flattened prosome, the robust second antenna, the three terminal seta of the maxillule, the slender maxilla, the armature of the maxilliped and the prominint beak-like process of leg 5 (Humes 1990).
Therefore, the new species is easily separated from its congeners by the combination of the following features: (1) ratio of length of claw to that of the last segment of the second antenna; (2) armature of the inner margin of the mandible; (3) armature of the maxillule and the maxilliped; (4) the seta II of the maxilla; and (5) basal swelling of leg 5. Furthermore, the description of this new Doridicola species is the first record of this genus associated with a scleractinian coral.

Description
Female. Body cyclopiform, slender with cephalothorax oval and cylindrical urosome ( Figure 6A, B). Body length 750 mm (650-790 mm) and width 420 mm (390-450 mm), based on four specimens. Ratio of length to width of prosome 1.19:1. Ratio of length of prosome to that of urosome 2.1:1. Prosome comprising cephalothorax fully incorporating first pedigerous somite and three free pedigerous somites. Urosome four-segmented comprising leg 5-bearing somite, genital double somite and two free abdominal subquadrate somites. Somite bearing leg 5 ( Figure 6D) wider than long, with some spinules on its lateral surface. Dorsal surface of free abdominal somites and posterior part of double somite ornamented with large, flattened epicuticular scales, arranged in irregular, overlapping rows ( Figures 6D, 10C). Posterior margins of all somites ornamented with hyaline frills with more or less serrated margins. Genital double somite about 1.25 times wider than long, bearing genital apertures, paired gonopores located laterally. Lateral margin of double somite ornamented with fringe of long spinules located about midway along double somite, posterior to gonopores level ( Figure 6C). Each genital area armed with one plumose seta. Integumental pores and sensilla present on urosomal somites ( Figure 6C). Caudal rami slightly longer than wide, ornamented dorsally with epicuticular scales; armed with six setae. Antennule 21-segmented ( Figures 6F, 6G); segmental fusion pattern as follows: 1(I), 2(II), 3(III), 4(IV), 5(V), 6(VI), 7(VII), 8(VIII), 9(IX-XII), 10(XIII), 11(XIV), 12(XV), 13(XVI), 14(XVII), 15(XVIII), 16(XIX), 17(XX), 18(XXI), 19(XXII-XXIII), 20(XXIV-XXV), 21(XXVI-XXVIII). Segments 1-8 each with two setae; segment 9 with eight setae; segment 10-17 each with two setae; segment 18 with two setae plus an aesthetasc; segment 19 with two setae; segment 20 with three setae; segment 21 with seven setae. Segment 10 (XIII) ( Figure 6G) reduced, partly overlapped by distal expansion of compound segment 9 (IX-XII). Antenna ( Figure 6E) biramous, 380 mm long with a small unarmed coxa ornamented with tuft of spinules and a large unarmed basis with fine spinule row. Exopod small, one-segmented, bearing two lateral and one apical setae. Endopod three-segmented; first segment elongated, ornamented with a row of long spinules; second segment produced distally on medial side but articulating with third segment proximally on lateral side and armed with one smooth seta, third segment armed with two short naked setae, and large terminal claw also ornamented with rows of fine spinules. Mandible ( Figure 7B) with twosegmented palp and stylet-like gnathobase. Stylet with denticulate margin subapically and located in oral cone. First segment of palp slender, unarmed but ornamented with a row of  spinules laterally; second segment with two terminal plumose setae. Oral cone long and slender, 293 mm long, formed by labrum and labium joined laterally, reaching nearly to the posterior margin of intercoxal plate of leg 2 ( Figure 7A, E). Maxillule bilobed ( Figure 7D); praecoxal endite more than four times longer than palp and more than three times wider than palp. Praecoxal endite armed with five distal setae (one of them smooth and short), ornamented with patch of long spinules distally and row of shorter spinules laterally. Palp armed with three terminal and one subterminal setae. Maxilla three-segmented ( Figure 7C). Praecoxa bearing long flaccid element medially ( Figure 10B), possibly an aesthetasc; coxa unarmed and claw-like basis bearing small hyaline process proximally in axil; armed with one very small seta at about one-half its length laterally; claw margins with row of minute spinules distally. Maxilliped five-segmented ( Figure 7F), first segment with small inner distal seta and patch of spinules. Second segment elongate and slender, with minute hyaline seta at midway of inner margin and rows of fine spinules distally. Third segment short, ornamented with two minute smooth setae and a patch of fine spinules medially. Fourth segment armed with two short setae, one of them smooth. Fifth segment with one terminal plumose seta and one claw-like seta, 105 mm long, ornamented with a lateral row of minute spinules.
Legs 1-4 biramous ( Figure 8A-D), with three-segmented protopods and threesegmented rami. Intercoxal sclerite present on legs 1-4, ornamented with patches of spinules on legs 1-3 and pair of processes on legs 1 and 2. Formula for armature as follows: Coxae of all legs ornamented with spinule rows laterally as figured. Inner coxal seta plumose in legs 1-3 and reduced and naked in leg 4. Except on leg 2, basal seta of all legs long and naked. Posterior surface of legs 1-4 ornamented with flattened epicuticular scales, arranged in irregular, overlapping rows ( Figure 10D). Lateral margins of exopodal segments with minute serrations; lateral margins of endopodal segment with row of setules.
Leg 5 ( Figure 6D) with elongated free segment. Three terminal setae, two of them plumose. Few minute spinules on both sides of free segment. Adjacent seta on body somite plumose. Leg 6 ( Figure 6D) represented by seta on genital area.
Leg 6 ( Figure 9A) forming large opercular plates closing off genital apertures, armed with two smooth setae, ornamented with rows of fine spinules.

