Influence of modern irrigation,drainage system and water management on spawning migration of mud loach, <Emphasis Type="Italic">Misgurnus anguillicaudatus</Emphasis> C.

We investigated the influence of human activities on the spawning migration of the mud loach, Misgurnus anguillicaudatus. Mud loaches migrate from rivers to paddy fields for spawning. In this study, the number of adult loaches in paddy fields showed a peak in late May and rapidly decreased in July. In early July, water is discharged from paddy fields in order to allow the fields to dry for a few weeks, and water is again supplied later. The spawning season of the loaches probably spans from mid May to August; this was concluded by observing physiological indicators such as the gonad somatic index, oocyte maturity and serum levels of testosterone, estradiol-17β and 17α, 20β-dihydroxy-4-pregnen-3-one. When the loaches mature, they migrated to paddy fields if they could swim upward through the drainage ditches. However, the drainage ditches of modern paddy fields were reconstructed to deepen the water levels. The artificial drought of the rice culture management led to a discharge of water and loaches from the paddy fields during their spawning season. These results indicate that modern rice cultivation causes obstruction of the migration route and loss of spawning grounds; consequently, loaches use limited space and period of the paddy field for their spawning.     

Movement and assemblage of fish in an artificial wetland and canal in a paddy fields area,in eastern Japan

This study, carried out in a paddy fields area in eastern Japan, investigated the timing and patterns of fish movement and assemblage in an artificial wetland and canal. The number of Misgurnus anguillicaudatus and Lefua echigonia immigrating and emigrating between the wetland and the canal accounted for 80–90 % of all the sampled fish. M. anguillicaudatus, L. echigonia, Pseudorasbora parva and Rhynchocypris lagowskii were dominant in the wetland. Immigration of mature M. anguillicaudatus and L. echigonia was detected between late winter and spring. The standard length of the two loach species in the wetland was smaller than that in the canal. These results confirm that wetlands play a role in spawning and nursery for the two species of loaches. The standard length of P. parva in the wetland was smaller than in the canal. This suggests that the wetland was a more suitable spawning and nursery area for this fish species than the canal. L. echigonia used the wetland as a spawning and nursery area, but previous studies reported that the loach did not use paddy fields near the wetland. This could be because the paddy fields were irrigated between June and September and this period did not largely overlap with the fish spawning season. Therefore, we conclude that the conservation and restoration of wetlands, where water is present throughout the year, will contribute toward the preservation of the fish population in a paddy fields area.     

Effect of loach consumption on the reproduction of giant water bug Kirkaldyia deyrolli: dietary selection, reproductive performance, and nutritional evaluation

Rice fields provide major habitats for lentic aquatic insects including the giant water bug Kirkaldyia (=Lethocerus) deyrolli (Heteroptera: Belostomatidae) in Japan. Previous researchers have emphasized that conserving populations of the frogs, Hyla japonicus and Rana nigromaculata, is very important for preserving K. deyrolli because these frogs were found to be a major component of the diet of K. deyrolli adults. However, these previous studies were carried out in rice fields with no loaches, Misgurnus anguillicaudatus, which were probably been inhabited by loaches in the past. A series of field surveys and laboratory experiments were conducted to determine the dietary preference of K. deyrolli adults for loaches, frogs, and aquatic insects; the reproductive effects of consuming these foods; and their protein content. In the rice fields, K. deyrolli adults ate loaches when they were available. When the three prey species (frog: R. nigromaculata, loach: M. anguillicaudatus, and dragonfly: Orthetrum albistylum speciosum) were supplied in laboratory conditions, K. deyrolli adults ate more loaches than frogs or dragonflies. In addition, K. deyrolli adults provided with loaches or frogs laid more egg masses within the limited breeding season than when provided with dragonflies. The edible parts of the loach bodies were the largest of the three prey types. It is possible that K. deyrolli adults have been forced to eat frogs due to reductions in the population density of loaches because modern rice cultivation obstructs loach migration routes and reduces their spawning grounds.     

