Koki Yano’s research while affiliated with National Institute for Basic Biology and other places

Long‐term biodiversity monitoring is necessary for conservation and management. In such circumstances, environmental DNA (eDNA) surveys can enable easy and effective biomonitoring of aquatic insects. However, previous studies of aquatic insects based on the mtDNA COI region have revealed incomplete taxonomic coverage, and frequent amplification of nontarget taxa (e.g., algae and diatoms). Additionally, it has been indicated that there are few reference sequences registered in databases for metabarcoding analysis of insects. Therefore, we developed a new primer set, MtInsects‐16S, for eDNA analyses of insects based on the mtDNA 16S rRNA region in a previous study. To address the insufficient database records of insect DNA sequences, we also constructed a comprehensive reference database of aquatic insects that occur in Kanagawa Prefecture, Japan. We conducted eDNA analyses at six sites in the two river systems (the Sagami‐gawa River and Sakawa‐gawa River systems), with three replicates per site, based on both the mtDNA COI and the 16S rRNA regions. These results were compared physical capture surveys at the same sites to examine the detection capability of eDNA for Insecta with using a well‐established database. Among the list of species which were collected by physical capture surveys, 74.9% were detected by MtInsects‐16S, whereas 40.1% were detected by the primer set of the mtDNA COI region, and also 80.0% were detected by the both primer sets. This study demonstrated that the application of eDNA analyses using the MtInsects‐16S primer set can be conducted with accuracy and reliability, provided that the reference DNA database is improved. The MtInsects‐16S region can be considered indispensable in eDNA analysis for aquatic insects.

Long-term biodiversity monitoring is necessary for the conservation and management of water resources. Notably, aquatic insects have been used as an indicator of water quality because they provide important basic information about freshwater ecosystems and water resources. Although environmental DNA (eDNA) surveys can enable easy and effective biomonitoring of aquatic insects, previous studies have not successfully detected all insect species, and there has been frequent amplification of nontarget taxa (e.g., algae and diatoms). Therefore, we developed a universal primers set, MtInsects-16S, for eDNA analyses of insects in the mtDNA 16SrRNA region. Furthermore, a well-established database of aquatic insects, especially the MtInsects-16S DNA region of Ephemeroptera, Plecoptera, and Trichoptera in Kanagawa Prefecture, which was the target area in this study, was constructed. Therefore, in this study, we conducted eDNA analyses using a universal primers set and using a well-established database. We conducted and compared capture surveys at the same sites to examine the detection capability of eDNA for Insecta. As a result, eDNA analyses using MtInsects-16S not only detected almost all of the captured species but also detected many more species without amplifying nontarget taxa. This study demonstrated the application of eDNA analyses with unprecedented accuracy and reliability. It was also shown that community structure by eDNA reflected a relatively narrow range at the water sampling point. Although the data accumulation for constructing locally specific databases is an urgent issue, using the MtInsects-16S region is expected to be a breakthrough in the metabarcoding of insects.

Floods affect the population structure of organisms that inhabit streams. In recent decades, the scale of floods has become larger due to climate change. Under these circumstances, on 12 October 2019, the largest typhoon in the history of observation in Japan struck the Japanese Archipelago. This typhoon caused heavy rainfall in various places, and the Chikuma-Shinano River System (Japan's largest) suffered great damage. Eight years before the large-scale disturbance in the river system, the population structure of the mayfly Isonychia japonica was studied in detail using quantitative sampling (population numbers and biomass) and by sequencing the mtDNA cytochrome c oxidase subunit I. To understand the impact of the flood on the population and genetic structures, we repeated the same research approximately 1 year after the flood. Direct comparison of sites before and after the flood revealed no significant changes between pre- and post-flood population genetic structure. This indicates high in situ resistance and/or resilience recovery of the populations to the disturbance. We hypothesize that this high resistance/resilience to flood disturbance is a result of strong selection for such traits in the rivers of the Japanese Archipelago, which are short, steep, flow rapidly and violently, and are strongly affected by floods.

DNA barcoding is a powerful tool that provides rapid, accurate, and automatable species identification using standardized genetic region(s), such as for revealing the existence of cryptic species and/or rare species in biodiversity monitoring. DNA barcoding techniques require the development of sets of universal PCR primers for DNA barcoding. We tried to develop universal primer sets, and succeeded in designing not only universal primer sets for DNA barcoding regions of almost all insects, which were designed to include a hypervariable site between highly conserved sites, but also primer sets for longer fragment sequences for registration in a database. We confirmed successful amplification for 14 orders, 43 families, and 68 species with DNA barcoding in the mtDNA 16S rRNA region, and for 13 orders, 42 families, and 66 species with DNA barcoding in the mtDNA 12S rRNA region, including Apterygota and Pterygota (Paleoptera, Polyneoptera, Paraneoptera, and Oligoneoptera). A key feature is that the DNA fragments of the DNA barcoding regions amplified by these primer sets are both short at about 200-bp, and longer fragment sequences will increase the level of data registration in the DNA database. In addition, we evaluated the sensitivity of these newly developed primers using Epeorus aesculus (Heptageniidae), which inhabits a relatively wide range of river systems. The results of this study revealed the existence of a cryptic species or an undescribed species. Such resulting database enhancements will provide opportunities for increasingly accurate assessment of biodiversity and genetic diversity.

