**1. Introduction**

In Japan, conservation and regeneration projects have been actively conducted for largesized birds such as the Japanese crested ibis, *Nipponia Nippon*, the oriental white stork, *Cico‐ nia boyciana* and the intermediate egret, *Ardea intermedia* (Photo 1) that inhabit rural areas [1, 2]. Many people are highly interested in these projects and a lot of information about growth and breeding for large-sized birds is broadcasted through television, radio and internet me‐ dia. In such a situation, a conspicuous topic has been found in recent months, that is, 2 indi‐ viduals of the Japanese crested ibis displayed beriberi symptom along with human being, because of overeating great favorite food that is the Dojo loach, *Misgurnus anguillicaudatus*. Their beriberi symptom appeared to be caused by eating the Dojo loach raw. The 2 individu‐ als were diagnosed as follows; this beriberi symptom occurred as vitamin B1 in the individ‐ ual bodies was destroyed by tiaminase enzyme contained in the Dojo loach. At present the two individuals may have completely recovered from the beriberi symptom through vita‐ min B1 supplementation by injection.

By the way, the presence of 10 or more loach species including the Dojo loach has been observed around paddy fields in rural areas, Japan. Most loach species appear to become food attractive for large-sized birds (Photo 1) [3] and one of the reasons is that the loach species cannot move as rapidly as swimming species such as the Japanese dace, *Tribolodon hakonensis* and the Ayu, *Plecoglossus altivelis altivelis*; hence large-sized birds are able to easily catch them. In addition, only the Dojo loach has been investigated, but nutrition contained in this loach was superior to other fish species; for instance, amount of calcium

in the Dojo was 9 times that of the Japanese ell, *Anguilla japonica*, and also the Dojo had the most amount of vitamin B2 in all fish [4, 5]. Actually these precise nutrient compo‐ nents may somewhat differ among the loach species, but their nutrient components could have to be fundamentally similar.

**Photo 1.** 2 individuals of the intermediate egret, *Ardea intermedia* that are finding individuals of many loach species as their food in paddy field (unpublished photo)

**Photo 2.** An adult of the Hotoke loach, *Lefua echigonia* (the above) with approximately 60 mm in body length and

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In recent years, because importance of ecosystem and biodiversity in rural areas has been deeply realized, various research activities have been carried out for conserving and recov‐ ering populations of the Hotoke loach. Distribution pattern and habitat characteristics of this loach were elucidated in some rural areas [15-18], manners of habitat utilization and migra‐ tion routes for the species were investigated [11, 12, 19, 20] and techniques of artificial prop‐ agation were developed with human chorionic gonadotropin [21-23]. Further, molecular analyses of phylogeography of the Hotoke loach using DNA sequences of mitochondrial genes revealed that populations of the species were evolutionally separated into a total of 7

Unfortunately, there is also another serious concern left in populations of the Hotoke loach. That is, as this loach has experienced, diminishment of population size may often cause to improve not only fragmentation among populations but also inbreeding among individuals. Such populations tend to have distinctly poor genetic diversity, occasionally threatened with extinction [30-33]. Usually, to evaluate genetic diversity including genetic population structure for such populations, polymorphism analysis has been performed using microsa‐ tellite loci in nuclear genome [33-35]. Only preliminary investigations, however, were imple‐ mented for populations the Hotoke loach [36-38], although microsatellite analyses have been

typical earth ditch (the bottom) where the loach inhabits around paddy field (unpublished photo)

genetic clades in Japan [24-29].

carried out for populations of several endangered species.

However, some of the loach species have confronted a kind of serious concerns, especially a decrease in their population size. In Japan, we have conducted many land consolidation projects for rising rice production and easing agricultural works in rural area since 1960s. In land consolidation projects, concrete canals, drops, diversion weirs, etc. have been installed around paddy fields as agricultural infrastructures; therefore not only fish populations and their habitats but also all of ecosystem and biodiversity in rural area have been extremely damaged [6-9].

The Hotoke loach, *Lefua echigonia* endemic to Japan (the above in Photo 2) has been well known as a representative loach species has been adversely impacted on its habitat due to land consolidation projects. Since populations of this loach have rapidly declined in some rural areas, consequently the Hotoke loach has been designated as an endangered species on the Red List of Japan [10]. Ecology of the loach is briefed as follows; this species is widely distributed across the Honshu Island from the Tohoku region to the Kinki region. They usu‐ ally inhabits earth canals and ditches around paddy fields into which ground water flows (the bottom in Photo 2) [11, 12]. The Hotoke loach often coexists with the Dojo loach in the habitat and geographic variations for this loach based on morphological characteristics is obscure [13, 14].

Genetic Diversity and Population Structure of the Hotoke Loach, *Lefua echigonia*, a Japanese Endangered Loach http://dx.doi.org/10.5772/53022 351

in the Dojo was 9 times that of the Japanese ell, *Anguilla japonica*, and also the Dojo had the most amount of vitamin B2 in all fish [4, 5]. Actually these precise nutrient compo‐ nents may somewhat differ among the loach species, but their nutrient components could

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

**Photo 1.** 2 individuals of the intermediate egret, *Ardea intermedia* that are finding individuals of many loach species

However, some of the loach species have confronted a kind of serious concerns, especially a decrease in their population size. In Japan, we have conducted many land consolidation projects for rising rice production and easing agricultural works in rural area since 1960s. In land consolidation projects, concrete canals, drops, diversion weirs, etc. have been installed around paddy fields as agricultural infrastructures; therefore not only fish populations and their habitats but also all of ecosystem and biodiversity in rural area have been extremely

The Hotoke loach, *Lefua echigonia* endemic to Japan (the above in Photo 2) has been well known as a representative loach species has been adversely impacted on its habitat due to land consolidation projects. Since populations of this loach have rapidly declined in some rural areas, consequently the Hotoke loach has been designated as an endangered species on the Red List of Japan [10]. Ecology of the loach is briefed as follows; this species is widely distributed across the Honshu Island from the Tohoku region to the Kinki region. They usu‐ ally inhabits earth canals and ditches around paddy fields into which ground water flows (the bottom in Photo 2) [11, 12]. The Hotoke loach often coexists with the Dojo loach in the habitat and geographic variations for this loach based on morphological characteristics is

have to be fundamentally similar.

