Zoological Science

We previously established two strains of Mongolian gerbil: a seizure-sensitive strain, established by selective inbreeding for motor seizures elicited by a stimulus called the S method and a seizure-resistant strain that does not exhibit inducible seizures. The behavior of the seizure-sensitive strain is characterized by a progressive increase in responsiveness to weekly application of the S method, from repetitive backward ear movements appearing after postnatal day 40, to a full-blown seizure, while the seizure-resistant strain is apparently unaffected by the stimulation. The difference between these two strains is presumably genetic. To determine the genetic factors underlying this difference, we first examined developmental changes in the stimulus-induced behavior of the F1 hybrids. When the S method was applied, most F1 hybrids had repetitive movements of the ears (and head) similar to the seizure-sensitive gerbils, but generalized seizures emerged considerably later than in seizure-sensitive gerbils. These results suggest that a half dose of the gene products involved renders most gerbils susceptible to the stimulus but is insufficient for the rapid accumulation of an as yet undefined change needed to spread the abnormal electrophysiologic activity to elicit generalized seizures.

We established a classical conditioning procedure for the cockroach, Periplaneta americana, by which odors were associated with reward or punishment. Cockroaches underwent differential conditioning trials in which peppermint odor was associated with sucrose solution and vanilla odor was associated with saline solution. Odor preference of cockroaches was tested by allowing them to choose between peppermint and vanilla sources. Cockroaches that had undergone one set of differential conditioning trials exhibited a significantly greater preference for peppermint odor than did untrained cockroaches. Memory formed by three sets of differential conditioning trials, with an inter-trial interval of 5 min, was retained at least 4 days after conditioning. This conditioning procedure was effective even for cockroaches that had been harnessed in plastic tubes. This study shows, for the first time in hemimetaborous insects, that both freely moving and harnessed insects are capable of forming olfactory memory by classical conditioning procedure. This procedure may be useful for future electrophysiological and pharmacological studies aimed at elucidation of neural mechanisms underlying olfactory learning and memory.

Certain proteins encoded by mitochondrial DNA (mt-DNA), including mt-ribosomal protein S4 (rps4), appear to play important roles in the initiation of cell differentiation. Partial disruption of rps4 in Dictyostelium discoideum Ax-2 cells by means of homologous recombination greatly impairs the progression of differentiation, while the the rps4 ^OE cells in which the rps4 mRNA was overexpressed in the extra-mitochondrial cytoplasm exhibit enhanced differentiation (Inazu et al., 1999). We have prepared a specific anti-RPS4 antibody and generated transformants (rps4 ^AS cells) by antisense-mediated gene inactivation of rps4. Surprisingly, in the rps4 ^AS cells the progress of differentiation was found to be markedly inhibited, suggesting that the antisense rps4 RNA synthesized in the extra-mitochondrial cytoplasm might be as effective as the partial disruption of rps4 gene. Immunostaining of the rps4 ^OE cells with the anti-RPS4 antibody demonstrated that the RPS4 protein synthesized in the extra-mitochondrial cytoplasm is capable of moving to the nucleus, as predicted by PSORTII. Taken together with the results obtained using immunostained Ax-2 cells, we propose a possible pathway of RPS4 translocation coupled with differentiation.

A survey to examine genetic variation among Hynobius naevius from four localities of Fukuoka Pref., northern Kyushu, Japan, resulted in the detection of two, sympatric, genetic types (A and B) that are clearly different in the allelic frequencies of four loci (ACOH-A, ACOH-B, ADH-A, and SOD-A) in each locality. Morphological investigations between the two genetic types also proved that they are clearly discriminated; the type A is about 75 mm in SVL, lacks mottling pattern on bluish purple dorsum, and possesses relatively short vomerine teeth series, while the type B is about 60 mm in SVL, and has light mottling on reddish purple ground color. These results strongly suggest that reproductive isolation occurs between these two types, and that they could be regarded as separate species. Populations from Toyota-cho, western Honshu, and Yabe-machi, central Kyushu, both close to Fukuoka Pref., were very similar to the types A and B, respectively. From these results, we consider that two evolutionary lineages that first evolved allopatrically in western Honshu and southern Kyushu secondarily contacted and became sympatric in the region of northern Kyushu.

Phylogeographic characteristics and population structure of Japanese wild boar (Sus scrofa leucomystax) were investigated using mitochondrial DNA (mtDNA) sequence data. Sixteen Japanese wild boar haplotypes detected from partial sequences of the mtDNA control region (574-bp) from 180 Japanese wild boar specimens from 10 local populations on Honshu, Shikoku, and Kyushu islands and 41 haplotypes from other S. scrofa were analyzed using the neighbor-joining method. The Japanese wild boars were more closely related to Northeast Asian wild boars from Mongolia than to the other Asian continental S. scrofa. The Japanese and Northeast Asian wild boars were not significantly distinguished by corrected average pairwise difference analysis. The ancestors of Japanese wild boars are suggested to have been part of the continental S. scrofa population that spread from Southeast to Northeast Asia during the Middle to Late Pleistocene. The Japanese wild boar mtDNA haplotype cladogram shows 95% parsimoniously plausible branch connections supporting three sympatric clades. Nested clade analysis indicates that these three clades are the result of distinct historical events or gene flow. The present population of Japanese wild boars may have been formed by a few independent migrations of distinct clades from the continent with subsequent mixing on the Japanese Islands.