Chronic Ankle Joint Instability Induces Ankle Sensorimotor Dysfunction: A Controlled Laboratory Study.

BACKGROUND: Chronic ankle instability (CAI) is a clinical sequela that causes the recurrence of ankle sprain by inducing ankle sensorimotor dysfunction. Animal models of CAI have recently shown that ankle ligament injuries mimicking an ankle sprain result in chronic loss of ankle sensorimotor function. However, the underlying mechanisms determining the pathogenesis of CAI remain unclear. HYPOTHESIS: Ankle instability after an ankle sprain leads to the degeneration of the mechanoreceptors, resulting in ankle sensorimotor dysfunction and the development of CAI. STUDY DESIGN: Controlled laboratory study. METHODS: Four-week-old male Wistar rats (N = 30) were divided into 2 groups: (1) the ankle joint instability (AJI) group with ankle instability induced by transecting the calcaneofibular ligament (n = 15) and (2) the sham group (n = 15). Ankle instability was assessed using the anterior drawer test and the talar tilt test at 4, 6, and 8 weeks after the operation (n = 5, for each group at each time point), and ankle sensorimotor function was assessed using behavioral tests, including ladder walking and balance beam tests, every 2 weeks during the postoperative period. Morphology and number of mechanoreceptors in the intact anterior talofibular ligament (ATFL) were histologically analyzed by immunofluorescence staining targeting the neurofilament medium chain and S100 proteins at 4, 6, and 8 weeks postoperatively (n = 5 per group). Sensory neurons that form mechanoreceptors were histologically analyzed using immunofluorescence staining targeting the mechanosensitive ion channel PIEZO2 at 8 weeks postoperatively (n = 5). RESULTS: Ankle sensorimotor function decreased over time in the AJI group, exhibiting decreased ankle instability compared with the sham group (P = .045). The number of mechanoreceptors in the ATFL was reduced (P < .001) and PIEZO2 expression in the sensory neurons decreased (P = .008) at 8 weeks postoperatively. The number of mechanoreceptors was negatively correlated with ankle sensorimotor dysfunction (P < .001). CONCLUSION: The AJI model demonstrated degeneration of the mechanoreceptors in the ATFL and decreased mechanosensitivity of the sensory neurons, which may contribute to CAI. CLINICAL RELEVANCE: Ankle instability causes degeneration of mechanoreceptors and decreases the mechanosensitivity of sensory neurons involved in the development of CAI. This finding emphasizes the importance of controlling ankle instability after ankle sprains to prevent recurrence and the onset of CAI.

Mild treadmill exercise inhibits cartilage degeneration via macrophages in an osteoarthritis mouse model.

Objective: We previously reported how treadmill exercise can suppress cartilage degeneration. Here, we examined the changes in macrophage dynamics in knee osteoarthritis (OA) during treadmill exercise and the effect of macrophage depletion. Design: OA mouse model, generated via anterior cruciate ligament transection (ACLT), was subjected to treadmill exercise of different intensities to investigate the effects on cartilage and synovium. In addition, clodronate liposomes, which deplete macrophages, were injected intra-articularly into the joint to examine the role of macrophages during treadmill exercise. Results: Cartilage degeneration was delayed by mild exercise, and concomitantly, an increase in anti-inflammatory factors in the synovium was observed, with a decrease in the M1 and increase in M2 macrophage ratio. On the contrary, high-intensity exercise led to the progress of cartilage degeneration and was associated with an increase in the M1 and a decrease in the M2 macrophage ratio. The clodronate liposome-induced reduction of synovial macrophages delayed cartilage degeneration. This phenotype was reversed by simultaneous treadmill exercise. Conclusions: Treadmill exercise, especially at high intensity, was detrimental to articular cartilage, whereas mild exercise reduced cartilage degeneration. Moreover, M2 macrophage response appeared necessary for the chondroprotective effect of treadmill exercise. This study indicates the importance of a more comprehensive analysis of the effects of treadmill exercise, not limited to the mechanical stress added directly to cartilage. Hence, our findings might help determine the type and intensity of prescribed exercise therapy for patients with knee OA.

