Seasonal change in the spatial utilization of host mussels in relation to ovipositor length by female rosy bitterling Rhodeus ocellatus kurumeus

The seasonal changes in ovipositor length and utilization patterns of mussels for oviposition in the rosy bitterling Rhodeus ocellatus kurumeus (Cyprinidae) were investigated in a field experiment and field surveys during the breeding period (April to August). The mean length of ovipositors at oviposition was short at the start (early April) and end (July) of the breeding period. Females with long ovipositor at oviposition were collected between mid‐April and June. Mark‐and‐recapture data showed that ovipositor length at oviposition changed rhythmically throughout the breeding period, shortening and lengthening as the female entered the spawning and resting phase. The density of rosy bitterling embryos in mussels increased between April and June, peaking in May, but decreasing in July. The position of eggs on mussel gills varied from close to the exhalant siphon to deeper inside the gill during April, and periodically thereafter. There was a positive correlation between ovipositor length at oviposition and the distance from exhalant siphon of mussels to eggs deposited by females, suggesting that ovipositor length at oviposition determined the position of eggs deposited on a mussel gill. Because dissolved oxygen in mussel gills decreased with the density of bitterling embryos, suitable positions for embryo survival in gills changed with embryo density. By changing ovipositor length at oviposition, females might be able to spawn their eggs in a position that maximizes embryo survival. Thus, plasticity in ovipositor length at oviposition may play an important role as an adaptation of rosy bitterling in utilizing mussels when their quality as a spawning substratum fluctuates seasonally.     

The bitterling–mussel symbiosis: a model for host‐parasite adaptation

Bitterlings (Acheilognatinae) are a monophyletic group of cyprinid fishes that lay their eggs in the gill chamber of freshwater mussels. They have evolved many behavioural, morphological and physiological adaptations to the symbiosis. Female bitterling develop a long ovipositor that insert into the exhalant siphon of a mussel and males fertilize the eggs by releasing sperm over the inhalant siphon of the mussel. Embryos hatch within 2 days but develop inside the mussel for further 3 to 6 weeks. Embryos are adapted to the low oxygen environment in the mussel's gill chamber. Both males and females discriminate among mussels in relation to their quality as host for developing embryos. On the other hand, mussels used for oviposition have larvae that obligate ectoparasites on fish. Here I review current knowledge on the status of the symbiosis, developmental and behavioural adaptations by bitterling and mussel and summarize costs and benefits to both symbionts. Further, I use a recent well‐resolved bitterling phylogeny to emphasize the potential of this model system to study the evolution of this symbiosis, which is a part of the ongoing study.     

A possible evolutionary lag in the relationship between freshwater mussels and European bitterling

The coevolutionary dynamics between European bitterling Rhodeus amarus and freshwater unionid mussels, which the former parasitize by laying eggs on their gills, were tested. In a series of experiments fish preferences and mussel responses were compared in parasites and hosts of recent (Europe) and ancient (Asia) sympatry. Rhodeus amarus readily oviposited on the gills of all mussel species tested. Fish that laid their eggs on the gills of Asian Anodonta woodiana, however, suffered a dramatic reduction in reproductive success compared to fish that oviposited on the gills of European mussels: Unio pictorum , Unio tumidus , Anodonta anatina and Anodonta cygnea . This difference was the result of egg ejection behaviour by mussels rather than the unsuitability of the internal gill environment for European bitterling embryo development. The ejection response of mussels with a long sympatry with European bitterling was considerably more pronounced than that of mussels with a substantially shorter sympatry. The data support a coevolutionary arms race between bitterling and mussels and point to an evolutionary lag in the relationship between R. amarus and its European mussel hosts.     

