The reproductive ecology of two subspecies of the bitterling Rhodeus atremius (Cyprinidae, Acheilognathinae)

The reproductive ecology of two endangered subspecies of the bitterling Rhodeus atremius was investigated in two rivers in Okayama and Fukuoka Prefectures, Japan. Several other bitterling species, R. ocellatus, Tanakia limbata, T. lanceolata, Acheilognathus tabira and A. rhombeus, are also found in these rivers. Spawning of both R. atremius subspecies was recorded between late March and August 2007, peaking between April and July. Females of R. atremius began to mature at small body size (minimum 25 mm). They had small clutch sizes (maximum 16 eggs) and developed a short ovipositor (mean 13.6 mm), which was used to deposit a bulb-shaped egg of comparatively large size (3.2 mm³, major and minor axes 2.8 and 1.5 mm). Clutch size, ovipositor length and egg size appear to be a function of female body size. Both the body size and ovipositor length of R. atremius with ripe ova and the size and shape of the eggs varied seasonally. Rhodeus a. suigensis achieved maturity at a smaller body size, had a longer ovipositor and produced smaller eggs, with the minor axis being wider relative to the major axis than those of R. a. atremius. Rhodeus atremius may be adapted for depositing small numbers of eggs in a much shallower position inside the gills of smaller host mussels than other bitterling species.     

Host mussel utilization of the Itasenpara bitterling (<Emphasis Type="Italic">Acheilognathus longipinnis</Emphasis>) in the Moo River in Himi,Japan

Bitterling are fishes that use freshwater mussels for oviposition. The pattern of mussel utilization of an endangered bitterling species, Acheilognathus longipinnis, which spawned in autumn, was investigated in a lowland river. Six Unio douglasiae nipponensis (1.5% of total 388 individuals) hosted A. longipinnis embryos consistently in their outer gills, and they inhabited areas along the river bank where mussel density was relatively high on a less compacted mud substrate. Unexpectedly low rates of host utilization and the rare occurrence of juvenile (shell length, <40 mm) hosts (<0.8% of total individuals) suggest the need for greater attention to the conservation of A. longipinnis.     

Ovipositor ultrastructure of the striped bitterling Acheilognathus yamatsutae (Teleostei: Acheilognathinae) during spawning season

The ovipositor of striped bitterling Acheilognathus yamatsutae was subjected to ultrastructure and histochemical analysis during spawning season using light and electron microscopy. Although the ovipositor of A. yamatsutae is a long cylindrical tube with smooth external surface, it was possible to confirm the presence of well-developed fingerprint structure using scanning electron microscopy. Internal aspect analysis of ovipositor revealed formation of 5–8 longitudinal folds. Cross section analysis revealed that the ovipositor is composed of an outer epithelial layer, a mid connective tissue layer, and an inner epithelial layer. The outer epithelial layer contains 7–9 cell layers composed mainly of epithelial and mucous cells. Result of AB–PAS (pH 2.5) and AF–AB reaction showed that mucous cells contained mainly acidic carboxylated mucosubstances. The connective tissue layer was loose and made mainly of collagen fibers and some muscle fibers, along with blood vessels and a small number of chromatophores. The inner epithelial layer, which is a single layer, is composed of columnar epithelia. Observation under transmission electron microscope enabled distinction of the outer epithelial layer into superficial, intermediate and basal layers. Although the types of cells in the superficial tissue layer were diverse, they all shared the development of glycocalyx covered microridges. The majority of epithelial cells in the intermediate layer were cuboidal shaped, while those in the basal layer were columnar. Two types (A and B) of secretory cells were observed in the outer epithelial layer. The connective tissue layer had two types of chromatophores including xantophore and melanophore, in addition to a well-developed nerve fiber bundles. Columnar epithelial cells, mitochondria-rich cells and rodlet cells were observed in the inner epithelial layer. Microvilli were well developed on the free surface of columnar epithelial cells.     