Predatory Potential of Freshwater Animals on an Invasive Agricultural Pest, the Apple Snail Pomacea canaliculata (Gastropoda: Ampullariidae), in Southern Japan

The apple snail Pomacea canaliculata is an invasive species and a serious pest of rice in many Asian countries. We studied predatory activities of various animals living in Japanese freshwater habitats, by keeping each individual of a potential predator species with 36 snails of various sizes for three days in the aquarium. Forty-six species were tested, and 26 in eight classes fed on small snails. A species of leech, crabs, the common carp, turtles, the mallard duck and the Norway rat attacked even adult snails of 20–30 mm in shell height. These findings will be helpful in identifying effective predators for biological control of the pest snail. In addition, most of the animals attacking snails are reported to be common in rivers or ponds, but few live in modernized paddy fields having little connections with natural water systems. This may be a reason why this snail maintains large populations in paddy fields but not in other freshwater habitats.     

Control the egg hatchling process of Pomacea canaliculata (Lamarck) by water spraying and submersion

Pomacea canaliculata (Lamarck) is an invasive snail species that has become a serious pest of rice and other hydrophytes. Usually it is aquatic but likes to lay its eggs higher than the waterline. In order to seek a feasible and efficient way to control the egg hatchling of this pest, here we systematically studied the effects of water spraying and submersion on its egg hatchling rates and durations. Our results demonstrated that water spraying and submersion could dramatically decrease the hatchling rates to maximal 5.8% and increase the hatchling duration up to 26.4 days on P. canaliculata. Not only the beginning time of water treatment, but also the frequencies of the water spraying is critical to control the hatchling rate and duration of P. canaliculata. Water submersion that began in 12 h after the eggs laid and lasted at least 48 h will significantly decrease the snails’ hatchling rates and extend the hatchling time. In addition, compared to spraying, the water submersion could achieve more remarkable effects. The capsule of the snail’s egg is able to withstand the water treatment. Based on our water spraying and submersion results, it can be inferred that 0–6 h after egg being laid, egg capsule precipitates are beginning to form, and this process will complete after 12–24 h. This special breeding characteristic of P. canaliculata makes the physical control by water treatment become feasible.     

Lethal and non-lethal effects of multiple indigenous predators on the invasive golden apple snail (Pomacea canaliculata)

1. We investigated the individual and combined effects of two predators (the climbing perch, Anabas testudineus, and the wetland crab, Esanthelphusa nimoafi) indigenous to wetlands in Laos, on the behaviour and survival of the invasive South American golden apple snail (Pomacea canaliculata). The snail is considered a pest, consuming large amounts of rice and other aquatic vegetation in the region. 2. Snail avoidance reactions to released predator chemical cues were investigated in aquaria while the effects of predators on a mixed snail population were studied in field enclosures that contained native aquatic plants (Salvinia cucullata, Ludwigia adscendens and Ipomoea aquatica). 3. In the aquaria experiment, neonate (2–3 mm) and medium‐sized snails (8–10 mm) responded to fish chemical cues by going to the surface, whereas adult snails (35–40 mm) went to the bottom. In contrast, no size class of snails reacted to chemical cues released by crabs. 4. In the field experiment, fish reduced the abundance of neonate snails, and crabs reduced the abundance of all size classes. The effect of the combined predators could not be predicted from the mortality rate observed in single predator treatments. The survival of neonate and medium‐sized snails was greater and of adults less than expected. The presence of predators did not affect egg production. Snails consumed significant amounts of plants despite the presence of predators. 5. Our findings suggest that some indigenous Asian predators have lethal and sublethal effects on P. canaliculata that depend on snail size and predator type. When in the presence of several predators the response of snails to one predator may either increase or decrease the vulnerability of snails to the others.     