THE BITTERLING–MUSSEL COEVOLUTIONARY RELATIONSHIP IN AREAS OF RECENT AND ANCIENT SYMPATRY

Host–parasite relationships are often characterized by the rapid evolution of parasite adaptations to exploit their host, and counteradaptations in the host to avoid the costs imposed by parasitism. Hence, the current coevolutionary state between a parasite and its hosts is predicted to vary according to the history of sympatry and local abundance of interacting species. We compared a unique reciprocal coevolutionary relationship of a fish, the European bitterling (Rhodeus amarus) and freshwater mussels (Unionidae) between areas of recent (Central Europe) and ancient (Turkey) sympatry. Bitterling parasitize freshwater mussels by laying their eggs in the gills of mussel and, in turn, mussel larvae (glochidia) parasitize the fish. We found that all bitterling from both regions avoided one mussel species. Preferences among other mussel species tended to be related to local mussel abundance rather than duration of sympatry. Individual fish were not consistent in their oviposition choices, precluding the evolution of host‐specific lineages. Mussels were demonstrated to have evolved strong defenses to bitterling parasitism in the area of ancient sympatry, but have no such defenses in the large areas of Europe where bitterling are currently invasive. Bitterling avoided glochidia infection irrespective of the duration of sympatry.     

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.     

Bayesian inference supports the host selection hypothesis in explaining adaptive host specificity by European bitterling

Generalist parasites have the capacity to infect multiple hosts. The temporal pattern of host specificity by generalist parasites is rarely studied, but is critical to understanding what variables underpin infection and thereby the impact of parasites on host species and the way they impose selection on hosts. Here, the temporal dynamics of infection of four species of freshwater mussel by European bitterling fish (Rhodeus amarus) was investigated over three spawning seasons. Bitterling lay their eggs in the gills of freshwater mussels, which suffer reduced growth, oxygen stress, gill damage and elevated mortality as a result of parasitism. The temporal pattern of infection of mussels by European bitterling in multiple populations was examined. Using a Bernoulli Generalized Additive Mixed Model with Bayesian inference it was demonstrated that one mussel species, Unio pictorum, was exploited over the entire bitterling spawning season. As the season progressed, bitterling showed a preference for other mussel species, which were inferior hosts. Temporal changes in host use reflected elevated density-dependent mortality in preferred hosts that were already infected. Plasticity in host specificity by bitterling conformed with the predictions of the host selection hypothesis. The relationship between bitterling and their host mussels differs qualitatively from that of avian brood parasites.     

Adaptive spatial utilization of host mussels by the Japanese rosy bitterling Rhodeus ocellatus kurumeus

The spatial pattern of resource utilization for oviposition in the Japanese rosy bitterling Rhodeus ocellatus kurumeus was investigated in two field experiments. The distribution of rosy bitterling eggs deposited in the four demibranchs of the gills of the test mussel species, Anodonta woodiana , differed with mussel reproductive state in pair spawnings, but not in group spawnings. In pair spawnings, female rosy bitterling may have had more time to select the site of oviposition in the gill in relation to the sex and reproductive state of the mussel, thereby maximizing embryo survival. Thus, the inner gill of female mussels may have been selected in preference to the outer gills to avoid mortalities of eggs due to the presence of the mussel's own embryos (glochidia) in the outer gill chambers. In male mussels, female rosy bitterling distributed their eggs equally among all parts of the mussel gill, thereby minimizing density-dependent mortality of embryos. During group spawnings, however, female rosy bitterling may have been more constrained in their decision making, ovipositing in the inner gills irrespective of mussel sex.     

