Confocal laser scanning microscopy (CLSM) as a new tool for morphological characterisation of both newly collected and museum voucher specimens of the Trypanorhyncha Diesing, 1863 (Platyhelminthes: Cestoda).

Taxonomic issues within Trypanorhyncha, e.g., the inaccurate light microscopic visualisation of the hook patterns, are solvable by confocal laser scanning microscopy (CLSM). We applied CLSM imaging to study Trygonicola macropora (Shipley et Hornell, 1906) and Dollfusiella michiae (Southwell, 1929) from Neotrygon caeruliopunctata Last, White et Séret from Bali, Indonesia. To illustrate the strength and limitations of CLSM, images of Otobothrium cysticum (Mayer, 1842) and Symbothriorhynchus tigaminacantha Palm, 2004, both permanent mounts from a collection, were also processed. The CLSM created image stacks of many layers, and edited with IMARIS Software, these layers resulted in three-dimensional images of the armature patterns and internal organs of both species. BABB (benzylalcohol and benzylbenzuolate) clearing was applied to T. macropora. We conclude that trypanorhynch cestodes stained with Mayer-Schuberg's acetic carmine permanently mounted in Canada balsam are suitable for CLSM, allowing detailed analyses of museum type-material as well as freshly collected and processed worms. BABB resulted in imaging the testes in detail, suggesting other stains to be used for CLSM in trypanorhynch cestode research. Application of CLSM for studies of other cestode groups is highly recommended.

Allopodocotyle palmi sp. nov. and Prosorhynchus maternus Bray & Justine, 2006 (Digenea: Opecoelidae & Bucephalidae) from the Orange-Spotted Grouper Epinephelus coioides (Hamilton, 1822) off Bali, Indonesia, Described Using Modern Techniques.

BACKGROUND: The most convincing species of Allopodocotyle Pritchard, 1966 (Digenea: Opecoelidae) are known overwhelmingly from groupers (Serranidae: Epinephelinae). Six species of Allopodocotyle have been reported, collectively, from species of Cromileptes Swainson, 1839, Epinephelus Bloch, 1793 and Plectropomus Oken, 1817. These are A. epinepheli (Yamaguti, 1942), A. heronensis Downie & Cribb, 2011, A. manteri (Saoud & Ramadan, 1984), A. mecopera (Manter, 1940), A. plectropomi (Manter, 1963) and A. serrani (Yamaguti, 1952). In addition, a not yet fully described and unnamed seventh species, morphologically and phylogenetically close to A. epinepheli, was isolated from the orange-spotted grouper Epinephelus coioides (Hamilton, 1822) off Bali, Indonesia in 2016. An eighth species, again from E. coioides off Bali is described herein. METHODS: Morphological and phylogenetic analyses justify the recognition of A. palmi sp. nov., which is also genetically different from the as yet unnamed congener from the same host and locality. For the first time, 3D confocal laser scanning microscopy was applied to study and distinguish Digenea taxonomically. We introduce the 'Palm pattern', a new simplified way to visualise morphometric differences of related digenean taxa. RESULTS: Allopodocotyle palmi sp. nov. is distinguished from its congeners that infect groupers by its elongate body with a size > 2.7 mm and diagonal testes. The ovary is located mainly, and the anterior testis completely, in the posterior half of the body; the uterine coils are in the fourth eighth of the body. The cirrus-sac is 0.75-1.4 (1.1) mm long, its posterior extremity is well separated from the anterior extent of the vitelline fields, just reaching the anterior border of uterine coils. In addition, Prosorhynchus maternus Bray & Justine, 2006 (Bucephalidae) was isolated from E. coioides, representing the first record in Indonesia and the third record for this fish species. CONCLUSION: The biodiversity research in Indonesia is enhanced with a new species description based on modern and newly applied techniques.