The Effects of Downhill Running and Maturation on Histological and Morphological Properties of Tendon and Enthesis in Mice.

To date, it remains unclear how overuse affects the tendons and entheses at different stages of maturation. Therefore, we evaluated histological and morphological changes in the tendons and entheses in adolescent (4-week-old) and adult mice (8-week-old) by performing flat-land and downhill running exercises. The mice were divided into the Sedentary, High Flat (flat-land high-speed running; concentric-contraction exercise), Low Down (downhill low-speed running; eccentric-contraction exercise), and High Down (downhill high-speed running; eccentric-contraction exercise) groups. Histological changes and inflammatory factor expressions were compared in the entheses and tendons after 4 weeks of exercise. Downhill, but not flat-land high-speed running, induced muscle-tendon complex hypertrophy in both adolescent and adult mice. Histological enthesis changes were induced in both groups during downhill running but were less pronounced in adult mice. Conversely, no significant cell aggregation or fiber orientation changes were observed in the tendon, but increased inflammatory factors were observed in both groups, with significantly higher expression in the tendons of adult mice. Downhill running induced histological and morphological enthesis changes and inflammatory factor increase in the tendons, regardless of running speed variations. These results may help elucidate the pathogenesis of enthesopathy and tendinopathy, which have different pathophysiologies despite having the same pathogenetic factors.

Eccentric contraction-dominant exercise leads to molecular biological changes in enthesis and enthesopathy-like morphological changes.

Entheses, which are tendon-to-bone attachment sites in the musculoskeletal system, play important roles in optimizing the mechanical stress and force transmitted from the muscle to the bone. Sports-related enthesopathy shows pathological features, including hyperplasia of the fibrocartilage (FC) region in the enthesis. The amount of exercise and type of muscle contraction during movement is involved in the pathogenesis of sports-related enthesopathy; however, the details of this condition are unclear. Here we examined the molecular pathways involved in the morphological changes of the muscle-tendon-enthesis complex and enthesis FC region in the supraspinatus muscle enthesis of mice under different exercise conditions. Following intervention, morphological changes in the muscle-tendon-enthesis complex were initiated in the eccentric contraction-dominant exercise group at 2 weeks, with activation of the transforming growth factor-ß (TGFß) superfamily pathway predicted by proteome and ingenuity pathway analyses. Histological and molecular biological analyses confirmed the activation of the TGFß/bone morphogenetic protein (BMP)-Smad pathway. The concentric contraction-dominant exercise group showed no change in the morphology of the muscle-tendon-enthesis complex or activation of the TGFß/BMP-Smad pathway, despite overuse exercise. Statement of Clinical Significance: These results suggest that eccentric contraction-dominant exercise induces sports-related enthesopathy-like morphological changes in the early stages as well as molecular biological changes, mainly in the transforming growth factor-ß superfamily pathway in enthesis. Statement of Clinical Significance: These results suggest that eccentric contraction-dominant exercise induces sports-related enthesopathy-like morphological changes in the early stages as well as molecular biological changes, mainly in the transforming growth factor-ß superfamily pathway in enthesis.

Effects of exercise on muscle reinnervation and plasticity of spinal circuits in rat sciatic nerve crush injury models with different numbers of crushes.

