Research Article |
Corresponding author: Rachel L. Welicky ( rwelicky@gmail.com ) Academic editor: Paula Beatriz Araujo
© 2017 Rachel L. Welicky, Kerry A. Hadfield, Paul C. Sikkel, Nico J. Smit.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Welicky RL, Hadfield KA, Sikkel PC, Smit NJ (2017) Molecular assessment of three species of Anilocra (Isopoda, Cymothoidae) ectoparasites from Caribbean coral reef fishes, with the description of Anilocra brillae sp. n. ZooKeys 663: 21-43. https://doi.org/10.3897/zookeys.663.11415
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A morphological review and molecular characterization of Anilocra haemuli Bunkley Williams & Williams, 1981, were completed using specimens collected from Haemulon flavolineatum Desmarest, 1823 (French grunt) and Epinephelus guttatus Linnaeus, 1758 (red hind). Molecular and morphological data suggest that the isopods parasitizing H. flavolineatum and E. guttatus are different species. The specimens collected from E. guttatus are recognized as a new species, Anilocra brillae sp. n. Differences between Anilocra brillae sp. n. and A. haemuli include but are not limited to the pleonites 1–3 of A. brillae sp. n. being wider than 4–5 and 4–5 subequal, whereas the pleonites 1–2 of A. haemuli are wider than 3–5, and 3–5 are subequal. The seventh pereopod of A. brillae sp. n. is proportionally larger, has more robust setae, and the setae are distributed more extensively over the articles when compared to A. haemuli. Additionally, this study provides the first genetic characterization of three Anilocra spp. from the Caribbean, and is based on mitochondrial cytochrome c oxidase subunit gene (COI) for A. haemuli from H. flavolineatum, A. brillae sp. n. from E. guttatus, and A. chromis Bunkley Williams & Williams, 1981 from Chromis multilineata Guichenot, 1853.
Anilocra haemuli , Anilocra chromis , brown chromis, Caribbean, coral reef, Cymothoidae , fish ectoparasite, French grunt, Isopoda , molecular analysis, new species, parasite, red hind, taxonomy
In the past half-century, taxonomic studies on the fish parasitic isopod genus Anilocra Leach, 1818, have reported nine species from the Caribbean (
To evaluate this claim a review of Anilocra haemuli morphology using specimens from both the Haemulidae and Serranidae families is warranted. The original description of A. haemuli was published before molecular approaches were used to aid in confirming the morphological classification of organisms. In the original description, careful attention was taken to describe A. haemuli as type specimens were collected from the same host and locality (Bunkley Williams and Willams 1981). Nevertheless, multiple morphologically similar species of Anilocra may have been identified as A. haemuli because there was no other method to verify if these specimens represented multiple species.
An increasing number of ecological studies are using Anilocra to understand trophic level dynamics (
In August 2016, Epinephelis guttatus Linnaeus, 1758, (family Serranidae) (n = 8) parasitized by a cymothoid isopod of the genus Anilocra were collected by free-divers using a modified cast net (
Of the specimens collected, genomic DNA was extracted from eight Anilocra from E. guttatus, seven A. haemuli from H. flavolineatum, and eight A. chromis from C. multilineata using a rapid DNA extraction method as described in the KAPA Express Extract Kit (Kapa Biosystems, Cape Town, South Africa). Polymerase chain reactions (PCR) were used to amplify a 710 basepair fragment of the mitochondrial cytochrome c oxidase subunit gene (COI) using the primer sets LCO 1490 and HCO 2198 (
Using BLASTn (Basic Local Alignment Search Tool; http://www.ncbi.nlm.nih.gov/blast), the obtained sequences were verified as belonging to the Isopoda. Pair-wise distance (p-distance) using the Kimura 2-parameter model and nucleotide differences were determined in MEGA7. Supplemental comparisons among the sequences of this study and those available for Anilocra sp. from GenBank were also determined. Newly-generated sequences for Anilocra spp. were deposited in GenBank under the accession numbers: A. haemuli: KY562752, KY562753, KY562754, KY562755, KY562756, KY562757, KY562758; A. brillae sp. n.: KY562744, KY562745, KY562746, KY562747, KY562748, KY562749, KY562750, KY562751; A. chromis: KY562736, KY562737, KY562738, KY562739, KY562740, KY562741, KY562742, KY562743.
