Male coloration signals direct benefits in the European bitterling (Rhodeus amarus)

Female mating preferences are frequently associated with exaggerated male sexual traits. In the European bitterling, Rhodeus amarus, a fish with a resource-based mating system, male coloration is not associated with indirect genetic benefits of female mate choice, and does not reliably signal spawning site quality. We tested a link between the extent of male carotenoid-based coloration and testis size and number of spermatozoa stripped from the testes. Male body size predicted spermatozoa number, but less reliably than the extent of male coloration. Male color was a highly significant predictor of spermatozoa number, with approximately 26 % of variance in the number of spermatozoa stripped from males predicted from male color after controlling for male body size. Body size, but not coloration, predicted teste size. Female bitterling often risk sperm limitation, especially during pair spawnings, and male nuptial coloration may be under direct selection through female mate choice as a signal of male fertilization efficiency.     

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.     

Range-wide population genetic structure of the European bitterling (Rhodeus amarus) based on microsatellite and mitochondrial DNA analysis

An understanding of recent evolutionary processes is essential for the successful conservation and management of contemporary populations, especially where they concern the introduction or invasion of species outside their natural range. However, the potentially negative implications of intraspecific introductions and invasions have attracted less attention, although they also represent a potential threat to biodiversity, and are commonly facilitated through human activities. The European bitterling (Rhodeus amarus) is a small cyprinid fish that decreased greatly in its distribution during the 1970s and 1980s and was subsequently included on many European conservation lists. This decline appears to have reversed, and the extent of its distribution now exceeds its former range. We used a combination of 12 microsatellite markers and cytochrome b sequences on a large data set (693 individuals) across the current range of the European bitterling to investigate possible scenarios for its colonization of Europe. We show that the inferred history of colonization of Europe was largely congruent between mitochondrial and nuclear markers. The most divergent mtDNA lineages occur in the Aegean region but probably are not reproductively isolated as the Aegean populations also displayed mtDNA haplotypes from other lineages and nuclear data indicated their close relationship to Danubian populations. Much of Europe is currently populated by descendants of two main lineages that came to natural secondary contact in western Europe. An approximate Bayesian computation analysis indicates different dates for admixture events among western and central European populations ranging from the last deglaciation (natural) to the last few centuries (human-assisted translocations).     

No evidence for host specialization or host-race formation in the European bitterling (Rhodeus amarus), a fish that parasitizes freshwater mussels

Coevolutionary relationships between parasites and hosts can elevate the rate of evolutionary changes owing to reciprocal adaptations between coevolving partners. Such relationships can result in the evolution of host specificity. Recent methodological advances have permitted the recognition of cryptic lineages, with important consequences for our understanding of biological diversity. We used the European bitterling (Rhodeus amarus), a freshwater fish that parasitizes unionid mussels, to investigate host specialization across regions of recent and ancient sympatry between coevolving partners. We combined genetic data (12 microsatellite and 2 mitochondrial markers) from five populations with experimental data for possible mechanisms of host species recognition (imprinting and conditioning). We found no strong evidence for the existence of cryptic lineages in R. amarus, though a small proportion of variation among individuals in an area of recent bitterling–mussel association was statistically significant in explaining host specificity. No other measures supported the existence of host‐specific lineages. Behavioural data revealed a weak effect of conditioning that biased behavioural preferences towards specific host species. Host imprinting had no effect on oviposition behaviour. Overall, we established that populations of R. amarus show limited potential for specialization, manifested as weak effects of host conditioning and genetic within‐population structure. Rhodeus amarus is the only species of mussel‐parasitizing fish in Europe, which contrasts with the species‐rich communities of bitterling in eastern Asia where several host‐specific bitterling occur. We discuss costs and constraints on the evolution of host‐specific lineages in our study system and more generally.     

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.     