Reproductive Ecology and Host Utilization of Four Sympatric Bitterling (Acheilognathinae,Cyprinidae) in a Lowland Reach of the Harai River in Mie,Japan

Bitterling are fishes that use freshwater mussels for oviposition. The reproductive ecology and pattern of mussel utilization of four sympatric species of bitterling, Acheilognathus rhombeus, A. tabira tabira, Tanakia lanceolata, and T. limbata, were investigated in a lowland river with seven sympatric mussel species. Three bitterling species are spring spawners with overlapping spawning seasons. A. rhombeus is an autumn-spawning species and is temporally isolated in its reproduction from the other species. Ovipositor length during oviposition of T. limbata and T. lanceolata was short, while those of A. tabira tabira and A. rhombeus were long. Most T. limbata inhabited near-shore areas, whereas the two other spring-spawning species were distributed across the entire river. All bitterling species used Inversidens brandti, Obovalis omiensis and Inversiunio jokohamensis as spawning hosts, but not the other mussel species available. T. lanceolata, A. tabira tabira and A. rhombeus showed spawning preferences for O. omiensis and I. brandti. However, T. limbata did not show clear preferences for any of the mussel species they used. A. t. tabira showed a significant preference for large I. brandti in offshore areas, while the other spring-spawning bitterling showed a preference for mussels inshore. These results are discussed in the context of reproductive resource partitioning.     

Life history and reproductive ecology of the endangered Itasenpara bitterling Acheilognathus longipinnis (Cyprinidae) in the Himi region,central Japan

The life history, reproductive ecology and habitat utilization of the Itasenpara (deepbody) bitterling Acheilognathus longipinnis were investigated in a lowland segment of the Moo River in Toyama Prefecture, central Honshu, Japan. Analysis of 1285 individuals revealed that the study population comprised a single size class, an age at maturation of 3 months and a life span of 1 year. On the basis of the growth pattern, the life cycle was divided into two stages: the juvenile stage, characterized by rapid growth, and the adult stage at which growth ceased. Spawning by A. longipinnis was recorded between early September and late October. Female A. longipinnis in the 0+ year age class began to mature when they reached a standard length (L_S) of 56·4 mm. Mature females had a large clutch size (maximum 273 eggs) and deposited highly adhesive and relatively large eggs (2·55 mm³; major axis, 3·12 mm; minor axis, 1·22 mm) via a short ovipositor (mean length, 21·5 mm) into freshwater mussels. The embryos remained in the gill cavities of the freshwater mussels (used as a spawning substratum) and emerged as juveniles (L_S, 9 mm). Habitat utilization during spawning was analysed using a generalized linear model. The best‐fit model showed that three environmental factors (freshwater mussel availability, water depth and vegetation cover) were important variables for habitat utilization by A. longipinnis. Shallow areas (water depth, 250–330 mm) created for rice paddy management and areas with an abundance of cover were particularly effective for predator avoidance. These results suggest that maintenance of water level fluctuations corresponding with rice cultivation and the abundance of vegetation on the river bank (particularly avoidance of concrete revetments) is essential for conservation of this species under current practices for rice cultivation in Japan.     

Factors affecting seasonal mortality of rosy bitterling (Rhodeus ocellatus kurumeus) embryos on the gills of their host mussel