Changes in chemical components in the freshwater apple snail, Pomacea canaliculata (Gastropoda: Ampullariidae), in relation to the development of its cold hardiness

The apple snail, Pomacea canaliculata, is an invasive freshwater snail. It increases its cold hardiness before winter. However, the physiological mechanism of cold hardiness in molluscs is poorly understood, especially in freshwater molluscs. In this study, we examined the changes in low molecular weight compounds, glycogen and lipids, in the body of P. canaliculata in association with the development of cold hardiness. When snails without cold hardiness were experimentally cold-acclimated, the amount of glycerol, glutamine, and carnosine increased, while glycogen and phenylalanine decreased. Overwintering cold-tolerant snails collected from a drained paddy field in November also showed increased glycerol in their bodies with decreasing glycogen concentration, compared to summer snails collected from a submerged field. Water content also decreased during the cold acclimation, although the water loss was minimal. These results indicate that the freshwater snail, P. canaliculata enhances cold hardiness by accumulation of some kinds of low molecular weight compounds in its body as some insects do. However, the actual function of each low molecular compound is still unknown.     

Biological control of the exotic invasive snail Pomacea canaliculata with the indigenous medicinal leech Whitmania pigra

The apple snail Pomacea canaliculata has been an invasive species in China for decades and causes enormous losses to agriculture. The predation capability of the leech Whitmania pigra on P. canaliculata was studied for its economic benefit and potential application. In the present study, the leech W. pigra exhibited a strong predatory capacity in controlling P. canaliculata under both laboratory and field conditions, and it showed no bias towards consuming smaller snails during the experiments. More than 80% of the 80 snails (of which half had reached maturity) were preyed upon by 20 developing leeches (2–7?g) in miniature rice fields over a 15-day period, and the number of rice seedlings damaged by P. canaliculata was decreased in the presence of W. pigra. In a separate experiment, 15 developing snails were exposed to caged leeches and conspecific snails for four weeks. The food intake and growth of P. canaliculata were inhibited under the predation risk imposed by W. pigra, but the feeding rate, food conversion efficiency and survival of P. canaliculata were not conspicuously influenced.     

Field observations of the alarm response to crushed conspecifics in the freshwater snail Pomacea canaliculata: effects of habitat,vegetation, and body size

The freshwater snail Pomacea canaliculata shows alarm responses to chemical cues released from injured conspecifics, but its behavioural responses in the field are unknown. We investigated effects of habitat (canals or paddy fields), vegetation, and body size on alarm responses in the field. Snails responded to crushed conspecifics within 4 min by burying themselves, but the proportions of self-buried snails were generally lower (0–28% depending on experiments) than those reported in the laboratory. Snails not only showed the alarm response, but also frequently fed on crushed conspecifics. There were no influences of habitat or body size on the proportions of individuals showing the alarm response. Nevertheless, in paddy fields with high-density vegetation a higher proportion of snails showed the alarm response than in paddy fields with low-density vegetation.     

Factors affecting the distribution and abundance of the commensal <Emphasis Type="Italic">Temnocephala iheringi</Emphasis> (Platyhelminthes: Temnocephalidae) among the southernmost populations of the apple snail <Emphasis Type="Italic">Pomacea canaliculata </Emphasis>(Mollusca: Ampullariidae)