Anilocra haemuli from Haemulon flavolineatum and Anilocra from Epinephelus guttatus were examined using material previously collected by Ernest Williams and Lucy Bunkley-Williams during 1976–1977 and 1983 and reported in
Comparative sequence analysis indicated that there were three distinct species present in the samples based on the host species, A. haemuli from H. flavolineatum, A. chromis from C. multilineata and another undescribed species of Anilocra from E. guttatus. The intraspecific divergence observed within species was as follows: A. haemuli, 1–6 nt (0.6%); A. sp. n., 1–4 nt (0.3%); and A. chromis, 1–6 nt (0.7%) (Suppl. materials
Anilocra
Canolira Leach, 1818: 350.
Epichthyes Herklots, 1870: 122.
A detailed diagnosis was given by
The type species for this genus is Anilocra cuvieri Leach, 1818, junior synonym of Anilocra physodes (Linnaeus, 1758) (see
The body of female Anilocra is dorsally symmetrical and strongly vaulted. The posterior margins of their cephalon are smooth and straight, and the rostrum is more blunt than pointed. The rostrum folds into the area between the antennula bases. The antennula is shorter than the antenna. The posterolateral margins of the pereonites are not produced. Coxae 1–3 are short, posteriorly rounded and do not form a rounded point posteriorly, whereas coxae 4–6 are longer, less rounded and more elongate than coxae 1–3, and form a rounded point posteriorly. The pereopods gradually increase in size towards the posterior.
In the Cymothoidae, the external-attaching genera include but are not limited to Anilocra, Nerocila Leach, 1818, Renocila Miers, 1880, Creniola Bruce, 1987, and Pleopodias Richardson, 1910. Anilocra can be distinguished from Nerocila by the posterior margin of the cephalon, which is conspicuously trilobed in Nerocila, whereas the posterior margin of the cephalon of Anilocra is not tri-lobed to weakly tri-lobed. The posterolateral pereonite margins of Nerocila are more produced, elongate and pointed than that of Anilocra. In the Caribbean, some species of Anilocra and Renocila share numerous similarities, but in Anilocra pereopod 6 is shorter in length than pereopod 7, whereas in Renocila pereopods 6 and 7 are of similar length. To date the genera Creniola and Pleopodias have not been reported from the Caribbean.
Anilocra
haemuli
(Part)
Holotype (female, TL, W unknown) subocular region of Haemulon flavolineatum (
All material from the subocular region of Haemulon flavolineatum. (TL, W, Voucher Number) Collected by EH and LB Williams: ♀ (32, 13,
Size intact (29, 13). Body weakly ovoid, 2–2.6 times as long as greatest width, dorsal surfaces smooth and polished in appearance, widest at pereonite 5, most narrow at pereonite 1, lateral margins mostly ovate posteriorly. Cephalon 0.5–0.7 times longer than wide, visible from dorsal view, weakly trapezoid shaped. Frontal margin rounded to form blunt rostrum or simple, not folded. Eyes oval with distinct margins, one eye width 0.1–0.2 times width of cephalon; one eye length 0.4–0.5 times length of cephalon. Pereonite 1 smooth, anterior border straight, anterolateral angle narrowly rounded, not produced. Posterior margins of pereonites smooth and slightly curved laterally. Coxae 2–3 wide; with posteroventral angles rounded; 4–7 rounded and curved; not extending past pereonite posterior margin. Pereonites 1–5 increasing in length and width; 6–7 decreasing in length and width; 1–4 narrower. Pleon with pleonite 1 wider than pleonites 2–5, visible in dorsal view; pleonites posterior margin 1–3 posteriorly weakly concave, 4–5 mostly straight. Pleonite 2 not overlapped by pereonite 7; posterolateral angles of pleonite 2 narrowly rounded. Pleonite 1 similar in form to pleonite 2. Pleonite 5 free, not overlapped by lateral margins of pleonite 4, posterior margin straight. Pleotelson 0.9 times as long as anterior width, dorsal surface smooth. Pleotelson lateral margins convex, posterior margin narrowly rounded.
