Environmental DNA Collected from Snow Tracks is Useful for Identification of Mammalian Species.

Noninvasive genetic analysis is being used increasingly in field surveys. However, detecting large and middle-sized mammals, such as Carnivora species, using noninvasive samples, such as scat or hair, is time- and labor-intensive due to their low densities and elusive behaviors. As snow tracks are the most frequently encountered natural signs of terrestrial mammals in winter, we employed several methods to recover environmental DNA (eDNA) from snow tracks. We performed both DNA metabarcoding and Sanger sequence analyses, in combination with universal primers on the mitochondrial 12S rRNA gene for mammals and taxon-specific primers on the mitochondrial NADH dehydrogenase subunit 2 gene for Martes species (martens and sables in Mustelidae). Snow samples of four Martes melampus tracks, one Cervus nippon track, one Vulpes vulpes track, and the track of an unidentified Carnivora species were collected from a snowfall area in Kyoto, Japan, in February 2018. Regarding DNA metabarcoding analyses, the sequences of three Carnivora species (M. melampus, V. vulpes, and Canis lupus familiaris) and a deer (C. nippon) were obtained from their respective snow tracks. Using Sanger sequencing, eDNA on snow tracks was recovered at the species level except for M. melampus using universal primers, while eDNA of M. melampus was sequenced using Martes-specific primers. Snow track surveys in combination with eDNA techniques could dramatically improve the efficiency of monitoring and conservation of mammals.

Rear-side localization of the centrosome in migrating neuroblastoma Neuro-2a cells and its roles in process elongation.

Axon elongation is usually performed by the migration of growth cones that leave axons. Axon microtubules are generated by enhanced polymerization of tubulin in the growth cones. Some kinds of neurons like cerebellar granule cells, however, generate axons as a result of migration of the cell body leaving axons at the rear. The mechanism to generate microtubules during such growth cone-independent elongation of axons is not well understood. To establish an experimental model to study this mechanism, we cultured neuroblastoma (Neuro-2a) cells on substrates that facilitate cell migration. When cultured on laminin-treated substrate, cells migrated actively and left processes at the rear. We investigated the role of the centrosome in this process formation. The centrosomes were always located at the base of the processes, i.e., at the rear side of the migrating cell body. Close observation of cytoskeletons revealed microtubules limited around the centrosomes, but concentrated at the periphery of the cells or within the processes. Microtubule regrowth experiments showed the ability of the centrosomes to nucleate microtubules. We thus examined the role of microtubule release from the centrosomes, by knocking down the expression of spastin, a microtubule-severing enzyme. Introducing siRNA for spastin into Neuro-2a cells reduced both the migration speed and the length of the processes. Taken together, Neuro-2a cells on laminin proved useful as a model to study the alternative type of axon elongation in which cell migration leaves axons at the rear. This model provided evidence for the involvement of microtubule release from centrosomes in the mechanisms for this type of process elongation.