Temnocephala iheringi is the most common temnocephalan inhabiting the mantle cavity of the apple snail Pomacea canaliculata, a freshwater neotropical gastropod that has become a serious rice pest in Southeastern Asia. T. iheringi has been recorded from Mato Grosso (Brazil) to water bodies associated with the Río de la Plata river (Argentina). During an extensive survey in the southern limit of the native area of P.␣canaliculata the presence of T. iheringi eggs was recorded in several apple snail populations, extending the known distribution of the commensal more than 400 km southwards. The aim of this study was to understand the factors affecting the distribution and abundance of T. iheringi among populations of P.␣canaliculata. Only 23% of the apple snail populations inhabiting streams harboured temnocephalans while the occurrence among lentic ones was 71%. T. iheringi was found mostly in populations of apple snails living in non-alkaline sites and where snails attaining sizes larger than 4 cm were very common. The prevalence of the temnocephalans in lentic populations was higher than 90%. The number of eggs on the shell (not including the umbilicus) ranged between 0 and 470 and was different among populations of P.␣canaliculata. The prevalence and number of eggs were lower in the lotic populations, except for a stream population immediately downstream of a lake with commensals. There was no difference between males and females of P. canaliculata neither in the prevalence nor in the number of eggs on the shell. The southernmost population of the world of P. canaliculata harbours commensals that tolerate cold winter water temperatures (4–5 °C) as well as its host. On the other hand, T. iheringi was found only in sites with bicarbonate concentrations lower than 6.6 meq l⁻¹, suggesting that the tolerance of the commensal is very much lower than that of the apple snail (up to 9.95 meq l⁻¹). The number of worms inside each snail or the life history variation of P. canaliculata could explain the influence of the size of the snails on the occurrence of T. iheringi. In the big-sized snails, where the number of commensals is higher, the probability of survival of at least one worm is also higher, specially during the hibernation period, when crawling and feeding are null and snails remain buried. On the other hand, P. canaliculata snails from lentic populations are generally bigger and mostly iteroparous, while those inhabiting streams are smaller and semelparous. In these populations the snails have access to mate only with snails of their same cohort, while in iteroparous populations they can copulate with individuals of other cohorts, allowing the inter-generation transmission of worms and the long term persistence of the population of commensals.     

The biological control of Pomacea canaliculata population by rice-duck mutualism in paddy fields

Duck has been used as a non-chemical control method against Pomacea canaliculata Lamarck, but little is known about its principles that underlie the control of snail populations. An indoor experiment was initially used to observe the predation potential of ducks, followed by replicated field trials. In the indoor studies, ducks effectively preyed on juvenile snails, but had a weak predatory effect on large snails and egg clusters. In the field, application of a rice-duck mutualism system significantly reduced the numbers of snails (especially number of immature individuals), number of snail egg clusters and snail damage to rice plants. The controlling effect was longer and more stable than the chemical application, resulting in a better yield than with the pentachlorophenol sodium and tea seed powder treatment. Our experimental results also suggested that the snail age structure in the rice-duck mutualism plots was shifted towards older snails by ducks preying, indicating a trend towards population decline, and ducks caused snails to oviposit on sites not ideal for hatchling establishment. Throughout the studies, it is suggested that a rice-duck mutualism system could be used for controlling P. canaliculata in organic rice production.     

Impact of invasive apple snails in Hong Kong on wetland macrophytes,nutrients, phytoplankton and filamentous algae

1. Grazing by invasive species can affect many aspects of an aquatic system, but most studies have focused on the direct effects on plants. We conducted mesocosm and laboratory experiments to examine the impact of the invasive apple snail Pomacea canaliculata on macrophytes, filamentous algae, nutrients and phytoplankton. 2. In a freshwater pond, we confined 500 g of Myriophyllum aquaticum or Eichhornia crassipes with 0, 2, 4 or 8 apple snails in 1 m × 1 m × 1 m enclosures for approximately 1 month. Apple snails grazed heavily on both species of macrophytes, with higher overall weight losses at higher snail densities. The damage patterns differed between the two macrophytes. In M. aquaticum, both leaves and stems suffered from substantial herbivory, whereas in E. crassipes, only the roots suffered significant weight reduction. 3. In addition to grazing on macrophytes, apple snails appeared to have controlled the growth of filamentous algae, as these did not develop in the snail treatments. The ability of P. canaliculata to control filamentous algae was supported by a laboratory experiment where the consumption was as high as 0.25 g g⁻¹ snail DW d⁻¹. Because of a lack of native herbivorous snails in the pond, the growth of filamentous algae (mainly Spirogyra sp.) reached 80.3 g m⁻², forming a spongy pond scum in the no‐apple snail control. Together with previous reports that apple snails could eat the juveniles and eggs of other freshwater snails, our results indicated that P. canaliculata could have out‐competed native herbivorous snails from the pond by predation on their juveniles or eggs. Alternatively, P. canaliculata might have out‐competed them by monopolisation of food resources. 4. Nitrogen and phosphorous concentrations remained low throughout both experiments and were not correlated with apple snail density. The treatment effects on chlorophyll a (Chl a) and phytoplankton composition varied in the two experiments. In the M. aquaticum experiment, with increasing snail density, Chl a increased, and the phytoplankton community became dominated by Cryptophyceae. In the E. crassipes experiment, Chl a level was independent of snail density, but with increasing snail density, the phytoplankton community became co‐dominated by Cryptophyceae, Chlorophyceae and Bacillariophyceae. 5. Given the multiple effects of P. canaliculata on wetland biodiversity and function, management strategies should be developed to prevent its further spread. In invaded wetlands, strategies should be developed to eradicate the apple snail and re‐introduce native snails which can control the development of filamentous algae.     

