Tadashi Aotsuka

Replication Origin of Mitochondrial DNA in Insects

The precise position of the replication origin (OR) of mtDNA was determined for insect species belonging to four different orders (four species of Drosophila, Bombyx mori, Triborium castaneum, and Locusta migratoria, which belong to Diptera, Lepidoptera, Coleoptera, and Orthoptera, respectively). Since the free 5′ ends of the DNA strands of mtDNA are interpreted as the OR, their positions were mapped at 1-nucleotide resolution within the A + T-rich region by using the ligation-mediated PCR method. In all species examined, the free 5′ ends were found within a very narrow range of several nucleotides in the A + T-rich region. For four species of Drosophila, B. mori, and T. castaneum, which belong to holometabolous insects, although the ORs were located at different positions, they were located immediately downstream of a series of thymine nucleotides, the so-called T-stretch. These results strongly indicate that the T-stretch is involved in the recognition of the OR of mtDNA at least among holometabolous insects. For L. migratoria (hemimetabolous insect), on the other hand, none of the long stretches of Ts was found in the upstream portion of the OR, suggesting that the regulatory sequences involved in the replication initiation process have changed through insect evolution.

Rapid isolation method of animal mitochondrial DNA by the alkaline lysis procedure

In recent years, the fields of evolutionary and population genetics have utilized techniques that were developed for direct analysis of variations in DNA sequences. Animal mitochondrial DNA (mtDNA) is of particular interest in these studies, due partly to the relative ease with which it can be isolated (Arise, 1986). Ultracentrifugation involving cesium chloride gradients is commonly used for the isolation of mtDNA (Lansman et al., 1981), however, expensive equipment and relatively long experimental times are required for this method. Simplified and more rapid isolation procedures were presented by Powell and Z6fiiga (1983) and Carr and Griffith (1987). In spite of these methods, the isolation of mtDNA is still laborious and is, therefore, becoming the limiting factor for the study of mtDNA in population biology, where numerous samples need to be analyzed for the particular characteristic of interest. We report here a simple and rapid procedure for the isolation of mtDNA from Drosophila. The method involves the alkaline lysis procedure, which is commonly used for the isolation of plasmid DNA (Maniatis et al., 1982).

Phylogeny of the Drosophila immigrans Species Group (Diptera: Drosophilidae) Based on Adh and Gpdh Sequences

Abstract The immigrans species group in the Drosophilinae is one of the representative species groups of Drosophila in East Asia. Although this group constitutes a significant part of the drosophilid fauna in the Old World, only a few species have been analyzed in previous molecular phylogenetic studies. To study the phylogeny of the immigrans group, we analyzed the nucleotide sequences of two nuclear genes, alcohol dehydrogenase (Adh) and glycerol-3-phosphate dehydrogenase (Gpdh), for 36 drosophilid species, including 12 species of the immigrans group. In the resultant phylogenetic trees, 10 species of the immigrans group (D. immigrans, D. formosana, D. ruberrima, D. albomicans, D. nasuta, D. neonasuta, D. pallidifrons, D. hypocausta, D. neohypocausta, D. siamana) consistently formed a clade (the immigrans group proper), although the phylogeny within this clade did not exactly correspond to the classification of species subgroups. However, D. annulipes and D. quadrilineata, both of which belong to the quadrilineata subgroup of the immigrans group, were not included in the immigrans group proper. Furthermore, we obtained the unexpected result that D. annulipes was included in a clade comprising Scaptomyza and Hawaiian Drosophila, together with D. maculinotata of the funebris group, although the phylogenetic relationships within this clade remain uncertain and need to be substantiated with further studies. Thus, according to the present study, the immigrans group is polyphyletic.

PCR Error and Molecular Population Genetics

The polymerase chain reaction (PCR) technique (Saiki et al., 1988) is a very useful tool for population genetic studies at the DNA level. However, there is an inherent problem in the PCR technique because the DNA fragments ampli® ed are subject to nucleotide substitution during the reaction processes. The rate and pattern of arti® cial substitution have already been estimated by several authors (e.g., Lundberg et al., 1991), and its in ̄ uence on phylogenetic studies has been also discussed (Kwiatowski et al., 1991). However, the in ̄ uence of PCR error on population genetic studies has not been well documented. Determinations of the sequences from PCR products were performed by two methods, cloning and direct sequencing. Assuming that the sequences obtained by the direct sequencing method are correct, we discuss the in ̄ uence of the error on the estimates of various parameters commonly used in molecular population genetics. In addition, we evaluate the efficiency of Pfu polymerase to improve the ® delity of PCR.

Molecular Phylogeny of Twelve Asian Species of Epilachnine Ladybird Beetles(Coleoptera, Coccinellidae)with Notes on the Direction of Host Shifts

Abstract We determined the nucleotide sequences of a part of the mitochondrial cytochrome c oxidase I gene (1,000 bp) for twelve species of Asian phytophagous ladybird beetles belonging to the genus Epilachna, and constructed molecular phylogenetic trees for ten “Henosepilachna” species, using two “Epilachna” species as outgroups. Based on the suggested phylogenetic trees, we discussed taxonomic issues and the direction of host shift in these epilachnines.