Host species preferences by bitterling, Rhodeus sericeus, spawning in freshwater mussels and consequences for offspring survival

Two hypotheses have been advanced to explain the evolution of host responses to parasites: the arms race-evolutionary lag and equilibrium hypotheses. We investigated predictions from these hypotheses based on interspecies host preferences and adaptations in an obligate spawning relationship between a freshwater fish, the European bitterling (Cyprinidae) and four species of freshwater mussels (Unionidae), which the fish use as hosts for their eggs. We found a significant trend in preference by the fish for mussels in the following order: Unio pictorum, U. tumidus, Anodonta anatina and A. cygnea. Male and female bitterling both showed this ranking and the clutch sizes deposited into each species also followed this trend. These host preferences proved to be adaptive in terms of egg ejection, which was lowest in the most preferred species (U. pictorum). Furthermore, these hierarchical host preferences were flexible, as females switched species when individuals of the preferred species ejected a greater number of eggs. The similarity in mussel defences between the U.K. population and a European population of ancient sympatry suggests that the absence of a defence in some mussel species may not be due to evolutionary lag. Mussel ejection behaviour may have reached an evolutionary equilibrium in each host species, or alternatively the fish may have evolved adaptive preferences that coincide with generalized mussel responses to foreign objects in their gills. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.     

An indirect effect of biological invasions: the effect of zebra mussel fouling on parasitisation of unionid mussels by bitterling fish

Invasive species represent a major threat with both direct and indirect effects on natural ecosystems, including effects on established and coevolved relationships. In a series of experiments, we examined how the interaction between two native species, a unionid mussel (Unio pictorum) and the European bitterling (Rhodeus amarus), a fish that parasitises unionids, was affected by the non-native zebra mussel (Dreissena polymorpha). The zebra mussel fouls hard substrates, including shells of living unionids, and its presence is often associated with a decrease in population density of native unionid mussels. Bitterling lay their eggs into live unionids and the embryos develop inside their gills. Using a range of zebra mussel densities, we demonstrated that zebra mussel fouling had a negative effect on the number of bitterling eggs inside the mussel host, with abundances of 5–10 zebra mussels (shell size 15–25 mm) per unionid critical for bitterling ability to utilise the host. In a further experiment, we found that bitterling did not discriminate between unfouled unionids and those fouled with five zebra mussels. Most ovipositions into fouled hosts, however, were unsuccessful as eggs failed to reach the unionid gills. We discuss implications of such unsuccessful ovipositions for bitterling recruitment and population dynamics.     

The European bitterling: a model for oviposition decision

For oviparous species, oviposition decisions can have significant fitness consequences. The European bitterling ( Rhodeus sericeus ) is a cyprinid fish that lays its eggs on the gills of freshwater mussels. Because bitterling use a discrete spawning site that can be readily manipulated, they are unusually amenable to field and laboratory studies aimed at understanding the adaptiveness and consequences of oviposition decisions. Here I present data demonstrating the adaptiveness of oviposition decisions by female bitterling, and link these decisions, using game‐theory models, with bitterling population dynamics. I explain the proximate cues used by females in making oviposition choices, and how these respond to environmental variables. I also show how the oviposition decisions of males relate to the risk of sperm competition in mussels, and propose that oviposition choices may represent an inter‐sexual conflict in bitterling. Finally, I present behavioural and genetic data from mesocosm experiments to show the implications of global and local male densities for female oviposition opportunities, and consider the consequences for bitterling population dynamics.     

An invasive species reverses the roles in a host-parasite relationship between bitterling fish and unionid mussels

The impact of multiple invading species can be magnified owing to mutual facilitation--termed 'invasional meltdown'--but invasive species can also be adversely affected by their interactions with other invaders. Using a unique reciprocal host-parasite relationship between a bitterling fish (Rhodeus amarus) and unionid mussels, we show that an invasive mussel reverses the roles in the relationship. Bitterling lay their eggs into mussel gills, and mussel larvae parasitize fish. Bitterling recently colonized Europe and parasitize all sympatric European mussels, but are unable to use a recently invasive mussel, Anodonta woodiana. The parasitic larvae of A. woodiana successfully develop on R. amarus, whereas larvae of European mussels are rejected by bitterling. This demonstrates that invading species may temporarily benefit from a coevolutionary lag by exploiting evolutionarily naive hosts, but the resulting relaxed selection may facilitate its exploitation by subsequent invading species, leading to unexpected consequences for established interspecific relationships.     