Antennula consisting of 7–8 articles; peduncle articles 1 and 2 distinct and articulated; article 2 0.8 times as long as article 1; article 3 0.9 times as long as wide, 0.4 times as long as combined lengths of articles 1 and 2; flagellum with 5 articles, extending to posterior margin of eye. Terminal article with 2 short simple terminal setae. Antenna consisting of 10 articles; article 3 1.6 times as long as article 2; article 4 1.2 times as long as wide, 1.5 times as long as article 3; article 5 1.3 times as long as wide, 1.1 times as long as article 4; flagellum with 5 articles, terminal article terminating in 5 short simple setae, extending to middle of pereonite 1. Mandibular molar process ending in an acute incisor; mandibular palp article 3 with 7 simple setae. Maxillula simple with 4 terminal robust setae. Maxilla mesial lobe partly fused to lateral lobe; lateral lobe with 2 recurved robust setae; mesial lobe with 2 recurved robust setae. Maxilliped weakly segmented, with lamellar oostegite lobe, article 3 with 3 small robust setae.
Pereopod 1 basis 1.7 times as long as greatest width; ischium 0.7 times as long as basis; merus proximal margin without bulbous protrusion; carpus with straight proximal margin; propodus 1.3 times as long as wide; dactylus stout, 2.7 times as long as propodus, 3.8 times as long as wide. Pereopod 2 propodus 2.1 times as long as wide; dactylus 2.2 as long as propodus. Pereopod 6 basis 2.6 times as long as greatest width; ischium 0.5 times as long as basis; propodus 1.3 times as long as wide; dactylus 2.5 times as long as propodus. Pereopod 7 basis 3.2 times as long as greatest width; ischium 0.7 times as long as basis, without protrusions; merus proximal margin without bulbous protrusion; merus 1.1 times as long as wide, 1.6 times as long as ischium; carpus 1.5 times as long as wide, 0.5 times as long as ischium, without bulbous protrusion; propodus 2.6 times as long as wide, 0.8 times as long as ischium; dactylus slender, 1.8 times as long as propodus, 5.0 times as long as wide. Pereopod 7 with few setae on propodus, carpus, and merus.
Pleopods without setae, exopod larger than endopod. Pleopod 1 exopod 1.5 times as long as wide, lateral margin weakly convex, distally narrowly rounded, medial margin weakly oblique, mesial margin weakly convex; endopod 1.6 times as long as wide, lateral margin weakly convex, distally narrowly rounded, mesial margin slightly convex; peduncle twice as wide as long, without retinaculae, pointed projection on lateral margin. Pleopods 2–5 similar to pleopod 1. Pleopods 3–5 endopods proximal borders do not extend below exopod to peduncle, fleshy lobes and medial lobes present. Peduncle lobes absent.
Uropod length equal length of pleotelson; peduncle 0.7–0.9 times longer than rami, lateral margin without setae; rami not extending beyond pleotelson, marginal setae absent, apices broadly rounded. Endopod apically rounded, 3.1–3.5 times as long as greatest width. Exopod not extending to end of endopod, 3.8–4.4 times as long as greatest width, apically rounded, lateral margin weakly convex, mesial margin weakly convex, terminating without setae.
Size (12, 6). Similar to female but smaller. Body 2.5 times as long as wide. Antennula bases separated, consisting of 8 articles, extending to posterior margin of eye. Antenna consisting of 10 articles, extending to middle of pereonite 1. Mandibular molar process ending in an acute incisor; mandibular palp article 3 with 11 simple setae. Maxillula simple with 4 terminal robust setae. Maxilla mesial lobe partly fused to lateral lobe; lateral lobe with 2 recurved robust setae; mesial lobe with 2 recurved robust setae. Maxilliped weakly segmented, with lamellar oostegite lobe, article 3 with 3 small recurved robust setae. Pereopod 7 with few small robust setae on carpus, merus and propodus. Pleopod 2 appendix masculina absent.