INTRODUCTION/AIMS: Motor function recovery is frequently poor after peripheral nerve injury. The effect of different numbers of nerve crushes and exercise on motor function recovery is unknown. We aimed to examine how different numbers of crushes of the rat sciatic nerve affects muscle reinnervation and plasticity of spinal circuits and the effect of exercise intervention. METHODS: Single and multiple sciatic nerve crush models with different numbers of crushes were created in rats. Treadmill exercise was performed at 10 m/min for 60 min, five times a week. Muscle reinnervation and synaptic changes in L4-5 motor neurons were examined by immunofluorescence staining. Behavioral tests were the sciatic functional index (SFI) and the pinprick tests. RESULTS: The percentage of soleus muscle reinnervation was not significantly increased by the presence of exercise in single or multiple crushes. Exercise after a single crush increased the contact of motor neurons with VGLUT1-containing structures (Exercised vs. Unexercised, 12.9% vs. 8.7%; p < .01), but after multiple crushes, it decreased with or without exercise (8.1% vs. 8.6%). Exercise after a single crush significantly improved SFI values from 14 to 24 days, and exercise after multiple crushes from 21 to 35 days (all p < .05). The pinprick test showed no difference in recovery depending on the number of crushes or whether or not exercised. DISCUSSION: Different numbers of sciatic nerve crushes affect muscle reinnervation and motor neuron synaptic changes differently, but motor function recovery may improve with exercise regardless of the number of crushes.

Structural and pathological changes in the enthesis are influenced by the muscle contraction type during exercise.

Mechanical stress is involved in the onset of sports-related enthesopathy. Although the amount of exercise undertaken is a recognized problem during disease onset, changes in muscle contraction type are also involved in the increase in mechanical stress during exercise. This study aimed to clarify the effects of increased mechanical stress associated with muscle contraction type and amount of exercise on enthesis. Twenty mice underwent treadmill exercise, and the muscle contraction type and overall load during exercise were adjusted by varying the angle and speed conditions. Histological analysis was used to the cross-sectional area of the muscle; area of the enthesis fibrocartilage (FC), and expression of inflammation-, degeneration-, and calcification-related factors in the FC area. In addition, the volume and structure of the bone and FC area were examined using microcomputer imaging. Molecular biological analysis was conducted to compare relative expression levels of inflammation and cytokine-related factors in tendons. The Overuse group, which increased the amount of exercise, showed no significant differences in parameters compared to the sedentary mice (Control group). The mice subjected to slow-speed downhill running (Misuse group) showed pathological changes compared to the Control and Overuse groups, despite the small amount of exercise. Thus, the enthesis FC area may be altered by local mechanical stress that would be increased by eccentric muscle contraction rather than by mechanical stress that increases with the overall amount of exercise. Clinical Significance: The muscle contraction type might be more involved in the onset of sports-related enthesopathy rather than the amount of exercise.

Treadmill Exercise after Controlled Abnormal Joint Movement Inhibits Cartilage Degeneration and Synovitis.

Cartilage degeneration is the main pathological component of knee osteoarthritis (OA), but no effective treatment for its control exists. Although exercise can inhibit OA, the abnormal joint movement with knee OA must be managed to perform exercise. Our aims were to determine how controlling abnormal joint movement and treadmill exercise can suppress cartilage degeneration, to analyze the tissues surrounding articular cartilage, and to clarify the effect of treatment. Twelve-week-old ICR mice (n = 24) underwent anterior cruciate ligament transection (ACL-T) surgery on their right knees and were divided into three groups as follows: ACL-T, animals in the walking group subjected to ACL-T; controlled abnormal joint movement (CAJM), and CAJM with exercise (CAJM + Ex) (n = 8/group). Walking-group animals were subjected to treadmill exercise 6 weeks after surgery, including walking for 18 m/min, 30 min/day, 3 days/week for 8 weeks. Safranin-O staining, hematoxylin-eosin staining, and immunohistochemical staining were performed. The OARSI (Osteoarthritis research Society international) score was lower in the CAJM group than in the ACL-T group and was even lower in the CAJM + Ex group. The CAJM group had a lower meniscal injury score than the ACL-T group, and the CAJM + Ex group demonstrated a less severe synovitis than the ACL-T and CAJM groups. The observed difference in the perichondrium tissue damage score depending on the intervention method suggests different therapeutic effects, that normalizing joint motion can solve local problems in the knee joint, and that the anti-inflammatory effect of treadmill exercise can suppress cartilage degeneration.