Distribution of the oriental weatherloach, Misgurnus anguillicaudatus, in paddy fields and its implications for conservation in Sado Island, Japan

The distribution of the oriental weatherloach, Misgurnus anguillicaudatus, together with related environmental factors were surveyed at 185 paddy field locations on Sado Island in Japan. This was associated with the reintroduction of the Japanese crested ibis, Nipponia nippon, which prefers to feed on the loach. Loach were found to be present at 90 locations (49%). Analysis with GIS, GLM and AIC revealed that positive factors for the loach distribution included the presence of an earth ditch, the connections at outlets and the proportion of paddy field area within an 800 m radius. Conversely, pumping-up water irrigation and flow irrigation affected loach distribution negatively. In an interview survey that was conducted concurrently, older farmers in the area recalled that the loach had once been distributed over almost the entire island (89/96, 93%) about half a century ago. The farmers also suggested that the impact of agricultural chemicals was one of the main reasons for loach reduction or local extinction. A map of the current potential loach distribution was made using the best fit model from GIS variables. The loach was expected to be distributed in the Kuninaka region with high probability (60–100%) and in patches in the Osado and Kosado regions with low–medium probability (10–50%). As a feasible scenario for the conservation of the loach, another predictive map of the loach distributions was made using a model fit based on the GIS variables and outlet connection, in which the all of the outlets were presumed to be connected to the ditches without gaps. In this case, the loach were expected to be distributed almost throughout the paddy fields with medium–high probability (40–100%). In another scenario that presumed the complete land consolidation of all the paddy fields, the probability of loach occurrence was low (0–30%) throughout the island with the exception of the island’s center in the Kuninaka region.     

Density-dependent growth and reproduction of the apple snail, Pomacea canaliculata: a density manipulation experiment in a paddy field

To examine density dependence in the survival, growth, and reproduction of Pomacea canaliculata, we conducted an experiment in which snail densities were manipulated in a paddy field. We released paint-marked snails of 15–20 mm shell height into 12 enclosures (pens) of 16 m² at one of five densities – 8, 16, 32, 64, or 128 snails per pen. The survival rate of released snails was 95% and was independent of snail density. The snail density had a significant effect on the growth and egg production of individual snails. This density dependence may have been caused by reduced food availability. The females at high density deposited fewer and smaller egg masses than those at low density, and consequently produced fewer eggs. The females at densities 8 and 16 deposited more than 3000 eggs per female, while the females at density 128 oviposited only 414 eggs. The total egg production per pen was, however, higher at higher snail density. The survival rates of juvenile snails were 21%–37% and were independent of adult density. The juvenile density was positively correlated with the total egg production per pen and hence was higher at higher adult density. However, the density of juveniles larger than 5 mm in shell height, i.e., juveniles that can survive an overwintering period, was not significantly different among density treatments. These results suggest that snail density after the overwintering period is independent of the density in the previous year. Thus, density dependence in growth and reproduction might regulate the population of P. canaliculata in paddies. Received: October 23, 1998 / Accepted: July 16, 1999     

Growth and survival of juvenile Pomacea canaliculata (Gastropoda: Ampullariidae) in plain streams associated to different land uses

Pomacea canaliculata is an apple snail that has become an invasive species in several countries. In this research, through two simultaneous experiments, we assessed the effects of different land uses (urban, agriculture, cattle grazing, farms and recreation) on the growth and survival of snails from native populations. The survival was not affected by the water quality of the three streams studied, whereas the growth of snails exposed to the water of the urban stream was impaired. Therefore, our results suggest that the growth rate of P. canaliculata could be useful for the assessment of a moderate urban impact.     