I investigated the seasonal change in factors affecting embryonic mortality in the rosy bitterling, Rhodeus ocellatus kurumeus, a freshwater fish that spawns on the gills of living unionid mussels. Research was conducted in a small pond during 1999 and 2001 in which bitterling were provided with Anodonta sp. mussels for spawning. Bitterling spawned between April and July, peaking mid–late May. Seasonal survival rate of bitterling embryos in their mussel hosts was unimodal, with a peak between late April and mid May (about 70% of total spawnings). In mid April, survival was about 50%. The lowest survival was from late May to July (0%). Losses of bitterling embryos from mussels were identified by ejections from the mussel host. Ejections were categorized as either ejections of live embryos, or ejections of embryos that died in the mussel and were subsequently expelled from the mussel. Ejection rates of live embryos were higher in the earlier part of the spawning period (early–mid April) and dead embryo ejections in the later period (after June). The ejection rate of live embryos was higher among younger embryos earlier in the season, probably because of the incomplete development of morphological and behavioural traits associated with maintaining the embryo inside the mussel gill chambers, and as a consequence of a more protracted developmental period at low temperatures making them more susceptible to ejection. The ejection rate of dead embryos was higher in older embryos later in the season, and in larger mussels and at high embryo densities. The survival of embryos in mussels was probably related to oxygen availability, with mortalities probably caused by asphyxiation. Increased embryo mortalities may arise through competition among embryos, between embryos and mussel, and ambient dissolved oxygen levels. The optimal period for bitterling to spawn may represent a balance between two opposing factors; with positive and negative effects of a seasonal rise in temperature directly affecting embryonic growth rate and oxygen availability.An erratum to this article can be found at This revised version was published online January 2005 with the correction of the authors name.     

Molecular cytogenetic study of the European bitterling Rhodeus amarus (Teleostei: Cyprinidae: Acheilognathinae)

The European bitterlings (Rhodeus amarus) from the Eastern locations were cytogenetically examined by conventional and molecular techniques. All analyzed individuals presented invariably the same chromosomal constitution of 2n = 48, with 8 metacentrics + 20 submetacentrics + 20 subtelo-acrocentrics and C-banding positive heterochromatin at the pericentromeric regions in most of the chromosomes. Moreover, some of the chromosomes had short arms entirely built with heterochromatin. GC-rich Ag-NORs (nucleolus organizer regions) were located at the short arms of two submetacentric chromosomes, and the length polymorphism of these regions was found. Multiple location of 28S rDNA sequences with fluorescence in situ hybridization signals was observed on the long and/or short arms of three submetacentric chromosomes including NOR regions and short arms of three to five acrocentric chromosomes in the studied fish. 5S rDNA sites were found on the short arms of two subtelocentric chromosomes, and telomeric repeats were localized at the ends of all chromosomes. Provided results have expanded our knowledge concerning genetic characteristics of the European bitterlings that may be profitable in the conservation programs of this endangered species.     

The reproductive ecology of the southern red tabira bitterling Acheilognathus tabira jordani in Japan

The life history, population and reproductive variables of the southern red tabira bitterling Acheilognathus tabira jordani were investigated in a lowland reach of the River Ohara in Shimane Prefecture, western Honshu, Japan. Acheilognathus t. jordani , like all other species of bitterling, lays its eggs on the gills of freshwater mussels. It was the only species of bitterling present in the study reach, and three species of bivalve mussel were available to it for spawning: Anemina arcaeformis, Anodonta lauta and Corbicula leana . Spawning by A.t. jordani was recorded between early April and early July in 2003 and began at a size of 38· 0 mm standard length ( L _S) in the 1+ age class. Ovipositor length ( L _OP) during oviposition was positively correlated with female L _S, and showed significant seasonal variation, with a mean ± s.d. L _OP of 27· 5 ± 5· 3 mm ranging from 16· 8 to 42· 0 mm during the spawning period, which was shorter than that of a previously studied A. t. tabira population. Eggs of this subspecies are relatively long and elliptic in shape, with a volume of c. 2· 4 mm³. Egg number correlated positively with female L _S and both egg shape and volume changed significantly with season. The population size of adults was estimated to be 850 individuals, and comprised age 0+ to 3+ individuals with L _S ranging from 12· 0 to 72· 2 mm. The population sex ratio was significantly female biased, with seven females: three males. Egg shape and size and L _OP during oviposition in the present A. tabira population may be the result of local adaptations to the mussel species utilized and no competition with other bitterling species for spawning sites.     

