Effectiveness of blocking primers and a peptide nucleic acid (PNA) clamp for 18S metabarcoding dietary analysis of herbivorous fish.

The structure of food webs and carbon flow in aquatic ecosystems can be better understood by studying contributing factors such as the diets of herbivorous fish. Metabarcoding using a high-throughput sequencer has recently been used to clarify prey organisms of various fish except herbivorous fish. Since sequences of predator fish have dominated in sequences obtained by metabarcoding, we investigated a method for suppressing the amplification of fish DNA by using a blocking primer or peptide nucleic acid (PNA) clamp to determine the prey organisms of herbivorous fish. We designed three blocking primers and one PNA clamp that anneal to fish-specific sequences and examined how efficient they were in suppressing DNA amplification in various herbivorous fish. The results showed that the PNA clamp completely suppressed fish DNA amplification, and one of the blocking primers suppressed fish DNA amplification but less efficiently than the PNA clamp. Finally, we conducted metabarcoding using mock community samples as templates to determine whether the blocking primer or the PNA clamp was effective in suppressing fish DNA amplification. The results showed that the PNA clamp suppressed 99.3%-99.9% of fish DNA amplification, whereas the blocking primer suppressed 3.3%-32.9%. Therefore, we propose the application of the PNA clamp for clarifying the prey organisms and food preferences of various herbivorous fish.

Marine protected area restricts demographic connectivity: Dissimilarity in a marine environment can function as a biological barrier.

The establishment of marine protected areas (MPAs) can often lead to environmental differences between MPAs and fishing zones. To determine the effects on marine dispersal of environmental dissimilarity between an MPA and fishing zone, we examined the abundance and recruitment patterns of two anemonefishes (Amphiprion frenatus and A. perideraion) that inhabit sea anemones in different management zones (i.e., an MPA and two fishing zones) by performing a field survey and a genetic parentage analysis. We found lower levels of abundance per anemone in the MPA compared to the fishing zones for both species (n = 1,525 anemones, p = .032). The parentage analysis also showed that lower numbers of fishes were recruited from the fishing zones and outside of the study area into each anemone in the MPA than into each anemone in the fishing zones (n = 1,525 anemones, p < .017). However, the number of self-recruit production per female did not differ between the MPA and fishing zones (n = 384 females, p = .516). Because the ocean currents around the study site were unlikely to cause a lower settlement intensity of larvae in the MPA, the ocean circulation was not considered crucial to the observed abundance and recruitment patterns. Instead, stronger top-down control and/or a lower density of host anemones in the MPA were potential factors for such patterns. Our results highlight the importance of dissimilarity in a marine environment as a factor that affects connectivity.

The Kuroshio current influences genetic diversity and population genetic structure of a tropical seagrass, Enhalus acoroides.

Information on genetic diversity and differentiation of seagrass populations is essential for the conservation of coastal ecosystems. However, little is known about the seagrasses in the Indo-West Pacific Ocean, where the world's highest diversity of seagrasses occurs. The influence of sea currents on these populations is also unknown. We estimated the genetic diversity and population genetic structure and identified reproductive features in Enhalus acoroides populations from the Yaeyama Islands, Hainan Island and the Philippines. The Philippines are situated at the centre of the E. acoroides range, Yaeyama and Hainan are peripheral populations, and the Yaeyama population is at the northern limit of the species range. The powerful Kuroshio Current flows from the Philippines to Yaeyama. Genetic analyses using nine microsatellite markers indicated that reproduction of E. acoroides is mostly sexual. Clonal diversity does not decrease in northern populations, although genetic diversity does. However, the genetic diversity of the Yaeyama populations is greater than that of the Hainan populations. Significant genetic differentiation among most populations was evident; however, the Yaeyama and north-east Philippines populations were genetically similar, despite being separated by ~1100 km. An assignment test suggested that recruitment occurs from the north-east Philippines to Yaeyama. The strong current in this region is probably responsible for the extant genetic diversity and recruitment patterns.

17-year change in species composition of mixed seagrass beds around Santiago Island, Bolinao, the northwestern Philippines.

Effects of fish culture can alter the adjacent ecosystems. This study compared seagrass species compositions in 2012 with those in 1995, when fish culture was less intensive compared to 2012 in the region. Observations were conducted at the same four sites around Santiago Island, Bolinao: (1) Silaqui Island, (2) Binaballian Loob, (3) Pislatan and (4) Santa Barbara, and by using the same methods as those of Bach et al. (1998). These sites were originally selected along a siltation gradient, ranging from Site 1, the most pristine, to Site 4, a heavily silted site. By 2012, fish culture had expanded around Sites 2, 3 and 4, where chlorophyll a (Chl a) was greater in 2012 than in 1995 by one order of magnitude. Enhalus acoroides and Cymodocea serrulata, which were recorded in 1995, were no longer present at Site 4, where both siltation and nutrient load are heavy.

Habitat use by fishes in coral reefs, seagrass beds and mangrove habitats in the Philippines.

Understanding the interconnectivity of organisms among different habitats is a key requirement for generating effective management plans in coastal ecosystems, particularly when determining component habitat structures in marine protected areas. To elucidate the patterns of habitat use by fishes among coral, seagrass, and mangrove habitats, and between natural and transplanted mangroves, visual censuses were conducted semiannually at two sites in the Philippines during September and March 2010-2012. In total, 265 species and 15,930 individuals were recorded. Species richness and abundance of fishes were significantly higher in coral reefs (234 species, 12,306 individuals) than in seagrass (38 species, 1,198 individuals) and mangrove (47 species, 2,426 individuals) habitats. Similarity tests revealed a highly significant difference among the three habitats. Fishes exhibited two different strategies for habitat use, inhabiting either a single (85.6% of recorded species) or several habitats (14.4%). Some fish that utilized multiple habitats, such as Lutjanus monostigma and Parupeneus barberinus, showed possible ontogenetic habitat shifts from mangroves and/or seagrass habitats to coral reefs. Moreover, over 20% of commercial fish species used multiple habitats, highlighting the importance of including different habitat types within marine protected areas to achieve efficient and effective resource management. Neither species richness nor abundance of fishes significantly differed between natural and transplanted mangroves. In addition, 14 fish species were recorded in a 20-year-old transplanted mangrove area, and over 90% of these species used multiple habitats, further demonstrating the key role of transplanted mangroves as a reef fish habitat in this region.

Development of 10 novel polymorphic microsatellite markers for the Indo-Pacific horned starfish, Protoreaster nodosus.

Ten novel polymorphic nuclear microsatellite simple sequence repeat (SSR) markers were isolated from an Indo-Pacific horned starfish, Protoreaster nodosus. The isolated SSR markers provided polymorphisms of 2-9 alleles per locus in three populations obtained from Indonesia and the Philippines. The expected and observed heterozygosities ranged from 0.049 to 0.691 and from 0.050 to 0.800, respectively. Pairwise FST values among three populations ranged from 0.018 to 0.050 (global FST=0.031). All P. nodosus individuals collected from three populations exhibited different genotypes for the ten identified SSR markers, indicating that P. nodosus reproduces sexually. Then the developed SSR markers will be useful for studying the population and conservation genetics of P. nodosus.