Etymology
The name of the species, astroidicola, is a combination of Astroides, the host name, and -icola from the Latin meaning ''inhabiting'', alluding to the relationship between the copepod and the coral.

Remarks
Eleven valid species of Asterocheres Boeck, 1859 have been reported as possessing a 21segmented antennule in the female. Three of them, A. suberitis Giesbrecht, 1897, A. violaceus Claus, 1889, and A. minutus Claus, 1889 (Boxshall and Huys 1994). Asterocheres urabensis, A. hirsutus, and the new species, A. astroidicola, possess a longer oral cone. In the first two species it reaches to the insertion of leg 1 and in the latter species the oral cone enlarges nearly to the intercoxal plate of leg 2. However, in a detailed comparison of A. astroidicola and the remaining species with a 21-segmented antennule, a number of additional differences can be discussed. Firstly, A. bulbosus, A. violaceus, and A. minutus have a one-segmented mandibular palp in contrast to the two-segmented mandibular palp present in the new species. As for A. tenuicornis, it differs from A. astroidicola by its very elongated caudal rami which is almost six times longer than wide.
From the point of view of body shape, A. reginae and A. jeanyeatmanae differ from the new species by their dorso-ventrally flattened prosome. Moreover, A. astroidicola can be distinguished from A. reginae by having one additional seta on the inner lobe of the maxillule and from A. jeanyeatmanae by having one distal additional seta on the free segment of the fifth leg. The extremely large inner lobe of the maxillule, at least four times longer than outer lobe, of A. astroidicola and A. hirsutus serves to separate them from the remaining four species, A. flustrae, A. suberitis, A. lunatus, and A. urabensis, which have a much smaller endite in the maxillule. Asterocheres suberitis and A. lunatus possess four terminal setae on the inner lobe of the maxillule in contrast with the five setae present in A. astroidicola. Asterocheres flustrae has a six-segmented maxilliped instead of the five-segmented maxilliped present in A. astroidicola. The caudal rami is 2.5 times longer than wide in A. hirsutus, whereas in A. astroidicola it is only slightly longer than wide.
Concerning hosts, except for A. suberitis and A. tenuicornis whose hosts are unknown, A. flustrae lives in association with the bryozoan Flustra foliacea L. and A. minutus and A. violaceus live in association with an echinoderm. The remaining species of this group, except for A. urabensis, are symbionts on sponges. Asterocheres urabensis and A. astroidicola are associated with scleractinian coral, Pocillopora damicornis (L.) and Astroides calycularis (Pallas, 1766), respectively. These two species are very similar but they can be distinguished by the following features: (1) the endopodal claw of the antenna; (2) the length of the oral cone; (3) the shape of the inner lobe of the maxillule; (4) the armature of the free segment of leg 5. The claw of the antenna is longer than the entire endopod in A. urabensis, while in the new species it is smaller and the oral cone is longer in A. astroidicola. The inner lobe of the maxillule is less than three times longer than the palp in A. urabensis, while that of A. astroidicola is more than four times longer and more than three times wider than the palp. The free segment of leg 5 has three smooth setae in A. urabensis and adjacent small seta on body somite 5 whereas A. astroidicola possesses two plumose setae and one smooth seta on leg 5 and a long adjacent seta on body somite 5 which reaches to the end of the free segment.

Remarks
The specimens from Tarifa differ slightly from those drawn by Giesbrecht (1899) in the second antenna, the maxillule, and the fourth leg. The setae ornamentation of the last endopodal segment of the second antenna is slightly dissimilar since the medial seta is shorter and has setules and the innermost seta is stronger than Giesbrecht's specimens. On the maxillule of our specimens the four setae of the endite are ornamented: the two outermost setae are densely plumose and of the two innermost setae, one is barbed and the other plumose laterally only. With respect to the legs, the only difference is in the length of the basis seta of leg 4 which is shorter in the specimens found in Tarifa.

Host
This species has been found free and caught either by surface-net at night (Brady 1880), or by dredging (Brady 1880;Thompson 1883). It was also discovered among seaweeds such as Laminaria saccharina and Sargassum (Sars 1915;Gotto 1993). The only invertebrate host known is the sponge, Spongelia fragilis var. ramosa. However, according to Canu (1892), this siphonostomatoid is unusually associated with sponges and ascidians. This is the first time that this species has been found associated with Cnidaria.