title: The Time Course and Neuroanatomy of Rhinophore Regeneration in the Nudibranch Berghia stephanieae
Creators: name: Maroyan, Ani
Publisher: name: University of Massachusetts Amherst
Year: 2021

description: Within five weeks, the nudibranch, Berghia stephanieae (Gastropoda, Mollusca) can regenerate a severed rhinophore, the main olfactory appendage, such that it is indistinguishable from a non-lesioned rhinophore. The rhinophore is a 2 mm long stalk with lateral sides covered in ridges and the distal third covered in fluorescent pigmentation. Its internal morphology is dominated by longitudinal musculature, overlaid by a complex neuronal plexus, which contains neurons that express various neuroactive substances including serotonin and small cardioactive peptide (SCP). Two large nerves originate in the rhinophore ganglion at the base of the rhinophore and run the length of the rhinophore. To quantify the regeneration of these external and internal characteristics, the rhinophore was entirely severed, with animals then sacrificed and dissected at one-day, three-days, five-days, seven-days, 21-days, and 35-days post-lesion. Immunohistochemical and neuronal tracing techniques were used to study the neuroanatomy of the regenerated structure. At each time point, brains, consisting of the cerebral, pleural, and pedal ganglia with attached rhinophores were quadruple stained with 4′,6-diamidino-2-phenylindole (DAPI) and phalloidin to label nuclei and actin respectively, and immunolabeled for SCP and serotonin. The length of regenerated rhinophores was measured and compared with the unlesioned side. There was a period of disorganization in the first five days, followed by rapid increase in size and a gradual slowing of growth from 21 days to 35 days. Ridge reappearance followed a similar trajectory, regrowing from the base of the rhinophore, rapidly after the first five days with gradual slowing and not matching the extent seen on unlesioned appendages. Fluorescent pigmentation on the distal part of the rhinophore was the slowest feature to return, first visible at 21-days post-lesion. These external features were still not equivalent to those seen in unlesioned rhinophores at 35 days. Internally, phalloidin labeling showed that the muscles were disorganized during the first week, but then regrew, matching the return of the external anatomy. Muscle fiber thickness did not vary between regenerated and unlesioned rhinophores. The SCP and serotonin immunoreactivity exhibited different patterns of regrowth. Serotonin fibers were present immediately after lesion at the tip of the rhinophore and increased in density over the rest of the rhinophore with increasing time post-lesion. The SCP plexus, in contrast, was not present until a week after lesion, and at later timepoints there was not a consistent pattern. Lastly, the projections into and from the brain were traced by application of neurobiotin to the cut ends of the rhinophore nerves. There was no obvious difference between regenerated and unlesioned sides five-weeks after the removal of one rhinophore; all major neural tracts were present, including those that traveled from the regenerated side contralaterally through the brain and up into the contralateral rhinophore ganglion. The neuroanatomy of Berghia’s brain and rhinophores thus suggests that after complete severance of one rhinophore, the animal is able to regrow the cells and fiber tracts in five weeks.