Applications

350

as their food in paddy field (unpublished photo)

damaged [6-9].

obscure [13, 14].

**Photo 2.** An adult of the Hotoke loach, *Lefua echigonia* (the above) with approximately 60 mm in body length and typical earth ditch (the bottom) where the loach inhabits around paddy field (unpublished photo)

In recent years, because importance of ecosystem and biodiversity in rural areas has been deeply realized, various research activities have been carried out for conserving and recov‐ ering populations of the Hotoke loach. Distribution pattern and habitat characteristics of this loach were elucidated in some rural areas [15-18], manners of habitat utilization and migra‐ tion routes for the species were investigated [11, 12, 19, 20] and techniques of artificial prop‐ agation were developed with human chorionic gonadotropin [21-23]. Further, molecular analyses of phylogeography of the Hotoke loach using DNA sequences of mitochondrial genes revealed that populations of the species were evolutionally separated into a total of 7 genetic clades in Japan [24-29].

Unfortunately, there is also another serious concern left in populations of the Hotoke loach. That is, as this loach has experienced, diminishment of population size may often cause to improve not only fragmentation among populations but also inbreeding among individuals. Such populations tend to have distinctly poor genetic diversity, occasionally threatened with extinction [30-33]. Usually, to evaluate genetic diversity including genetic population structure for such populations, polymorphism analysis has been performed using microsa‐ tellite loci in nuclear genome [33-35]. Only preliminary investigations, however, were imple‐ mented for populations the Hotoke loach [36-38], although microsatellite analyses have been carried out for populations of several endangered species.

Genetic properties of microsatellite loci are briefed as follows (Fig. 1). These loci are repeat‐ ing sequences of 2 to 6 base pairs of DNA, for instance CACA…, CTCTCT… and CAT‐ CAT… Microsatellites that are typically neutral and co-dominant are used as molecular markers in genetics for kinship, population and other studies, because of often presenting high levels of inter- and intra-specific polymorphism [33-35]. Especially, CA nucleotide re‐ peats appear to be very frequent in human and other genomes and present every few 10,000 to 100,000 base pairs. A repeat size in a locus is treated as an allele and a pair of repeat sizes which are inherited from both of parents is used as genotypes at a locus for a diploid organ‐ ism. Heterozygous describes a genotype consisting of two different sizes (alleles), while ho‐ mozygous does it consisting of two identical ones (Fig. 1).

**Figure 2.** Analysis flow chart of microsatellite loci in this chapter (unpublished figure)

**2.1. Development of microsatellite loci**

ally improved based on the latest studies [39, 40].

[43] and using the pooled biotinylated probes (CA)12 and (CT)12.

**2. Development and characterization of microsatellite loci**

In Section 2, a total of 19 novel microsatellite loci for the Hotoke loach were isolated with an individual obtained in the Shitada R., Chiba Pref. and characterized using 32 individuals collected from the Koise R., Ibaraki Pref. The following development procedure [36] is parti‐

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A sample of this loach was collected from an agricultural canal in the Shitada R., Chiba Pref. in 2005 and preserved in 99% EtOH, and then stored at -30 °C. Genomic DNA was extracted from single caudal fin clip, approximately 5 mm × 5 mm, using a standard phenol-chloro‐ form procedure [41]. Microsatellite enriched libraries were developed following the previ‐ ous study [42] with some modifications. Briefly extracted DNA was digested with RsaI (New England Biolabs) and then ligated to SuperSNX linkers (SuperSNX24 Forward: 5'-GTT TAA GGC CTA GCT AGC AGA ATC-3' and SuperSNX24+4P Reverse: 5'-phosphate-GAT TCT GCT AGC TAG GCC TTA AAC AAA A-3'). Linker-ligated DNA was enriched for mi‐ crosatellites using streptavidin-coated magnetic beads (Dynal) treated with a blocking step

Recovered DNA was amplified by the polymerase chain reaction (PCR) and PCR products were cloned using a TOPO-TA Cloning Kit (Invitrogen) following the manufacture's proto‐ col. A total of 192 positive clones were sequenced on a 3130*xl* Genetic Analyzer (Applied Bi‐ osystems; ABI) using BigDye Terminator kit version 3.1 (ABI) and resultant sequences were

**Figure 1.** Scheme of microsatellite loci in nuclear genome DNA (unpublished figure)

In this chapter, to detect the existence of the above serious genetic issues, we carried out a series of analysis for genetic diversity and population structure in population of the Hotoke loach (Fig. 2). Novel microsatellite loci applied in this loach were developed and character‐ ized in Section 2. Using these developed loci, genetic diversity and population structure were investigated for populations in the upper Kokai River along with adjacent rivers, the southeast part of Tochigi Prefecture as a case study in Section 3. Technical terms related to population and conservation genetics are often used in the sections; thus, details of mean‐ ings of these terms are able to be known by references cited in the end of this chapter.

**Figure 2.** Analysis flow chart of microsatellite loci in this chapter (unpublished figure)