Rhodeus pseudosericeus sp. nov., a new bitterling from South Korea (Cyprinidae, Acheilognathinae)

A new bitterling, Rhodeus pseudosericeus sp. nov., is described on the basis of 31 specimens from five localities included in the Namhan River system, South Korea. The new species is distinguished from other Rhodeus species by the following combination of characters: branched dorsal fin rays 9–10 (mode 9); branched anal fin rays 9–11 (mode 10); longest simple ray of dorsal fin strong and stiff, distally segmented; pelvic fin rays i, 6–7; iris of males blackish; dorsal and anal fins of males grayish in breeding season; karyotype with 2n = 48 (8m + 20sm + 20st). Rhodeus pseudosericeus sp. nov. is similar to Rhodeus sericeus sericeus in the number of pelvic fin and branched dorsal fin rays and the melanophores present on the dorsal fin membrane, but differs from the latter in having a greater body depth, more branched anal fin rays, fewer vertebrae, a lower number of scales in the lateral series, and differing male nuptial coloration. Received: June 30, 2000 / Revised: February 21, 2001 / Accepted: March 6, 2001     

Variation in male reproductive traits among three bitterling fishes (Acheilognathinae: Cyprinidae) in relation to the mating system

Male traits that correlate with fertilization success include testis size and structure, ejaculate size, ejaculation frequency, and sperm motility. Two hypotheses potentially explain interspecific differences in these traits: sperm competition and sperm limitation. We examined variation in six traits associated with fertilization success in three closely‐related species of bitterling fish; the European bitterling (Rhodeus amarus), the Chinese rose bitterling (Rhodeus ocellatus), and the Chinese bitterling (Rhodeus sinensis). Interspecific differences indicated that the three study species have evolved different sperm allocation strategies. Rhodeus amarus displayed the most developed reproductive apparatus with a number of traits associated with both high levels of sperm production and fertilization efficiency. Rhodeus ocellatus and R. sinensis appear to have more comparable sperm allocation strategies, although relative testis size and spermatozoa head : tail ratio were greater in R. sinensis, suggesting that sperm competition risk may be higher in this species. All three species possessed an unusually well developed sperm duct with evidence of mucin production, which greatly extends the longevity of sperm and, consequently, the period over which fertilization can occur. We discuss these findings in the context of differences in the mating systems of the species examined, and relate the results obtained to differences in the temporal and spatial clustering of fertilizations. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 103 , 622–632.     

Histological structure of the female gonads and ovipositor of the European bitterling,Rhodeus amarus (Bloch, 1782) (Cyprinidae: Acheilognathinae)

We investigated the histological structure of the female gonads and ovipositor of the European bitterling, Rhodeus amarus. The base of the ovipositor was formed by the conical organ or ‘eminence’. Based on the structure of the conical organ, including a special distribution of collagenous and muscular fibrils, a well‐developed vascular system and numerous scyphoid mucous cells in the internal epithelium, this organ was used for temporary storage of oocytes during the spawning period and for their movement along the ovipositor. An extensive network of blood vessels, muscular fibrils and numerous collagenous fibrils in the connective tissue of the ovipositor may make a functional contribution to the ovipositor by making it firmer during egg laying. Mucous cells were detected in the medial and distal regions of the ovipositor, which may play a role in facilitating insertion of the ovipositor into the exhalant siphon of a mussel during oviposition. European bitterling are batch spawners, and the female spawns eggs in clutches at intervals during the breeding season, which were visible as three distinct cohorts of oocytes in the ovary.     

