V.N. Ivanenko 1 , Mary Wakeford 2 , Julian Caley 2

1. New data on the diversity, host specificity and distribution of crustacean copepods associated with stony corals (Cnidaria: Anthozoa: Scleractinia) of the Indo-Pacific coral reefs V.N. Ivanenko1, Mary Wakeford2, Julian Caley2 1Department of Invertebrate Zoology, Moscow State University, Russia 2Australian Institute of Marine Science, Australia

2. Diversity of Crustacean Copepods (insects of the Sea) 11 orders Number of valid species 15 000+ Presumed number of species: 60 000-100 000 to 1 million • ~2.5% of known copepod species are barcoded • Most of the barcoded copepods are planktonic or fish parasites

3. Diversity of Symbiotic Copepoda • ~ 2000 species of symbiotic copepods associate with invertebrate hosts (Annelida, Cnidaria, Crustacea, Mollusca, Porifera, Echinodermata, Urochordata, etc) • This number represents only a small fraction (~1%) of the species living with marine invertebrates (Humes, 1994)

4. Diversity of Copepoda living on/in scleractinian corals (from original database) • 900+ records of copepods from scleractinian corals (the first record was in 1960) • 275 species 89 genera, 14 families of copepods • 158 species 52 genera 15 families of scleractinian corals • Copepoda associates with most examined colonies of scleractinian corals • Most scleractinian species have not yet been examined for copepod associates • No barcoded copepods living on scleractinian corals were known till our project

5. Common Copepoda associate with ScleractiniaBody length 0.4-3 mmObligate ecto- or endosymbionts Use their coral host as a habitat and source of food • Poecilostomatoida: • Lichomolgidea • Anchimolgidae 90+ sp • Rhynchomolgidae 30+ sp • Xarifidae 85+ species • Siphonostomatoida: • Asterocheridae 40+ sp • Corallomyzontidae 6+ sp • Harpacticoida: • Tegastidae 4 sp

6. Multiple association and abundance of Copepoda (Humes, 1994) • Copepoda are found in multiple association with scleractinian hosts • Copepoda living on corals can be abundant. • In one fragment ofAcropora hyacinthus 30 x 30 cm there were 1250 copepods Acropora hyacinthus Heron Island, 2010

7. Material(May – Dec 2010, 262 colonies of stony corals) Vietnam, October 2010 48 colonies Lizard Island Sep 2010 96 colonies • . Vietnam-Russia Tropical Centre, Na Trang Ningaloo Reef May 2010 48 colonies Heron Island Nov 2010 69 colonies CReefs project, AIMS

8. Corals were collected by SCUBA (70+ dives, max depth 30m) • All corals were photographed alive underwater • Pieces of coral colonies were isolated in plastic bags and brought to the surface Ningaloo Reef, May 2010 Lizard Island, Sep 2010 Heron Island, Nov 2010

10. Material and Methods • In the lab the small amount of ethanol (~10%) were added to the plactic bag with the coral hosts • 20-30 munities later the corals wereheavily shaked in the solution which was filtered through a fine net. • The obtained residue was sorted under the dissecting microscope • The copepods were photographed and preserved in 95% ethanol. • In addition DNA subsamples of the scleractinian corals were preserved and their skeletons were cleaned by bleaching powder, dried and labeled skeletons of the coral hosts were photographed and deposited to the Museum of Tropical Queensland

11. CReefs Database (AIMS) Field and taxonomic data obtained from the copepods and their hosts, including underwater photographs of the living corals and their skeletons, are stored in an the MS Access database. The data entry interface consists of a series of forms organised into tabs. The three major tabs are Sites, Samples and Specimens. Specimens contains four sub-forms- Specimen Details, Lot, Images and DNA. The database comes with a utility called IrfanView which greatly improves the speed at which large image files are processed.

13. DNA extraction, PCR amplification, and DNA sequencing Sequence analysis was carried out at the Canadian Centre for DNA Barcoding using standard barcoding protocols Compete specimen of morphospecies represented by several specimens in the sample (body length 0. 5-1.5 mm) were used forDNA extraction. Two methods of DNA extraction were tested: • 1) Standard automated CCDB bind-wash-elute protocol (Ivanova et al. 2006) and • 2) HotSHOT alkaline lysis (Montero-Pau et al. 2008) in combination with voucher recovery protocol (Porco et al. 2010). For PCR amplification and sequencing, LCO1490 and HCO2198 primers (Folmer, 1994) were used.

14. Methods of analysis • Sequence data were managed using the Barcode of Life Data System (BOLD) • Analysis of genetic similarity was performed using MEGA version 5.0 • Trees were built using the NJ algorithm with the K2P model. • jMOTU 1.0.7, a Java program, was used for clustering sequences into Molecular Operational Taxonomic Units (MOTU) (Blaxter et al., 2005).

15. ResultsI. New collection of copepods and their coral hosts • In total 258 colonies of 83species of scleractinian corals representing 28 genera and 15 families were collected • Washing of colonies discovered many copepods on most collected colonies(1 to 100s of specimens of up to 10morphospecies of copepods on one colony). Fig. Photos from the database: colonies of scleractinian corals investigated for symbiotic copepods, Lizard Island, GBR, 2010.