Genetic diversity in the Japanese rosy bitterling, Rhodeus ocellatus kurumeus (Cyprinidae)

Comparison of meristic characters (pored lateral line scales, vertebrae, and fin rays), and PCR-RFLP analysis in the D-loop and ND1 regions of mitochondrial DNA were performed to estimate the genetic diversity in local populations of the Japanese rosy bitterling, Rhodeus ocellatus kurumeus. In terms of meristic characters, the Fukuoka population was the largest in both range and variance of the number of pored lateral line scales and vertebrae (abdominal and caudal), and Osaka was the second, whereas the Kagawa population showed the smallest range and variance in these characters. In PCR-RFLP analysis, 11 haplotypes (3 in Fukuoka, 2 in Okayama, 2 in Kagawa, and 4 in Osaka) were observed, and nucleotide sequence divergence (NSD) was approximately two times larger in ND1 (mean, 0.61%) than in D-loop (mean, 0.31%). In the neighbor-joining (NJ) tree, based upon the NSD value in ND1, haplotypes were arranged into four clades, which corresponded to the locality of each haplotype. The Fukuoka population was conspicuously apart from the other populations (mean, 0.90% in NSD), but the remaining three showed a similar genetic distance with each other (mean, 0.48%–0.52% in NSD). In haplotype diversity of mtDNA, half the stations in Osaka and all in Kagawa were monomorphic. Especially, two haplotypes endemic to Kagawa were randomly distributed, irrespective of drainages. Rhodeus o. kurumeus in Fukuoka inhabits small rivers and creeks (open water systems), while that in Kagawa and Osaka lives in small ponds (closed water systems). Taking the information of morphology, mtDNA, and habitat into consideration, the low genetic diversity in Kagawa and Osaka populations of R. o. kurumeus is thought to be mainly the result of the isolation of their habitat. Received: January 14, 2001 / Revised: June 14, 2001 / Accepted: July 1, 2001     

Trends of a bitterling (Rhodeus sericeus) population in a man-made ditch network

This study describes the trends of a bitterling population in the Bourgneuf Marsh in relation to human activities. Although there were no bitterling during the period 1987-91, the species rapidly colonized the ditch network sampled (density reaching 61.7 +/- 47.0 individuals/100 m2 and frequency of occurrence around 70-75%) during a 5-year period (1997-2001). Clearance operations probably favoured the expansion of this vulnerable fish species because of positive associations of bitterling with large and deep ditches that had been cleared in the last 2-3 years and characterized by a small cover of submerged plants. Management recommendations are discussed for conserving the bitterling population.     

Molecular characterization and expression analysis of a peroxiredoxin 1 cDNA from Korean rose bitterling (Rhodeus uyekii)

Peroxiredoxins (Prxs), also known as natural killer cell enhancing factors in fish, role as antioxidant proteins and participate in a variety of biological processes, including H₂O₂-mediated cell signaling, molecular chaperoning, and mitochondrial function. In this study, we isolated and characterized a Prx 1 cDNA from the Korean rose bitterling Rhodeus uyekii, and designated it RuPrx 1. The RuPrx 1 cDNA encodes a 197-amino-acid polypeptide that belongs to the class of typical 2-Cys Prxs that contain peroxidatic and resolving cysteines. The deduced RuPrx 1 protein shows strong homology (77.38–92.89 %) with Prx 1 proteins from other species, including fish, amphibians, and mammals, and it is most closely related to rainbow smelt Prx 1. RuPrx 1 mRNA was ubiquitously detected in all tested tissues and its expression was comparatively high in the brain, intestine, kidney, liver, ovary, stomach, and testis. Expression of RuPrx 1 mRNA in liver peaked 3 h post-infection with Aeromonas hydrophila and decreased 24 h post-infection while the expression in intestine decreased 24 h post-infection. These results suggest that RuPrx 1 is conserved through evolution and may play roles similar to its mammalian counterparts.     

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.     

Taste attractiveness of different hydrobionts for roach Rutilus rutilus, bitterling Rhodeus sericeus amarus, and rainbow trout Oncorhynchus mykiss