Spatio-temporal patterns of the decline of freshwater mussels in the Little South Fork Cumberland River,USA

The Little South Fork Cumberland River, Kentucky and Tennessee, USA, was a globally important conservation refugium for freshwater mussels (Mollusca: Unionidae) because it supported an intact example (26 species) of the unique Cumberland River mussel fauna including imperiled species. We used previous surveys and our 1997–1998 survey to reconstruct the historical fauna, to describe spatio-temporal patterns of density and number of species, and to evaluate the probable sequence and cause of observed mussel declines. We were specifically interested in better understanding how mussel assemblages respond to chronic disturbances, and how these changes manifest in persistence patterns. Density and numbers of species declined steadily from 1981 to 1998, but declines occurred first in the lower river (early 1980s), followed by declines in the upper river (late 1980s to early 1990s). Of the total species recorded from the Little South Fork, 17 (65%) are seemingly extirpated and five others appear near extirpation. Declines are associated with at least two, temporally distinct major insults. Lower river declines are associated with surface mining, whereas, oil extraction activities are implicated in upper river declines. Regardless of causal factors, species persistence was primarily a function of predecline population size with only the most numerous and widespread species surviving. At this time, the river appears lost as a conservation refugium for mussels despite its remoteness, predominantly forested watershed, and several layers of existing statutory and regulatory environmental safeguards. We suggest that the river could be restored and mussels reintroduced if an interagency task force is formed to identify and mitigate specific stressors now affecting most mussel species in the river.Nomenclature: Turgeon et al. (1998).     

Genetic variation of the cytochrome b gene in the rosy bitterling, Rhodeus ocellatus (Cyprinidae) in Japan

According to conventional views, the rosy bitterling, Rhodeus ocellatus, comprises two subspecies, R. ocellatus kurumeus and R. ocellatus ocellatus, the former being native to Japan whereas the latter was introduced into Japan from China during World War II. To examine the genetic structure of Japanese R. ocellatus, part of the mitochondrial cytochrome b gene from 48 individuals collected from various locations in Japan was sequenced. Three major mitochondrial lineages were found. Based on historical evidence, two of these represent R. ocellatus ocellatus and the third R. ocellatus kurumeus. The existence of two distinct lineages of R. ocellatus ocellatus in Japan suggests at least two colonizations. Some local populations comprised purely R. ocellatus kurumeus, but those from Kashima and Ogori included both subspecies. Because the proportion of R. ocellatus ocellatus in Kashima increased from 1994 to 1995, invasion by R. ocellatus ocellatus into R. ocellatus kurumeus habitats is apparently in progress. Received: April 30, 1999 / Revised: March 22, 2000 / Accepted: December 20, 2000     

Effects of temperature on free‐embryonic diapause in the autumn‐spawning bitterling Acheilognathus rhombeus(Teleostei: Cyprinidae)

In bitterling Acheilognathus rhombeus , developmental arrest always occurred at stage D of the free‐embryonic phase, regardless of incubation temperature. Developmental arrest was terminated only by a cold treatment at 4° C for 60–90 days, initiated 10 days post‐hatching. After the termination of developmental arrest, free‐embryos became larvae c . 6 months after hatching, regardless of the time of initiation and duration of the cold treatment. In hybridization experiments between A. rhombeus and several species of spring‐spawning bitterlings, free‐embryos became free‐swimming larvae within 60 days after hatching in all experiments. Developmental arrest was not observed in any of the hybrids, regardless of parental sex. These results suggest that free‐embryonic diapause in A. rhombeus is not induced by environmental factors, such as cold, but by genetic factors, which are recessive to those in spring‐spawning bitterlings. Free‐embryonic diapause in A. rhombeus appears to be an adaptation to winter, which might have evolved with reproduction in autumn among autumn‐spawning bitterling species. This is the only report of free‐embryonic diapause after hatching in fishes, and only the second example of diapause in fishes, along with annual killifishes (Rivulidae).     