Effect of Various Types of Muscle Contraction with Different Running Conditions on Mouse Humerus Morphology.

How various types of muscle contraction during exercises affect bone formation remains unclear. This study aimed to determine how exercises with different muscle contraction types affect bone morphology. In total, 20 mice were used and divided into four groups: Control, Level, Down Slow, and Down. Different types of muscle contraction were induced by changing the running angle of the treadmill. After the intervention, micro-computed tomography (Micro-CT), tartrate-resistant acid phosphatase/alkaline phosphatase (ALP) staining, and immunohistochemical staining were used to analyze the humerus head, tendon-to-bone attachment, and humerus diaphyseal region. Micro-CT found that the volume ratio of the humeral head, the volume of the tendon-to-bone attachment region, and the area of the humeral diaphyseal region increased in the Down group. However, no difference was detected in bone morphology between the Level and Down Slow groups. In addition, histology showed activation of ALP in the subarticular subchondral region in the Down Slow and Down groups and the fibrocartilage region in the tendon-to-bone attachment. Moreover, Osterix increased predominantly in the Down Slow and Down groups.Overall bone morphological changes in the humerus occur only when overuse is added to EC-dominant activity. Furthermore, different type of muscle contractile activities might promote bone formation in a site-specific manner.

Spatiotemporal distribution of juvenile chum salmon in Otsuchi Bay, Iwate, Japan, inferred from environmental DNA.

To understand the ecology of juvenile chum salmon during early marine life after their downstream migration, we developed a quantitative PCR-based environmental DNA (eDNA) method specific for chum salmon and investigated the spatiotemporal distribution of eDNA in Otsuchi Bay, Iwate, Japan. Indoor aquarium experiments demonstrated the following characteristics of chum salmon eDNA: (1) the eDNA shedding and degradation were time- and water temperature-dependent and the bacterial abundance could contribute to the eDNA decay, (2) fecal discharge may not be the main source of eDNA, and (3) a strong positive Pearson correlation was found between the number of juveniles and the eDNA amounts. As we discovered strong PCR inhibition from the seawater samples of the bay, we optimized the eDNA assay protocol for natural seawater samples by adding a further purification step and modification of PCR mixture. The intensive eDNA analysis in the spring of 2017 and 2018 indicated that juvenile chum salmon initially inhabited in shallow waters in the shorefront area and then spread over the bay from January to June. The eDNA data also pointed out that outmigration of juvenile chum salmon to open ocean temporarily suspended in April, possibly being associated with the dynamics of the Oyashio Current as suggested by a previous observation. The eDNA method thus enables us large-scale and comprehensive surveys without affecting populations to understand the spatiotemporal dynamics of juvenile chum salmon.

Changes in ovarian gene expression profiles and plasma hormone levels in maturing European eel (Anguilla anguilla); Biomarkers for broodstock selection.

Complete sexual maturation of European eels (Anguilla anguilla) in captivity can only be achieved via injections with gonadotropins. For female eels this procedure takes 4-6months and the response ranges from "unresponsive" to final maturation and ovulation. Reproductive success could be significantly increased via early selection of responders based on predictive markers and minimally invasive sampling methods. To get a better understanding of the genetic background of ovarian maturation of the European eel we performed a pilot deep-sequencing transcriptome analysis of ovarian tissue derived from a yellow eel, a prepubertal silver eel and a post-spawning matured eel. Two key players in steroidogenesis were strongly correlated with advanced sexual maturation, namely P450c17 and liver receptor homolog-1, suggesting that blood plasma steroids might qualify as minimally invasive markers for early detection of responders. Since the predictive value of plasma sex steroid levels for final maturation of the European eel had not yet been carefully examined, we performed an extensive artificial maturation trial. Farmed silver eels were treated with pituitary extracts and sampled at multiple time intervals. Expression of steroidogenesis-related genes in ovarian tissue of responding and non-responding eels after four weekly injections with pituitary extract was compared using a custom-built microarray and RNAseq. Increased expression of 17ß-hsd1 was strongly linked to sexual maturation. Blood plasma levels of sex steroids were measured using ELISAs. We show that a 2.5-fold increase in blood-plasma estradiol level after 4 weekly pituitary extract injections is a strong predictor of final sexual maturation of female European eel.