Comparative study of the taste attractiveness of different aquatic invertebrates (daphnids Daphnia longispina and D. pulex, larvae of Chironomidae, whirligig Gyrinus marinus, water skaters Gerris spp.) and plants (duckweed Lemna minor, filamentous alga Cladophora sp.) for roach Rutilus rutilus, bitterling Rhodeus sericeus amarus, and rainbow trout Oncorhynchus mykiss has been performed. Roach’s taste preferences for agar-agar pellets, containing the aqueous extracts of the organisms under study, varies from maximum (daphnids, filamentous alga, larvae of chironomids) to minimum (duckweed) or it can be absent (whirligig). Different taste preferences to pellets containing the extracts of aquatic organisms has been also found in bitterling (daphnids, larvae of chironomids, water skaters) but not in rainbow trout (daphnids, water skaters). It has been suggested that whirligigs and water skaters do not contain any deterrent substances ensuring chemical defense from predators. It has been demonstrated that intraoral gustatory reception of food items follows two alternative behavioral stereotypes that are different from each other in the time during which food items are retained in the oral cavity and the number of manipulations (subsequent grasps of a food item) during the perception process. We discuss the importance of gustatory reception as the major sensory mechanism that ensures feeding selectivity of fish and decreases interspecific competition over food.     

Genetic markers distinguishing between the two subspecies of the rosy bitterling,Rhodeus ocellatus (Cyprinidae)

Eleven populations of the rosy bitterling,Rhodeus ocellatus, from different localities in Japan, were genetically compared at 16 protein-coding loci using starch-gel electrophoresis. Two loci,Ldh-2 andPgdh, were demonstrated as diagnostic markers for the identification of two subspecies;R. ocellatus kurumeus, which is native to Japan, andR. ocellatus ocellatus, which was introduced from China. The two subspecies were distinguished by the complete substitution of different alleles between them. Population ofR. ocellatus kurumeus occurring in Yao City, Osaka, and in Kanzaki, Saga Prefecture were genetically closely related to each other (genetic distance: D=0.056) but distantly so toR. ocellatus ocellatus from Saitama Prefecture (D=0.202 or 0.265). Electrophoretic analyses also elucidated the existence of hybrid populations of the two subspecies. The populations ofR. ocellatus kurumeus in Yao City had lower genetic variability and a lower incidence of white coloration on the ventral fins than populations of the same in Saga Prefecture. The present study strongly implies that the introduction of the foreign freshwater fishes with subspecific differentiation, into the original range of indigenous subspecies, should be averted not to bring the genetic pollution.     

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     

Sex determination system of the rosy bitterling, Rhodeus ocellatus ocellatus

The rosy bitterling, Rhodeus ocellatus ocellatus, is the only fish species known in which artificial triploids are always male, regardless of the kind of polyploidization technique used. In order to elucidate the genetic sex determination system of the rosy bitterling, two kinds of gynogenesis were carried out: retention of the second polar body (GRSPB) and suppression of the first cleavage (GSFC). The sex ratio of progeny was nearly 7:1 (:) for both GRSPB and GSFC, while those of control and parental fish were almost 1:1. In backcrosses of female progeny by GRSPB and normal diploid males, male progeny were observed at low frequency (one or two individuals in each experiment), except in one experiment where the appearance rate of males was about 50%. From results of gynogenesis and backcrosses, the following conclusions can be made. The genetic sex determination system of the rosy bitterling is a heterogametic female system (ZW). Survival rate of superfemales (WW), produced by gynogenesis, is much lower than that of males (ZW).There is a possibility that crossovers between sex determining genes and a centromere occur in the first meiosis. With repect to the mechanism of unisexuality (male) of artificial triploids of the rosy bitterling, only males (ZZZ and ZZW) are presumed viable, while females (ZWW) are probably inviable.     

Rose bitterling (Rhodeus ocellatus) embryos parasitize freshwater mussels by competing for nutrients and oxygen

Spence, R. and Smith, C. 2011. Rose bitterling (Rhodeus ocellatus) embryos parasitize freshwater mussels by competing for nutrients and oxygen. —Acta Zoologica (Stockholm) 00 : 1–6. Understanding how parasites inflict fitness costs on their hosts is a key question in host–parasite biology. Rose bitterling (Rhodeus ocellatus) are small cyprinid fish that place their eggs in the gills of living freshwater mussels. The embryos complete their development inside the mussel gill and emerge as free‐swimming larvae after approximately 4 weeks. Bitterling show a range of specialized adaptations for using mussels as a spawning substrate, and the presence of bitterling embryos has been shown to retard the growth of mussels. We compared the development and survival of embryos incubated in either mussels or Petri dishes and exposed to either nutrient‐rich or nutrient‐poor pond water. Embryonic development rate was significantly faster in Petri dishes, probably as a result of oxygen limitation in mussel gills. Embryo survival rate was significantly higher in nutrient‐rich than filtered water, suggesting that the embryos obtained a nutritional benefit prior to emergence from the host. The results imply that bitterling embryos compete for oxygen and nutrients with their host mussel, as well as each other, and in this way, impose a growth cost on mussels.     

