The taxonomy of Dactylogyrus Diesing, 1850 (Monogenea: Dactylogyridae), like that of most monopisthocotyleans, relies heavily on the morphology of sclerites of the posterior attachment organ (haptor). However, the associated neuromusculature is essentially unknown and, therefore, the aim of this study was to use confocal microscopy to examine the haptoral neuromusculature in two species of Dactylogyrus: D. amphibothrium Wagener, 1857 and D. crucifer Wagener, 1857. The monogeneans were stained with phalloidin for muscle and with antibodies for FMRFamide and 5HT and confocal reflectance microscopy was used to visualise the sclerites (i.e. anchors, marginal hooks and bars). Both species had a similar architecture of the anchoral musculature, with a pair of extrinsic muscles, two interconnecting muscles and muscles attached to the haptoral wall, anchoral openings and a connecting bar. The musculature of most marginal hooks consisted of retractors and protractors inserted on the proximal ends of the hooks. The two species differed significantly in the musculature of the accessory ventral bar: D. crucifer had a four-rayed bar with an elaborate musculature associated with the marginal hooks and D. amphibothrium had a rod-shaped bar with simple musculature. Patterns of neurotransmitter immunoreactivity (IR) in the haptor were similar in both species: RFamide-IR cells were clustered in a pair of pre-anchoral ganglia interconnected by a neurite bundle, 5HT-IR cells formed a ventral loop projecting neurites to the marginal hooks. The functional roles of haptoral muscles and patterns of neurotransmitters are discussed. In D. crucifer, the confocal reflectance microscopy revealed an additional sclerite (accessory dorsal bar) that had never been described previously in this or any other species of Dactylogyrus, suggesting that the reflected-light technique might be useful in identifying sclerites undetectable by conventional methods.

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Abstract:The taxonomy of Diplectanum Diesing, 1858, a genus of monopisthocotylean monogeneans, remains unsettled and needs to be revised based on new morphological criteria. Recent studies in monopisthocotyleans have shown that the muscle arrangement in the posterior attachment organ (haptor) differs between congeneric species and can be used as an additional criterion in genus-level taxonomy. To explore the possibility of using the haptoral musculature and nervous system in the taxonomy of Diplectanum, we conducted a detailed confocal-microscopy study of three species of Diplectanum (D. aculeatum Parona et Perugia, 1889, D. sciaenae van Beneden et Hesse, 1863 and D. similis Bychowsky, 1957) with phalloidin staining for muscle and indirect immunostaining for 5HT and FMRFamide. A further goal was to clarify the functional mechanics of the haptor and the role of its essential components (squamodiscs and anchors) in attachment to the host. The system of connecting bars and gaffing anchors was found to have a complex musculature consisting of 23 muscles in D. aculeatum and D. sciaenae, and 21 muscles in D. similis. The squamodiscs were shown to be operated by several groups of muscles attached primarily to the area termed the squamodisc fulcrum. Most of the haptoral musculature is identical in D. aculeatum and D. sciaenae and these species differ only in the presence of a muscle sheath around the tissue strand between the squamodiscs in D. sciaenae and in the different patterns of superficial squamodisc muscles. Diplectanum similis shows more significant differences from the other two species: besides lacking two of the haptoral muscles, it also differs in the shapes and arrangement of several other muscles. The nervous system of all three species conforms to the general pattern typical for the Dactylogyroidea and shows little variation between species.

The posterior attachment organs of monogeneans known as haptors show exceptional morphological plasticity and play a major role in monogenean taxonomy. Our knowledge of haptoral musculature, however, is still very limited and further study of this musculature in different groups of monogeneans may be instrumental in improving their taxonomic system and furthering our knowledge of their modes of attachment. This study used phalloidin staining in conjunction with confocal microscopy to examine haptoral musculature in three species of Ligophorus (L. llewelyni Dmitrieva et al., 2007, L. pilengas Sarabeev et Balbuena, 2004 and L. kaohsianghsieni (Gusev, 1962) Gusev, 1985), a member of the monopisthocotylean family Ancyrocephalidae sensu Bychovsky et Nagibina, 1978. Sclerotised structures (anchors, connecting bars and marginal hooks) were visualised simultaneously with muscles by capturing laser light reflected off the surfaces of these sclerites. The haptoral musculature was shown to be identical in L. pilengas and L. llewelyni and essentially similar between these two species and L. kaohsianghsieni, but the latter species had also some notable differences. Common to all three species were thick extrinsic muscles arising from the inner roots of the anchors, a muscle bundle connecting the inner roots of the opposite ventral anchors, muscles running from the outer roots of the anchors to the body wall, muscles originating from the dorsal bar and inserting on the anchoral openings and muscles connecting the bars with anchoral roots. Ligophorus llewelyni and L. pilengas have a more elaborate haptoral musculature than L. kaohsianghsieni with some additional muscles, tendon-like muscle extensions and a strong bracket-shaped muscle presumably functioning as a catch-muscle for the dorsal anchors. Functional roles of individual haptoral muscles in attachment are discussed and the architecture of haptoral musculature in Ligophorus is compared with that of other ancyrocephalids.