Novel microsatellite marker development from the unassembled genome sequence data of the marbled flounder Pseudopleuronectes yokohamae.

Various genome-scale data have been increasingly published in diverged species, but they can be reused for other purposes by re-analyzing in other ways. As a case study to utilize the published genome data, we developed microsatellite markers from the genome sequence data (assembled contigs and unassembled reads) of the marbled flounder Pseudopleuronectes yokohamae. No microsatellites were identified in the contig sequences, whereas the computer software found 781,773 sequences containing microsatellites with di- to hexa-nucleotide motif in the unassembled reads. For 86,732 unique sequences among them, a total of 331,368 primer pairs were designed. Screening based on PCR amplification, polymorphisms and accurate genotyping resulted in sixteen primer sets, which were later characterized using 45 samples collected in Onagawa Bay, Miyagi, Japan. The presence of null alleles was suggested at four loci in the studied population but no evidence of allelic dropout was found. The observed number of alleles and heterozygosity was 2-20 and 0-0.88889, respectively, indicating polymorphisms and usefulness for population genetic analyses of this species. In addition, a large number of the microsatellite primers developed in this study are potentially applicable also for kinship estimation, individual fingerprint and linkage map construction.

Genomic Resources Notes accepted 1 February 2015 - 31 March 2015.

This article documents the public availability of (i) raw transcriptome sequence data, assembled contigs and BLAST hits of the Antarctic plant Colobanthus quitensis grown in two different climatic conditions, (ii) the draft genome sequence data (raw reads, assembled contigs and unassembled reads) and RAD-tag read data of the marbled flounder Pseudopleuronectes yokohamae, (iii) transcriptome resources from four white campion (Silene latifolia) individuals from two morphologically divergent populations and (iv) nuclear DNA markers from 454 sequencing of reduced representation libraries (RRL) based on amplified fragment length polymorphism (AFLP) PCR products of four species of ants in the genus Tetramorium.

Identification of molecular markers in pectoral fin to predict artificial maturation of female European eels (Anguilla anguilla).

The European eel is a critically endangered species that cannot be reproduced in captivity yet. Artificial maturation of female European eels can be achieved via a laborious and expensive procedure, including weekly injections with pituitary extracts for up to 6 months. The success rate is highly variable and a minimally invasive method for early selection of responsive eels would prevent the unnecessary and lengthy treatment of non-responding individuals. Since sexual maturation of European eels is accompanied by morphological changes of the pectoral fin, we examined whether fin could be used to monitor the response to the hormone treatment. Farmed eels were subjected to weekly injections with pituitary extracts and representative groups were sampled at 0 and 14-18 weeks of hormone treatment. Responders and non-responders were identified based on the gonado-somatic index. Transcriptomes of pectoral fin samples obtained at the start and end of the trial were mapped using Illumina RNAseq. Responders showed 384 and non-responders only 54 differentially expressed genes. Highly stringent selection based on minimum expression levels and fold-changes and a manual re-annotation round yielded 23 up-regulated and 21 down-regulated maturation marker genes. The up-regulated markers belong to five categories: proteases, skin/mucus structural proteins, steroid hormone signaling, tyrosine/dopamine metabolism and lipid metabolism. The down-regulated markers are either blood markers or lectin-related genes. In conclusion, pectoral fin transcriptomes are a rich source of indicator markers for monitoring hormone induced sexual maturation of female European eels. In addition, these markers provide important new insight into several fundamental processes in eel biology.

First draft genome sequence of the Japanese eel, Anguilla japonica.