Off the coast of southern Florida (USA) and throughout the Caribbean (
Known from Haemulon flavolineatum (Desmarest, 1823), H. aurolineatum (Cuvier, 1830), H. carbonarium (Poey, 1860), H. chrysargyreum (Günther, 1859), H. macrostomum (Günther, 1859) H. plumieri (Lacépède, 1801), H. sciurus (Shaw, 1803). Host records previously reported and which should be verified in the future: Cephalopholis cruentaus (Lacepède, 1802; formerly reported and classified as Epinephelus cruentatus, Lacepède, 1802) , C. fulva (Linnaeus, 1758; formerly reported and classified as Epinephelus fulvus Linnaeus, 1758), Epinephelus guttatus (Linnaeus, 1758), Paranthias furcifer (Valenciennes, 1828), Mycteroperca rubra (Bloch, 1793), M. bonaci (Poey, 1860), and Orthopristis ruber (Cuvier, 1830).
The description of A. haemuli from H. flavolineatum given above is in agreement with the original description in
Anilocra haemuli from H. flavolineatum can be distinguished from all other Caribbean species based on the morphological and/or site attachment differences among species that were reported in
Anilocra
haemuli
(Part) of
All material from the subocular region of Epinephelus guttatus.
Holotype. Ovigerous ♀ (38, 17,
Paratype. Ovigerous ♀ dissected (39, 15,
Collected by EH and LB Williams: ♀ (33, 13,
Ovigerous female.Size (38, 17). Body ovoid, 2.1–2.4 times as long as greatest width, dorsal surfaces smooth and polished in appearance, widest at pereonite 5, most narrow at pereonite 1, lateral margins mostly posteriorly ovate. Cephalon 0.5–0.7 times longer than wide, visible from dorsal view, trapezoid shaped. Frontal margin rounded to form blunt rostrum, not folded. Eyes oval with distinct margins, one eye width 0.1 times width of cephalon; one eye length 0.5–0.6 times length of cephalon. Pereonite 1 smooth, anterior border straight, anterolateral angle narrowly rounded, not produced. Posterior margins of pereonites smooth and slightly curved laterally. Coxae 2–3 wide with posteroventral angles rounded; 4–7 with narrowly produced point, curved; not extending past pereonite posterior margin. Pereonites 1–5 increasing in length and width; 6–7 decreasing in length and width; 5 and 6 subequal in width, 1–4 narrower. Pleon with pleonite 1 most wide, visible in dorsal view; pleonites posterior margin smooth, 1–4 posteriorly concave, 5 straight. Pleonite 2 not overlapped by pereonite 7; posterolateral angles of pleonite 2 narrowly rounded. Pleonite 1 differ in form to pleonite 4 and 5, similar to pleonite 2 and 3. Pleonite 5 equal width to pleonite 4, not overlapped by lateral margins of pleonite 4, posterolateral angles narrowly rounded, posterior margin straight. Pleotelson 1.1–1.4 times as long as anterior width, dorsal surface smooth, lateral margins convex, posterior margin converging to weak caudomedial point.
Antennula bases separated, shorter than antenna, consisting of 7–9 articles; peduncle articles 1 and 2 distinct and articulated; article 2 1.5 times as long as article 1; article 3 0.9 times as long as wide, 0.5 times as long as combined lengths of articles 1 and 2; flagellum with 4 articles, extending to posterior margin of eye. Terminal article terminating in 1 short simple seta. Antenna comprised of 9–10 articles, peduncle article 3 1.5 times as long as article 2; article 4 1.3 times as long as wide, 1.1 times as long as article 3; article 5 1.6 times as long as wide, 1.1 times as long as article 4; flagellum with 4 articles, terminal article with 5 short simple setae, extending to posterior of pereonite 1. Mandibular molar process ending in an acute incisor; mandibular palp article 3 with 8 simple setae. Maxillula simple with 4 terminal robust setae. Maxilla mesial lobe partly fused to lateral lobe; lateral lobe with 2 recurved robust setae; mesial lobe with 1 recurved robust seta. Maxilliped weakly segmented, with lamellar oostegite lobe, article 3 with 3 recurved robust setae.