DNA barcoding is a powerful tool that provides rapid, accurate, and automatable species identification by using standardized genetic region(s). It can be a powerful tool in various fields of biology such as for revealing the existence of cryptic species and/or rare species and in environmental science such as when monitoring river biota. Biodiversity reduction in recent times has become one of the most serious environmental issues on a worldwide scale. DNA barcoding techniques require the development of sets of universal PCR primers for DNA metabarcoding. We tried to develop universal primer sets for the DNA barcoding of all insect groups. In this study, we succeeded in designing not only universal primer sets for DNA barcoding regions of almost all insects, which were designed to include a hypervariable site between highly conserved sites, but also primer sets for longer fragment sequences for registration in a database. We confirmed successful amplification for 14 orders, 43 families, and 68 species with DNA barcoding in the mtDNA 16S rRNA region, and for 13 orders, 42 families, and 66 species with DNA barcoding in the mtDNA 12S rRNA region. A key feature is that the DNA fragments of the DNA barcoding regions amplified by these primer sets are both short at about 200-bp, and longer fragment sequences will increase the level of data registration in the DNA database. Such resulting database enhancements will serve as a powerful tool for increasingly accurate assessment of biodiversity and genetic diversity.

The intermediate stages of speciation are important for understanding the processes involved in the creation of biodiversity, and also comprise a number of interesting phenomena. However, difficulties are associated with dividing clear speciation stages because speciation is a continuous process. Therefore, the elucidation of speciation is an interesting and important task in evolutionary biology. We herein present an example of a species in an intermediate stage of speciation using the giant water bug Appasus japonicus (Heteroptera, Belostomatidae) that was investigated using mating experiments and phylogenetic analyses of the mtDNA COI (658 bp) and 16S rRNA (435 bp) regions, and nDNA SSR (13 loci) and its genome-wide SNPs (11,241 SNPs). The results of our phylogenetic analyses based on their mtDNA dataset and the genome-wide SNPs dataset strongly supported the paraphyly of the Japanese populations. Therefore, it is suggested that their ancestral lineage which being distributed in the Japanese Archipelago subsequently migrated to the Eurasian Continent (i.e., “back-dispersal” occurred). Furthermore, the results of the mating experiments suggested that among A. japonicus, even between closely related lineages, pre-mating reproductive isolation has been established by the differentiation of copulatory organ morphologies. In contrast, pre-mating reproductive isolation is not established in the absence of the differentiation of copulatory organ morphologies, even if genetic differentiation is prominent. These results suggested that their phylogenetic distance does not predict pre-mating reproductive isolation. Furthermore, in the present study, we present a clear example of pre-mating reproductive isolation driving speciation between closely related lineages.

In August 2017, an outbreak of the stick insect, Ramulus mikado (Rehn, 1904), was observed in the Akashina area in Central Japan. This stick insect is a wingless, non‐flying species that inhabits East Asia. Although it seems that the occurrence of many individuals emerging together had been observed in the 2 or 3 years prior, the outbreak in 2017 was on a far larger scale, and was therefore not comparable to anything observed previously. Indeed, it was also observed that the stick insects fed on almost all leaves of the large Japanese zelkova trees until the trees were bare. Additionally, mass gatherings of the stick insects on the walls of houses in the area were observed. In Japan, there have been no other reports of outbreaks of R. mikado, thus this represents a unique case. In the Akashina area, the population's sex ratio is heavily biased towards females, as is the case with other populations, indicating that they reproduce parthenogenetically. However, among our observations, one individual of the 724 specimens collected in the present study was in fact a male. As there have only ever been about 10 observed cases of R. mikado males, the male individual collected in our field research constitutes a valuable record. We observed an outbreak of the stick insect Ramulus mikado in the Akashina area, Central Japan. Relationship with an insect parasite fungus and a record of male R. mikado are also reported.

We developed microsatellite markers for Appasus japonicus (Heteroptera: Belostomatidae). This belostomatid bug is distributed in East Asia (Japanese Archipelago, Korean Peninsula and mainland China) and often listed as an endangered species in the Red List or the Red Data Book at the national and local level in Japan. Here, we describe twenty novel polymorphic microsatellite loci developed for A. japonicus, and marker suitability was evaluated using 56 individuals from four A. japonicus populations (Nagano, Hiroshima and Yamaguchi prefectures, Japan, and Chungcheongnam-do, Korea). The number of alleles per locus ranged from 1 to 12 (mean = 2.5), and the average observed and expected heterozygosity and fixation index per locus were 0.270, 0.323 and 0.153, respectively. In addition, a population structure analysis was conducted using the software STRUCTURE, and its result suggested that the 20 markers described here will be useful for investigating the genetic structure of A. japonicus populations, which should contribute to population genetics studies of this species.

Oviparous, ovoviviparous and viviparous reproduction are interesting subjects for understanding animals' evolutionary pathways and adaptation to their life history and habitat conditions. In this study, we examined the reproductive mode of the ovoviviparous mayfly Cloeon dipterum, particularly comparing embryogenesis between hand-pairing and unmated females' common oviduct. Our study suggested that the high developmental rate of C. dipterum observed in a recent study could be ascribed to their absorption of unfertilized eggs. The developmental rates of hand-paired females were almost 100%, while their egg-bearing numbers were lower than those of virgin females. Thus, such reduced egg numbers suggest the maternal absorption of unfertilized eggs. This trait is thought to have evolved with the ovoviviparous characteristics of C. dipterum. We identified the basis of the irregularity of this species exhibiting such a high (i.e., 100%) developmental rate in our previous recent study.