Molecular characterization of a tandem-repeat galectin-9 (RuGlec9) from Korean rose bitterling (Rhodeus uyekii)

Galectin-9 is a b-galactoside-binding lectin that regulates many cellular functions, ranging from cell adhesion to pathogen recognition. We isolated and characterized the cDNA of tandem-repeat galectin-9 (RuGlec9) from the Korean rose bitterling (Rhodeus uyekii), an endemic Korean fish belonging to the Acheilognathinae subfamily of the Cyprinidae family. RuGlec9 cDNA is 1486 bp long and encodes a polypeptide of 323 amino acids containing two carbohydrate-recognition domains connected by a linker peptide. The deduced amino acid sequence of RuGlec9 shows 45-84% amino acid sequence identity to other galectin-9 sequences, including those from mammals and fish. RuGlec9 appeared in a large cluster with other galectin-9 sequences from fish and is more closely related to galectin-9 from Danio rerio than to those of other fish and mammals. RuGlec9 mRNA was expressed highly in the testis, spleen, intestine, stomach, and liver, and moderately in the brain, kidney, ovary, and gills of normal Korean rose bitterling. RuGlec9 mRNA expression in the spleen was increased by lipopolysaccharide. These results suggest that RuGlec9 plays a role in innate immunity in Korean rose bitterling.     

The role of the European bitterling (<Emphasis Type="Italic">Rhodeus amarus,</Emphasis> Cyprinidae) in parasite accumulation and transmission in riverine ecosystems

In aquatic ecosystems, fish play a key role in parasite accumulation and transmission to predacious animals. In the present study, realized on seven populations of a small cyprinid fish species, the European bitterling Rhodeus amarus, we investigated (1) the role of the European bitterling as a potential intermediate or paratenic host, (2) the ability of the fish to accumulate parasites with similar final host group, and (3) its significance as a potential source of parasite infection in the ecosystem in respect to habitat characteristics. A total of 36 parasite species were recorded; 31 species (90% of all parasite specimens) were classified as endoparasites. Most of the endoparasites were found in the larval life stage, using bitterling as an intermediate or paratenic host. In particular, parasite community structure showed significantly higher proportions of allogenic parasites in comparison with autogenic. The supposed co-occurrence of parasite species with identical final host groups showed only a weak association. The adjacent reservoir areas were a significant determinant of both the total and infracommunity parasite species richness and for the mean parasite abundance. No relationship between the distance of sampling site from the adjacent reservoir and parasite community characteristics was found. As a small-sized fish with a wide distribution range and high local abundances, the European bitterling can represent a natural prey for a wide range of piscivorous predators. Due to its susceptibility to the number of larval endoparasites, this fish species may therefore fulfill the role as important transmitter of parasites to their final hosts.     

Minute tubercles on the skin surface of larvae in the Korean endemic bitterling, Rhodeus pseudosericeus (Pisces, Cyprinidae)

The morphology and distribution of the minute tubercles on the skin surface of larvae in Korean bitterling, Rhodeus pseudosericeus, were observed during larval development. Just after hatching, the epidermis of the larvae consists of a thin single cell layer having smaller basophilic flat or round‐flattened basal cells. As the larvae grow, the epidermis contains more small flat cells and large epidermal cells that are round or hemisphere‐shaped. These large unicellular epidermal cells, called minute tubercles, consist of more or less homogeneous cytoplasm that is PAS (Periodic acid‐Schiff method) positive. They are more densely distributed in the wing‐like yolk sac projection. Vestigial minute tubercles occur in the body region and the caudal fin‐fold region. These minute tubercles grow in number and height from 6 to 8 days after hatching onward. However, they become reduced in height and number as the larvae develop. At day 31 after hatching (i.e. free‐swimming stage), minute tubercles no longer exist on the larval skin. The sequence of occurrence and gradual disappearance of these cell structures are described and histologically documented for comparative purposes of beta, taxnomomic and environmental studies.