Pereopod 1 basis 1.8 times as long as greatest width; ischium 0.23 times as long as basis; merus proximal margin without bulbous protrusion; carpus with straight proximal margin; propodus 1.9 times as long as wide; dactylus moderately slender, 1.8 times as long as propodus, 3.7 times as long as wide. Pereopod 2 propodus 1.7 as long as wide; dactylus 2.7 times as long as propodus, 4.9 times as long as wide. Pereopods gradually increasing in size towards posterior. Pereopod 6 basis 1.7 times as long as greatest width; ischium 0.7 times as long as basis; propodus 1.5 times as long as wide, dactylus 2.3 times as long as propodus, 3.8 times as long as wide. Pereopod 7 basis 3.0 times as long as greatest width; ischium 0.7 times as long as basis, without protrusions; merus proximal margin without bulbous protrusion, 2.0 times as long as wide, 0.7 times as long as ischium; carpus 1.5 times as long as wide, 0.6 times as long as ischium, without bulbous protrusion; propodus 3.2 times as long as wide, 0.8 times as long as ischium; dactylus moderately slender, 0.9 times as long as propodus, 3.5 times as long as wide. Pereopod 7 with many setae on propodus, carpus, and merus.
Pleopods without setae, exopod larger than endopod. Pleopod 1 exopod 1.2 times as long as wide, lateral margin weakly convex, distally narrowly rounded, medial margin weakly oblique, mesial margin weakly convex; endopod 1.8 times as long as wide, lateral margin weakly convex, distally narrowly rounded, mesial margin slightly convex, peduncle 2.2 times as wide as long, with pointed projection on lateral margin. Pleopods 2–5 similar to pleopod 1. Pleopods 3–5 endopods proximal borders do not extend below exopod to peduncle, fleshy lobes and medial lobes present. Peduncle lobes absent.
Uropod more than half the length of pleotelson, peduncle 0.7 times longer than rami, peduncle lateral margin without setae; rami not extending beyond pleotelson, marginal setae absent, apices broadly rounded. Endopod apically rounded, 2.2 times as long as greatest width, lateral margin weakly convex, mesial margin weakly convex, terminating without setae. Exopod not extending to end of endopod, 2.6 times as long as greatest width, apically rounded, lateral margin convex, mesial margin weakly convex or weakly concave, terminating without setae.
Size (11, 6). Similar to female but smaller. Body 2.6 times as long as wide. Antennula bases separated, consisting of 8 articles, extending to middle of eye. Antenna consisting of 10 articles, extending to middle of pereonite 1. Mandibular molar process ending in an acute incisor; mandibular palp article 2 with 2 simple setae, article 3 with 7 simple setae. Maxillula simple with 4 terminal robust setae. Maxilla mesial lobe partly fused to lateral lobe; lateral lobe with 2 recurved robust setae; mesial lobe with 2 recurved robust setae. Maxilliped weakly segmented, with lamellar oostegite lobe, article 3 with 3 recurved robust setae. Pereopod 7 with several small robust setae on carpus, merus and propodus. Pleopod 2 appendix masculina absent.
This species is named in honor of Elizabeth R. Brill for her dedication to studying the ecology of A. haemuli, and for collecting many of the A. haemuli and A. brillae sp. n. specimens used in this study.
Known from St. John and St. Thomas, USVI, Guana Island, BVI, and islands of Puerto Rico, Spanish Virgin Islands. Expected distribution throughout the Caribbean Sea, where fish of the Serranidae family inhabit.
Known from Epinephelus guttatus (Linnaeus, 1758).