The Japanese eel is a much appreciated research object and very important for Asian aquaculture; however, its genomic resources are still limited. We have used a streamlined bioinformatics pipeline for the de novo assembly of the genome sequence of the Japanese eel from raw Illumina sequence reads. The total assembled genome has a size of 1.15 Gbp, which is divided over 323,776 scaffolds with an N50 of 52,849 bp, a minimum scaffold size of 200 bp and a maximum scaffold size of 1.14 Mbp. Direct comparison of a representative set of scaffolds revealed that all the Hox genes and their intergenic distances are almost perfectly conserved between the European and the Japanese eel. The first draft genome sequence of an organism strongly catalyzes research progress in multiple fields. Therefore, the Japanese eel genome sequence will provide a rich resource of data for all scientists working on this important fish species.

Genomics in eels--towards aquaculture and biology.

Freshwater eels (genus Anguilla), especially the species inhabiting the temperate areas such as the European, American and Japanese eels, are important aquaculture species. Although artificial reproduction has been attempted since the 1930s and large numbers of studies have been conducted, it has not yet fully succeeded. Problems in eel artificial breeding are highly diverse, for instance, lack of basic information about reproduction in nature, no appropriate food for larvae, high mortality, and high individual variation in adults in response to maturation induction. Over the last decade, genomic data have been obtained for a variety of aquatic organisms. Recent technological advances in sequencing and computation now enable the accumulation of genomic information even for non-model species. The draft genome of the European eel Anguilla anguilla has been recently determined using Illumina technology and transcriptomic data based on next generation sequencing have been emerging. Extensive genomic information will facilitate many aspects of the artificial reproduction of eels. Here, we review the progress in genome-wide studies of eels, including additional analysis of the European eel genome data, and discuss future directions and implications of genomic data for aquaculture.

Primitive duplicate Hox clusters in the European eel's genome.

The enigmatic life cycle and elongated body of the European eel (Anguilla anguilla L., 1758) have long motivated scientific enquiry. Recently, eel research has gained in urgency, as the population has dwindled to the point of critical endangerment. We have assembled a draft genome in order to facilitate advances in all provinces of eel biology. Here, we use the genome to investigate the eel's complement of the Hox developmental transcription factors. We show that unlike any other teleost fish, the eel retains fully populated, duplicate Hox clusters, which originated at the teleost-specific genome duplication. Using mRNA-sequencing and in situ hybridizations, we demonstrate that all copies are expressed in early embryos. Theories of vertebrate evolution predict that the retention of functional, duplicate Hox genes can give rise to additional developmental complexity, which is not immediately apparent in the adult. However, the key morphological innovation elsewhere in the eel's life history coincides with the evolutionary origin of its Hox repertoire.

Within-population structure highlighted by differential introgression across semipermeable barriers to gene flow in Anguilla marmorata.

In the marine environment, differential gene exchange between partially reproductively isolated taxa can result in introgression that extends over long distances due to high larval dispersal potential. However, the degree to which this process contributes to interlocus variance of genetic differentiation within introgressed populations remains unclear. Using a genome-scan approach in the Indo-Pacific eel Anguilla marmorata, we investigated the degree of interpopulation genetic differentiation, the rate of introgression, and within-population genetic patterns at 858 AFLP markers genotyped in 1117 individuals. Three divergent populations were identified based on clustering analysis. Genetic assignments of individuals revealed the existence of different types of hybrids that tended to co-occur with parental genotypes in three population contact zones. Highly variable levels of genetic differentiation were found between populations across the AFLP markers, and reduced rates of introgression were shown at some highly differentiated loci. Gene flow across semipermeable genetic barriers was shown to generate spatial introgression patterns at some loci which define within-population structure over long distances. These results suggest that differential introgression in subdivided populations may be relevant when interpreting spatial variation patterns displayed by outlying loci in other marine fish populations.

Deep-ocean origin of the freshwater eels.