Phylogenetic Position of the Subgenus Lordiphosa of the Genus Drosophila (Diptera: Drosophilidae) Inferred from Alcohol Dehydrogenase (Adh) Gene Sequences

Abstract. We analyzed the phylogenetic relationship between the species of Lordiphosa and other Drosophilidae using alcohol dehydrogenase (Adh) gene sequences. The phylogenetic trees consistently show that the four species Drosophila kurokawai, D. collinella, D. stackelbergi, and D. clarofinis, which include three species groups of Lordiphosa, form a monophyletic clade. This clade is placed as a sister group to the willistoni and saltans groups of Sophophora. On the other hand, three species of Lordiphosa, D. tenuicauda, D. pseudotenuicauda, and D. acutissima, all of which belong to the tenuicauda group, are not shown to be related to the major Lordiphosa lineage. In the phylogenetic trees, these species are included into the clade comprised of Drosophila and Hirtodrosophila, although it remains uncertain whether the tenuicauda group is a monophyletic group or not. These results indicate that Lordiphosa is polyphyletic and that most of the members of the subgenus have a close relationship to the neotropical groups of Sophophora. The above conclusion is compatible with the hypothesis of Okada (Mushi [1963] 37:79–100) and Lastovka and Máca (Acta Ent Bohemoslov [1978] 75:404–420) that Lordiphosa is most closely related to Sophophora; in contrast, our results contradict the hypothesis of Grimaldi (Bull Am Mus Nat Hist [1990] 197:1–139) that Lordiphosa is a sister group to the genus Scaptomyza.

A novel chimeric gene, siren, with retroposed promoter sequence in the Drosophila bipectinata complex.

Retrotransposons often produce a copy of host genes by their reverse transcriptase activity operating on host gene transcripts. Since transcripts normally do not contain promoter, a retroposed gene copy usually becomes a retropseudogene. However, in Drosophila bipectinata and a closely related species we found a new chimeric gene, whose promoter was likely produced by retroposition. This chimeric gene, named siren, consists of a tandem duplicate of Adh and a retroposed fragment of CG11779 containing the promoter and a partial intron in addition to the first exon. We found that this unusual structure of a retroposed fragment was obtained by retroposition of nanos, which overlaps with CG11779 on the complementary strand. The potential of retroposition to produce a copy of promoter and intron sequences in the context of gene overlapping was demonstrated.

Two Cryptic Species of the Phytophagous Ladybird Beetle Epilachna vigintioctopunctata (Coleoptera: Coccinellidae) Detected by Analyses of Mitochondrial DNA and Karyotypes, and Crossing Experiments

Abstract Analyses of a part of mitochondrial cytochrome c oxidase I gene sequences (645 bp) for seventeen individuals of Epilachna vigintioctopunctata (Fabricius) from eight localities in east and southeast Asia revealed that the populations are divided into two genetically distinct groups (Chiba, Tokyo, Naha, Iriomote, Bangkok vs. Kuala Lumpur, Padang, Bogor). The number of nucleotide substitutions between sequences of different groups was 57–60, while that between sequences within each group was 1–8. Karyotypes of the two groups were also distinctly different. Crossing experiments showed that there exist strong postmating barriers between the two groups: eggs obtained from between-group crossings usually did not hatch, whereas more than 90% of eggs from within-group crossings hatched. It is concluded that E. vigintioctopunctata, a notorious pest of solanaceous crops in Asia and Australia, is composed of at least two reproductively isolated biological species that probably occupy different geographic ranges.

Drosophila subpulchrella, a new species of the Drosophila suzukii species subgroup from Japan and China (Diptera: Drosophilidae)

Drosophila (Sophophora) subpulchrella Takamori and Watabe, sp. nov., of the D. suzukii subgroup in the D. melanogaster species group, is described from Japan and southern China, and compared with its sibling species, D. pulchrella Tan et al. distributed in the Yun‐Gui Highland, south‐western China. The results of cross‐experiments show a complete pre‐mating isolation between D. subpulchrella and D. pulchrella.

Sexual dimorphism and courtship behavior in Drosophila prolongata

Sexual dimorphism is often derived from sexual selection. In sexually dimorphic Drosophila species, exaggerated male structures are used for specific behaviors in male-to-male competition or courtship toward females. In Drosophila prolongata, a member of the melanogaster species group, males have enlarged forelegs whereas females do not. However, the adaptive role of the enlarged forelegs is unclear because little is known about the behavior of D. prolongata. In this study, the courtship behavior of D. prolongata was investigated in comparison with closely related species. Males of D. prolongata use their forelegs in a specific behavior, “leg vibration”, in which the male vigorously vibrates the female’s abdomen by extending his forelegs from in front of her. Leg vibration was observed immediately before “attempting copulation”, indicating that it has an adaptive role in the mating process. In contrast, leg vibration was not observed in closely related species. Because the large forelegs are necessary to accomplish leg vibration, it was suggested that the sexual dimorphism of D. prolongata forelegs is currently under the influence of sexual selection in courtship behavior.