Detection of hybridization between two loach species (<Emphasis Type="Italic">Paramisgurnus dabryanus</Emphasis> and <Emphasis Type="Italic">Misgurnus anguillicaudatus</Emphasis>) in wild populations

Artificial interspecific hybrids between large scale loach P. dabryanus and tetraploid pond loach M. anguillicaudatus (Cobitidae, Cypriniformes) are viable. To detect the occurrence of possible natural hybridization, genetic analyses by using microsatellite markers were performed for natural populations of large scale loach and pond loach, the reciprocal laboratory hybrids, and “supposed hybrids” with ambiguous morphology. The fertility of the artificial hybrids was also tested. At one diagnostic microsatellite (Mac50), one out of 20 “supposed hybrids” was identified to be F₁ hybrid between the two loach species because it had the same genotype as that of the laboratory hybrids. The triploid hybrids between the two species were confirmed to be female-sterile. The results show that rare hybridization has occurred between diploid large scale loach and tetraploid pond loach in nature although it may have little effect in genetic introgression. This study is helpful for fish conservation and encourages further investigation on natural hybridization and introgression of loaches.     

Morphological comparison and DNA barcoding of four closely related species in the genera Misgurnus and Paramisgurnus (Cypriniformes: Cobitidae)

To evaluate the feasibility of morphological and genetic identification of the closely related species in the genera Misgurnus and Paramisgurnus, the morphological characters of four species in these genera and DNA barcoding of five loaches (P. dabryanus, M. anguillicaudatus, M. bipartitus, M. mohoity, and Barbatula toni) were investigated. Twelve morphological characters were measured in 542 individuals to perform the comparative analysis. Among these characters, only the caudal peduncle length (L_CP) revealed significant difference (P < 0.05) among these four species. The clustering based on morphological characters formed two clusters (P. dabryanus and M. anguillicaudatus; M. bipartitus and M. mohoity). A total of 186 COI fragments for the five loaches investigated were sequenced and analyzed. The results showed that interspecific K2P distance was much higher than intraspecific distance within the five species. Bayesian inference of phylogeny showed that individuals of these species were divided into five specific clades. Meanwhile, the COI fragments exhibited 22 character attributes for the differentiation of the five loach species based on character-based method. Our results suggested that DNA barcoding based on COI can be used as an efficient identifier of these five loach species; the combination of distance-based method, Bayesian inference and character-based approach provides higher resolution of identification at species level.     

The role of spotted green pufferfish Tetraodon nigroviridis in controlling golden apple snail Pomacea canaliculata: an effective biological control approach involving a new agent

The golden apple snail Pomacea canaliculata is invasive in South China and poses serious harm to agricultural production and aquatic ecosystems. In the present study, we investigated the predatory potential of the spotted green pufferfish Tetraodon nigroviridis (50?mm in body length) on snails of various sizes in fresh and brackish water (4.5 ppt salinity). The survival and damage rates of 50 snails were estimated at 1, 3, 5 and 7 days. The survival rate of snails, including 10 adult snails (20–35?mm shell height), decreased less than 25% in the presence of pufferfish, and this decrease was more profound in brackish than in fresh water. Approximately 98% of the snails died in brackish water in the presence of only four pufferfish. The pufferfish also inflicted a high rate of snail injury resulting from attacks involving bites. In addition, the pufferfish affected the behaviour of the snails, with more than 95% of snails exhibiting tightly closed opercula. Thus, the results of this study suggest that pufferfish are effective for the biological control of golden apple snails.     