Of more than 800 species of eels of the order Anguilliformes, only freshwater eels (genus Anguilla with 16 species plus three subspecies) spend most of their lives in freshwater during their catadromous life cycle. Nevertheless, because their spawning areas are located offshore in the open ocean, they migrate back to their specific breeding places in the ocean, often located thousands of kilometres away. The evolutionary origin of such enigmatic behaviour, however, remains elusive because of the uncertain phylogenetic position of freshwater eels within the principally marine anguilliforms. Here, we show strong evidence for a deep oceanic origin of the freshwater eels, based on the phylogenetic analysis of whole mitochondrial genome sequences from 56 species representing all of the 19 anguilliform families. The freshwater eels occupy an apical position within the anguilliforms, forming a highly supported monophyletic group with various oceanic midwater eel species. Moreover, reconstruction of the growth habitats on the resulting tree unequivocally indicates an origination of the freshwater eels from the midwater of the deep ocean. This shows significant concordance with the recent collection of mature adults of the Japanese eel in the upper midwater of the Pacific, suggesting that they have retained their evolutionary origin as a behavioural trait in their spawning areas.

Seasonality of spawning by tropical anguillid eels around Sulawesi Island, Indonesia.

Remarkably little is known about the life histories of the many tropical anguillid eels distributed across the Indo-Pacific region, and since the Danish expedition to study eels in the region in 1928 and 1929, research on these eels has only begun again in recent years. Sampling for anguillid leptocephali in the Indonesian Seas has been carried out recently to learn about the spawning ecology and larval distributions of tropical eels there. The leptocephali of Anguilla marmorata, Anguilla bicolor pacifica, Anguilla borneensis, Anguilla interioris, and Anguilla celebesensis were collected around Sulawesi Island both in May 2001 and October of 2002. The development of genetic identification techniques has enabled these leptocephali to be identified to species, and their distributions and sizes during different seasons indicated that there are differing life history patterns among sympatric species in the region. A. celebesensis was found to have been spawning in Tomini Bay of northeastern Sulawesi Island in March and April 2001, but apparently, no spawning had occurred in the late summer and fall of 2002. Studies on anguillid glass eels have suggested that tropical anguillids may spawn throughout much of year, but our research on leptocephali in Tomini Bay and data on the downstream migration of tropical anguillids in the major tributary to Tomini Bay indicate that A. celebesensis may have a distinct seasonal pattern of spawning possibly related to the regional monsoon cycles. This is the first evidence of seasonality of spawning in tropical anguillid eels whose life histories are only just beginning to be revealed.

Multiple population structure of the giant mottled eel, Anguilla marmorata.

The population structure of the giant mottled eel, Anguilla marmorata, was investigated with mitochondrial and microsatellite DNA analyses using 449 specimens from 13 localities throughout the species range. Control region F-statistics indicated the North Pacific (Japan, Taiwan, Philippines, Sulawesi), South Pacific (Tahiti, Fiji, New Caledonia, Papua New Guinea), eastern Indian Ocean (Sumatra), western Indian Ocean (Réunion, Madagascar), Ambon, and Guam regions were significantly different (Phi(ST) = 0.131-0.698, P < 0.05) while only a few differences were observed between localities within the South Pacific. These regions were roughly clustered in the neighbour-joining tree, although Ambon individuals were mainly divided into North and South Pacific groups. Analysis with eight microsatellite loci showed almost identical results to those of the control region, except no genetic difference was observed between the western and eastern Indian Ocean (F(ST) = 0.009, P > 0.05). The Bayesian cluster analysis of the microsatellite data detected two genetic groups. One included four North Pacific localities, and the other included eight localities in the South Pacific, Indian Ocean, and Guam, but Ambon individuals were evenly assigned to these two groups. These results showed that A. marmorata has four genetically different populations (North Pacific, South Pacific, Indian Ocean, Guam region). The North Pacific population is fully panmictic whereas the South Pacific and Indian Ocean populations have a metapopulation structure. Interestingly, Guam was suggested to be inhabited by a reproductive population restricted to that region, and the individuals from the North and South Pacific populations co-exist in Ambon.