Previously, A. brillae sp. n. was identified as A. haemuli. Compared to A. haemuli, the outer margins of the cephalon and pereonites 1–4 of A. brillae sp. n. form a more pronounced trapezoid shape and the remaining portion of the body is ovoid. A. brillae sp. n has more strongly narrowed pleonites than A. haemuli. Pleonites 1–3 of A. brillae sp. n. are wider than 4–5 and 4–5 are subequal, whereas the pleonites 1–2 of A. haemuli are wider than 3–5, and 3–5 are subequal. Pleonite 5 is more posteriorly rounded in A. brillae sp. n, but this is somewhat variable among individuals. Another more variable feature is A. brillae sp. n. has a more caudomedially pointed pleotelson than A. haemuli. Typically, the seventh pereopod of A. brillae sp. n. is proportionally larger, has more robust setae, and the setae are distributed more extensively over the articles when compared to A. haemuli. The antennula peduncle of A. brillae sp. n. is regularly observed as shorter and more robust than that of A. haemuli. With respect to attachment, both species infest the subocular region, and if infested by two parasites, one parasite typically attaches under each eye. Infestation by a third A. brillae sp. n. on a single host seems to occur with more frequency than tertiary infestation by A. haemuli on a single host. The third parasite is typically attached between the eyes on the head of the host, or adjacent to one of the other parasites (RLW, pers obs).
Anilocra brillae sp. n. can be distinguished from all other Caribbean species except Anilocra haemuli using the same morphological comparisons described between A. haemuli and other Anilocra spp. given in
The results of this study provide the first reliable COI sequences for species of Anilocra, and confirm that A. haemuli from H. flavolineatum is morphologically and genetically different than the Anilocra specimens collected from E. guttatus, and are here described as A. brillae sp. n. Our morphological data suggest there are two different species given the number of differences consistently observed, and our molecular analyses demonstrate a 4% difference between A. haemuli and A. brillae sp. n. This difference is less than half of that observed between A. brillae sp. n. and A. chromis, which are more conspicuously morphologically different. Our supplemental analyses were conducted utilizing the available Anilocra sp. COI sequences on GenBank, and there was a high level of interspecific divergence of these sequences compared with our dataset. The large differences in interspecific divergence between the specimens of this study and those provided on GenBank may be explained by the fact that the GenBank specimens may have been misidentified or not identified at all, as no morphological identification was described in
Anilocra spp. have been reported to influence the fitness (Adlard and Lester 2004,
This study exemplifies that there is an incomplete but growing knowledge of cymothoid life histories, genetics, and morphology, and how these disciplines relate to host-parasite ecology. Continued efforts to conduct studies in these disciplines are necessary to better understand one of the least understood parasite families.
The financial assistance of the National Research Foundation (NRF) of South Africa supported this research and is hereby acknowledged (NRF project IFR2011040100022, NJ Smit, PI). Opinions expressed, and conclusions arrived at, are those of the authors and are not necessarily those of the NRF. Financial assistance for KA Hadfield and RL Welicky from the Claude Leon Foundation of South Africa for this research is acknowledged. A portion of the fieldwork reported herein was supported by the U.S. National Science Foundation (OCE-121615, PC Sikkel, PI) and Puerto Rico Sea Grant (project number R-31-1-14, PC Sikkel, PI). The Falconwood Corporation supported work at Guana Island. Drs. Bert and Lucy Williams are acknowledged for the specimens they collected during 1976–1977 and 1983 that were used in this study. This is the first in a series of publications emanating from the Williams’ parasitic isopods collection hosted by the NWU-Water Research Group. We thank C. Cook and E.C. Netherlands for their assistance with the molecular component of this study, and O. Kudlai and C. Baillie for their feedback on the preparation of this manuscript. We also thank the Sikkel lab, the staff of the Virgin Islands Environmental Resource Station, US National Parks Service (St. John), and Guana Island for logistic support, and E.R. Brill for field and collections assistance. This is contribution number 173 from the Center for Marine and Environmental Studies, University of the Virgin Islands and contribution number 179 from the NWU-Water Research Group.
Basepair differences of Anilocra spp.
Data type: statistical data
Explanation note: The number identifier in the horizontal header column represents the number and corresponding species listed in the vertical column header.
Kimura 2-Parameter (K2P) distance of Anilocra spp.
Data type: statistical data
Explanation note: K2P distance expressed in percent. The number identifier in the horizontal header column represents the number and corresponding species listed in the vertical column header.