Timing of juvenile emergence from host mussels in the Japanese rosy bitterling, <Emphasis Type="Italic">Rhodeus ocellatus kurumeus</Emphasis>

The diel rhythm of juvenile emergence from host mussels in the Japanese rosy bitterling, Rhodeus ocellatus kurumeus, was investigated in a small pond in Osaka prefecture, Japan. The emergence of juvenile bitterling from host mussels occurred principally at night, with a peak in the early part of the night. The pattern of juvenile bitterling emergence was discussed in the context of an adaptive response to diurnal predation.     

Complete mitochondrial genome of Acheilognathus signifer (Cypriniformes, Cyprinidae): comparison of light-strand replication origins from six Acheilognathus species

Acheilognathus signifer (Cypriniformes, Cyprinidae) is an endemic, endangered fresh water fish species in the Korean peninsula. In this study, the mitochondrial genome of A. signifer was completely sequenced, which is 16,566 bp in length. The characteristics of the complete mitochondrial genome were described in detail.     

Development of European bitterling in the gills of freshwater mussels

The development of bitterling embryos within the unique environment of a freshwater mussel's gills requires a departure from typical cyprinid embryological development. Eggs are large (2·6×1·7 mm), illustrating the low risk of predation, and elliptical; a response to unionid gill morphology and a way of increasing the transfer rates of respiratory and excretory products to and from the tissues. The yolk sac develops elongated lateral processes during early ontogeny; these secure the embryo into the host's interlamellar space. Once the larva is capable of movement (8·2 mm) the lateral processes are lost and the larva becomes less dependent of the host's gills for the provision of oxygen. Hatching (3·3 mm) and pigmentation of the blood (6·4 mm) occur relatively early; this may increase the rate of oxygen supply to the tissues. Pigmentation of the eyes and appearance of the melanophores occurs relatively late (7·4 mm and 7·9 mm, respectively); embryos are not required to detect or avoid predators. Bitterling larvae generally emerge from the host once the yolk sac has been consumed (10·5 mm); this may mark a change in respiratory and nutritional requirements.     

A proximate cue for oviposition site choice in the bitterling (Rhodeus sericeus)

1. We investigated two possible proximate cues used for oviposition site choice by females of the bitterling ( Rhodeus sericeus ), a freshwater fish that spawns on the gills of live unionid mussels. The two cues were the flow velocity and/or oxygen content of water emerging from the exhalant siphon of a mussel.
2. Field observations showed that female bitterling always inspected the exhalant siphons of mussels before they spawned in them. Siphon inspection was not always a prelude to spawning and it may serve as a means of assessing mussel quality. Female skimming behaviour, swimming over a mussel without spawning, may also be used to assess mussel quality, although the mechanism for this is unclear.
3. Measurements of the flow velocity of water emerging from the exhalant siphons of four mussel species ( Anodonta anatina, A. cygnea, Unio pictorum and U. tumidus ) showed a significant difference among species, with U. tumidus having the highest mean flow velocity and U. pictorum the lowest.
4. Measurements of the change in oxygen concentration of water entering a mussel inhalant siphon and leaving its exhalant siphon in field and laboratory studies showed a significant difference among the four mussel species, with A. cygnea exhibiting a significantly higher change in oxygen concentration than the other species.
5. The presence of bitterling embryos in the gills of a mussel significantly increased its oxygen consumption whereas larval glochidia had no significant effect. We discuss oxygen availability as a possible proximate cue for oviposition site choice in bitterling.     

Features of reproductive biology in females of bitterlings (Cyprinidae: Acheilognathinae) from the Amur River basin

Data on morphological and functional characteristics of the ovipositor, conical organ, and ovaries in four species of bitterlings (Russian bitterling Acanthorodeus asmussii, Khanka spiny bitterling A. chankaensis, bitterling Rhodeus sericeus, and Amur bitterling R. amurensis) from the Amur River basin are obtained and reviewed for the first time. In all studied fish species, several generations of sex cells develop at the same time, which leads to releasing of oocytes in batches. The specificity of Amur bitterling, bitterling, and Russian bitterling to selection of mollusks for egg deposition is determined.