16. II Preliminary results of DNA barcoding of copepods from scleractinian corals • DNA barcodes were generated from 119of 190 selected for DNA barcoding specimens • 25% morphospecies (=comparative units) are lost. • There was no significant difference in barcode recovery between the two methods of DNA extraction (Standard automated CCDB bind-wash-elute protocol and HotSHOT alkaline lysis) • However using HotSHOT alkaline lysis in combination with voucher recovery protocol allowed 80%(instead of 10%) vouchers to be preserved in a condition suitable for morphological studies . Sequence Length Distribution - COI-5P

17. Siphonostomatoida • Accumulation Curves of DNA barcoded copepods associated • with scleractinian corals of Lizard Island shows: • The number of DNA barcodes of copepods increases more rapidly for the order Poecilostomatoida than Siphonostomatoida. • Further investigation of copepods associated with corals will providemore data about molecular diversity of copepods. Poecilostomatoida (obtained from BOLD)

18. Siphonostomatoida Poecilostomatoida Harpacticoida Cyclopoida Calanoida Unrooted COI tree for DNA barcoded copepods (BOLD) including new data reconstructed by Neighbor-Joining with Kimura-2-Parameter (K-2-P) distances

19. 66 Taxonomic Units The average value of intraspecies distinction of copepods was 1.56 % Interspecies distinctions of all copepods investigated during the project varies within 10.2 % to 45.9 % and is characterized by a normal distribution.

20. Clustering of sequences by jMOTU program was conducted at cutoffs from 0 to 30 bases. The number of MOTU dropping from 96 (0 base difference) to 66 (at 11+ bases or ~ 1.75+ % difference) The sequences included in each MOTU correspond to the results obtained by Neighbor-Joining analysis (2% threshold values) 96 MOTU 11+ bases or 1.75% difference 66 MOTU

21. Host Specificity and Multiple Association of Copepods from Corals • Only one species of host have all but one randomly selected copepods (60 MOTUs, 109 specimens) from randomly selected 26 species of corals (33of 96 colonies from Lizard Island) representing 17 genera and 10 families • Different multiple (2-5) associations of MOTUs on one species of a coral host are found (not all potential species of copepods was sent for the test barcoding Fig. Frequency histogram illustrating the number of host species of corals used by on or more MOTUs

22. Host Specificity and Multiple Associations in Xarifidae (Poecilostomatoida) Fig. CO1 NJ tree for 5 MOTUsof Xarifia from from 4 colonies (Xarifidae are found in 55 colonies)

23. Host Specificity and Multiple Associations in Asterocheridae + Corallomyzontidae (Siphonostomatoida) Fig. CO1 NJ tree for40 specimens, 11 MOTU of copepods from 8 species of 11 colonies of corals

24. Our preliminary results show: • Effectives of DNA barcoding for investigation of symbiotic copepods from scleractinian corals • High diversity, species level host specificity and multiple association of symbiotic copepods • Necessity of DNA barcoding of the remaining collection of copepods • Necessity of DNA barcoding of the coral hosts • Necessity of better preservation of copepods during DNA extraction

25. Funding • The samples from Australia were collected as part of the CReefs Australia project. • The samples from Vietnam were collected as part of a project of Vietnam-Russia Tropical Centre (Trung tâm nhiệt đới Việt Nga) . • Molecular data were obtained at the Canadian Center for DNA Barcoding funded by the Government of Canada through Genome Canada and the Ontario Genomics Institute (2008-OGI-ICI-03). • Research of VNI was supported by grants from Ministry of Education and Science of the Russian Federation, Russian Foundation for Basic Research, and Smithsonian Institution. • The conference trip of VNI was supported by the Russian Foundation for Basic Research. Acknowledges • Chad Buxton (Museum of Tropical Queensland) as well as Mary Wakeford (Australian Institute of Marine Science) were the great diving buddies of VNI during sampling of corals in Australia. • Chad Walter (Smithsonian Institution) for taking part in preparation database on Copepoda from scleractinians • Natalia Ivanova and Alex Borisenko (Canadian Center for DNA Barcoding) helped to obtain and analyse molecular data.

27. Confocal microscopy of a copepod nauplius (exuvia, exoskeleton) in a permanent glycerin slide. Soft tissues were: - removed without damaging the external anatomy by immersing for 10 min in a K-protease digestive medium from QIAGEN - staining procedure with a solution of Yellow Fuchsin. Ivanenko et al. 2011

28. Lizard Island • The northern Great Barrier Reef, 270 km north of Cairns, Queensland. • Lizard Island Research Stationoperated by the Australian Museum. Creef’s team, September, 2010

29. Female Poecilostomatoida Xarifidae Humes 1960 Body length: 0.5-3 mm Diversity: 85+ species in 5 genera Distribution: Indian Ocean and West Pacific Ecology: all species live in the coral polyps urosome Fig. Female ofXarifia scutipes sp. from Goniopora tenuidens, in 3 m,Moluccas, from A.G. Humes and M. Dojiri, 1983 caudal ramus egg sac YU-RONG CHENG National Taiwan University

30. Harpacticoida • Tegastidae • Body length: 0.5 mm • Distribution: worldwide • Ecological comments: • 4 species are found on scleractinians of Indo-Pacific: • T. georgei from Stylophora sp. and Pocillopora sp., Red Sea; • T. acroporanus from Acropora Jlorida, MarshallIslands; • T. gemmeus from Cyphastreaocellina and Montipora verrucosa, Hawaii; • T. paulipes from Pocillopora verrucosaat Moorea, Society Islands. • Tegastids are known as Red Bugs of badly destroying colonies of cultivating scleractinians corals

31. Frequency histogram of the genetic variation • The average value of intraspecies distinction of copepods was 1.56 % • Interspecies distinctions of all copepods investigated during the project varies within 10.2 % to 45.9 % and is characterized by a normal distribution. Fig. Frequency histogram of the genetic variation of 119 specimens of 66MOTUs of copepods from Scleractinian corals at Lizard Island, GBR, 2010). (p-dist %). The gapbetween distribution of intraspecific variation and interspecific divergence is arrowed.