Comparative and evolutionary analysis in natural diploid and tetraploid weather loach Misgurnus anguillicaudatus based on cytochrome b sequence data in central China

To obtain the phylogenetic relationship between diploid and tetraploid Misgurnus anguillicaudatus, the mitochondrial cyt b gene in the diploid and tetraploid weather loach were isolated and sequenced. The DNA sequences were analyzed using MEGA 3.0 software to determine the phylogenetic relationship. Forty-five variable sites among cyt b gene sequences and 18 amino acid substitutions occurred within the diploid and tetraploid loaches as deduced from the nucleotide sequences analysis of the cyt b gene. The nucleotide pairwise distance between diploid and tetraploid loach ranged from 0.001 to 0.025. Phylogenetic analysis revealed evolutionary relationships between diploid and tetraploid loach. Our results indicated a significant difference between diploid and tetraploid loach about the cyt b gene. AMOVA analysis indicated that there were no significant genetic variations within diploid loaches (Fst = 0.2529, P > 0.05) and within tetraploid loaches (Fst = 0.0564, P > 0.05), neither. However, significant genetic differences were found between diploid and tetraploid loaches (Fst = 0.7634, P < 0.05). Thus, it is concluded that no reproductive isolation was found within the same cytotypes of different localities, but there was reproductive isolation between these two cytotypes. The diploid loach existed before the tetraploid loach in nature. The present study is the first to describe the phylogenetic relationships of natural polyploidy weather loach using mtDNA cyt b gene.     

Evidence of a secondary interspecific mitochondrial DNA introgression in the pond loach Misgurnus dabryanus (Teleostei: Cobitidae) population introduced in Japan

The pond loach Misgurnus dabryanus is a freshwater fish with a distribution range spanning the eastern part of the Asian continent, the Korean Peninsula, and Taiwan. The pond loach was transplanted to the Japanese archipelago through the co-inclusion with dojo loach (Misgurnus anguillicaudatus species complex) populations, which were imported live from China for food materials, and it is currently distributed widely across Japan. A previous mitochondrial DNA (mtDNA) analysis revealed that a pond loach population in Ehime Prefecture (Shikoku Island, Japan) included two highly diverged mtDNA groups (Groups I and II). To examine the origin of these two distinct forms of mtDNA within the Japanese pond loach population, we performed phylogenetic analyses using sequences based on the mtDNA of cytochrome oxidase b (cyt b) and the nuclear DNA recombination activating gene 1 (RAG-1). We also conducted a random amplified polymorphic DNA (RAPD) analysis to examine the establishment of reproductive isolation between sympatric pond loaches with two different mtDNA groups. Our mtDNA phylogenetic results indicated that the two diverged pond loach mtDNA sequences showed polyphyletic relationships among Misgurnus species and its related genus Cobitis. In contrast, there were no clear divergence in nuclear DNA among the pond loaches irrespective of their mtDNA groups, and they all formed monomorphic clades in the phylogenetic relationships among the species. The discrepancy between the mtDNA and nuclear DNA genes support that the existence of two diverged forms of DNA within the pond loach population could be attributed to past mtDNA introgressions from other species rather than convergent evolution. Previous mtDNA phylogenetic studies among Cobitidae revealed that the dojo loach also consisted of two genetically diverged polyphyletic clades: an original Misgurnus mtDNA and an introgressed mtDNA from Cobitis species. In our mtDNA result, the Group II haplotype of the pond loach was included in the mtDNA from the introgressed dojo loach. This suggested that the Group II haplotype was derived from introgressed dojo loach mtDNA. The close relationships between the introgressed dojo loach and the pond loach mtDNA indicated that this secondary introgression had recently occurred via hybridization in a recent artificial aquaculture or transportation process. Common RAG-1 alleles and RAPD bands were shared between the sympatric pond loaches with original and introgressed mtDNAs. This indicates that the introgressed mtDNA haplotype is included as one of the polymorphic genotypes within the pond loach populations, and does not represent existence of different cryptic species.