Effects of forest cover on richness of threatened fish species in Japan.

Estuaries--one of the most vulnerable ecosystems globally--face anthropogenic threats, including biodiversity loss and the collapse of sustainable fisheries. Determining the factors contributing to the maintenance of estuarine biodiversity, especially that of fish, is vital for promoting estuarine conservation and sustainability. We used environmental DNA metabarcoding analysis to determine fish species composition in 22 estuaries around Japan and measured watershed-scale land-use factors (e.g., population size, urban area percentage, and forest area percentage). We sought to test the hypothesis that the richness of the most vulnerable estuarine fish species (i.e., registered by the Japanese Ministry of the Environment in the national species red-list) is determined by watershed-scale land-use factors. The richness of such species was greater, where forest cover was highest; thus, forest cover contributes to their conservation. The proportion of agriculture cover was associated with low species richness of red-listed fishes (redundancy analysis, adjusted R2 = 43.9% of total variance, df = 5, F = 5.3843, p = 0.0001). The number of red-listed species increased from 3 to 11 along a watershed land-use gradient ranging from a high proportion of agriculture cover to a large proportion of forest cover. Furthermore, the results showed that throughout Japan all the examined watersheds that were covered by >74.8% forest had more than the average (6.7 species per site) richness of red-listed fish species. This result can be attributed to the already high average forest cover in Japan of 67.2%. Our results demonstrate how the land use of watersheds can affect the coastal sea environment and its biodiversity and suggest that proper forest management in conjunction with land-use management may be of prime importance for threatened fish species and coastal ecosystems in general.

Efectos de la Cobertura Forestal sobre la Riqueza de Especies Amenazadas de Peces en Japón Resumen Los estuarios-uno de los ecosistemas más vulnerables a nivel mundial-enfrentan amenazas causadas por el hombre, incluyendo la pérdida de biodiversidad y el colapso de las pesquerías sustentables. La determinación de los factores que contribuyen al mantenimiento de la biodiversidad estuarina, especialmente la de los peces, es vital para fomentar la conservación y sustentabilidad estuarinas. Usamos un análisis de metasecuenciación de ADN ambiental para determinar la composición de especies de peces en 22 estuarios de Japón y medimos los factores del uso de suelo a nivel de cuenca (p. ej.: tamaño poblacional, porcentaje de área urbana y porcentaje de área forestal). Buscamos probar la hipótesis de que la riqueza de las especies de peces más vulnerables (es decir, aquellas registradas por el Ministerio Japonés del Ambiente en la lista roja de especies a nivel nacional) está determinada por los factores de uso de suelo a nivel de cuenca. La riqueza de dichas especies fue mayor en donde la cobertura forestal era la más alta; por lo tanto, la cobertura forestal contribuye a la conservación de estas especies. La proporción de la cobertura agrícola estuvo asociada con una baja riqueza de especies de peces en la lista roja (análisis de redundancia, R2 ajustada = 43.9% de la varianza total, gl = 5, F = 5.3843, p = 0.0001). El número de especies en la lista roja incrementó de 3 a 11 a lo largo del gradiente de uso de suelo de la cuenca, yendo desde una proporción alta de cobertura agrícola a una proporción alta de cobertura forestal. Además, los resultados mostraron que, en Japón, todas las cuencas analizadas que contaban con una cobertura forestal >74.8% tenían mayor riqueza de especies de peces en la lista roja que el promedio (6.7 especies por sitio). Este resultado puede atribuirse a la ya de por sí elevada cobertura forestal de Japón (67.2%). Nuestros resultados demuestran cómo el uso de las cuencas puede afectar al ambiente costero marino y a su biodiversidad y sugiere que la gestión adecuada de los bosques en conjunto con el manejo del uso de suelo puede ser de suma importancia para las especies amenazadas de peces y los ecosistemas costeros en general.

Evaluation of fish biodiversity in estuaries using environmental DNA metabarcoding.

Biodiversity is an important parameter for the evaluation of the extant environmental conditions. Here, we used environmental DNA (eDNA) metabarcoding to investigate fish biodiversity in five different estuaries in Japan. Water samples for eDNA were collected from river mouths and adjacent coastal areas of two estuaries with high degrees of development (the Tama and Miya Rivers) and three estuaries with relatively low degrees of development (the Aka, Takatsu, and Sendai Rivers). A total of 182 fish species across 67 families were detected. Among them, 11 species occurred in all the rivers studied. Rare fishes including endangered species were successfully detected in rich natural rivers. Biodiversity was the highest in the Sendai River and lowest in the Tama River, reflecting the degree of human development along each river. Even though nutrient concentration was low in both the Aka and Sendai Rivers, the latter exhibited greater diversity, including many tropical or subtropical species, owing to its more southern location. Species composition detected by eDNA varied among rivers, reflecting the distribution and migration of fishes. Our results are in accordance with the ecology of each fish species and environmental conditions of each river.

Establishment of MDCKII cell monolayer with metabolic activity by CYP3A4 transduced with recombinant adenovirus.

  This study aims to establish an in vitro system that can assess intestinal first-pass metabolism of CYP3A4 substrate drugs using adenoviral transduction. Madin-Darby canine kidney II (MDCKII) cells were used as a model of intestinal epithelial cells. Recombinant adenovirus expressing green fluorescent protein (AdGFP) and CYP3A4 (AdCYP3A4) was used as vectors. On day 2 after seeding MDCKII cells onto a semipermeable membrane, cells were infected with each adenovirus vector at various MOIs (multiplicities of infection) ranging from 0 to 200. On day 5, cell monolayers were used for drug transport study. The expression of GFP in monolayers of MDCKII cells transduced with AdGFP increased MOI-dependently and adenoviral infection showed no effect on the membrane permeability of drugs. The metabolite formation rate of midazolam, a CYP3A4 substrate, in the permeation process of a monolayer linearly increased with an increase in the MOI of AdCYP3A4. When the period that vectors and cells were located adjacent to each other was prolonged, the rate increased 2-fold compared with that calculated from a result with a monolayer obtained from a shorter period of adjacency. This study indicates that monolayers of MDCKII cells transduced with AdCYP3A4 have the potential to enable estimation of the first-pass metabolism by CYP3A4 in the intestinal absorption process.