Drieschia sp. MicroCT-Scans for 3D reconstruction

Annelids are predominantly found along the seafloor, but over time have colonised a vast diversity of habitats, such as the water column, where different modes of locomotion are necessary. Yet, little is known about their potential muscular adaptation to the continuously swimming required in the water column. The musculature and motility were examined for five scale worm species of Polynoidae (Aphroditiformia, Annelida) found in shallow waters, deep sea and caves that exhibit crawling, occasional swimming or continuous swimming, respectively. Their parapodial musculature was reconstructed using microCT and computational 3D analyses and the muscular functions interpreted from video recordings of their locomotion. Since most benthic annelids are able to swim for short distances using body and parapodial muscle movements, suitable musculature for swimming and a pelagic lifestyle is already present. Our results also indicate that rather than rearrangements or addition of muscles, a shift to a pelagic lifestyle is mainly accompanied by structural loss of muscle bundles and density, as well as elongation of extrinsic dorsal and ventral parapodial muscles. In addition, our study documents clear differences in locomotion and muscular arrangement among closely related annelids with different lifestyles as well as points to myoanatomical adaptations for accessing the water column.
Methods
All microCT scans were made at the Smithsonian National Museum of Natural History, Washington D.C.,USA. Scans were obtained from at least two individuals from each species for a resolution of 102.0 um/voxel for G. jameensis; 112.6um/voxel for P. iliffei; 71.208um/voxel for M. longipalpa; 103.3um/voxel for H. imbricata and 63.9um/voxel for Branchipolynoe sp.Specimens not already fixed in ethanol were dehydrated through a dilution series to70% ethanol. Postprocessing was done over a 2-3 step process fromthe initial fixative to DI-water, followed by an additional 3-5 incremental steps from water to 70% ethanol. Once in 70% ethanol, all animals were placed in either individual vials or 6-wellplates covered with parafilm containing 0.3-0.6% PTA (phosphotungstic acid)in 70% ethanol. Samples were kept at roomtemperature with gentle rocking from 5-15 days. Fresh PTA was exchanged every 3-4 days. After staining, samples were washed with 70% ethanol over 2-3 days at room temperature with gentle rocking and regular rinsing. After rinsing, samples were prepared for scanning by being individually placed in pipettetips filled with either 70% ethanol or with 0.5% low melt agarose made with DI-water. Tips werepre-sealedat the bottom by melting over a flame before adding the sample, ethanol or agarose. Melted paraffin wasused to seal the tops of the pipette tips.To create a mount for the specimens, a single pipette tip was cut inhalf and hot-glued to ~2 mm carbon rod, then in turn, each specimen within their own pipette tip, was nested within the halved pipette tip and secured using dental wax. All specimens were scanned using thenano tube (180kV) on theGE Phoenix v|tome|x M 240/180kV Dual TubemicroCT machine.