Divergence of ovipositor length and egg shape in a brood parasitic bitterling fish through the use of different mussel hosts

Bitterling fishes deposit their eggs on the gills of living mussels using a long ovipositor. We examined whether ovipositor length (OL) and egg shape correlated with differences in host mussel species in the family Unionidae among populations of the tabira bitterling (Acheilognathus tabira) in Japan. Bitterling populations that use mussels in the sub-family Anodontinae possessed longer ovipositors and more elongated eggs than those using mussels of Unioninae, as expected from the difference in host size between the sub-families (anodontine mussels are larger than unionine mussels). Based on a robust phylogeny of A. tabira populations, we demonstrated that the evolution of both OL and egg shape were correlated with host differences, but not with each other, suggesting that these traits have been selected for independently. Our study demonstrates how adaptive traits for brood parasitism may diverge with host shift due to different host availability and/or interspecific competition for hosts.     

Females solicit sneakers to improve fertilization success in the bitterling fish (Rhodeus sericeus)

In most species there is greater variance in reproductive fitness among males than females. A consequence is the evolution of alternative male mating tactics, including sneaking behaviour whereby males attempt to mate with females courted by other males, which could undermine female choice. In bitterling (Rhodeus sericeus), fish that lay their eggs on the gills of living freshwater mussels, sneaking is common and successful. Here we show that female bitterling can significantly improve their fertility with an increase in the number of spawning partners. Females perform conspicuous behaviours associated with spawning more frequently close to sneakers, and spawn more eggs close to high-quality sneakers. This is the first time that females have been shown to engage in behaviour that increases the probability of sneaking, and raises the possibility of a sexual conflict over the number and source of ejaculates during spawning in this species.     

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.     

Reproductive ecology of the striped bitterling Acheilognathus cyanostigma (Cyprinidae: Acheilognathinae)

The reproductive ecology of the striped bitterling Acheilognathus cyanostigma was investigated in a small pond in Mie Prefecture, central Honshu, Japan. A. cyanostigma was the only species of bitterling in the study pond, where only a single species of mussel Anodonta woodiana was present. Spawning of A. cyanostigma was recorded between early April and early July, peaking between late April and mid-June in 2003. Ovipositor length during the spawning period ranged from 19.2 to 42.8 mm, and was positively correlated with female body length, but did not show significant seasonal variation. The eggs are elliptical with a volume of approximately 1.5 mm³. Egg size correlates positively with female body size, and both egg shape and volume changed significantly with season. The embryos were located on host mussel gills approximately 30 mm from the exhalant siphon and were found more frequently on the inner rather than the outer mussel gills. The possible ecological significance of these observations is discussed in the context of the adaptations of A. cyanostigma for utilizing mussels for oviposition.     

The endangered thick-shelled river mussel (<Emphasis Type="Italic">Unio crassus</Emphasis>): a new host species for the European bitterling (<Emphasis Type="Italic">Rhodeus amarus</Emphasis>)

Large freshwater mussels (Unionoida) are declining throughout the world. The European bitterling Rhodeus amarus (Bloch, 1782) female spawns its eggs inside the unionids’ shells, where fertilisation and further embryonic development take place; thus its reproduction depends fully on the presence of large freshwater mussels. Unio crassus, previously regarded as one of the most numerous unionids in Europe, is now listed in the IUCN Red Data List as being globally endangered. Despite its previous prevalence, it was never reported as a host for the bitterling. A large population of U. crassus was studied in small river at the ?wi?tokrzyskie Mts (Poland), where also electrofishing was conducted. In each bitterling territory located on the study plots, we found individuals of U. crassus, with the bitterling eggs or larvae developing on mussel’s gills. That proves that this species can be also used by the bitterling for reproduction. We suggest that this relationship has not been reported to date due to the mussels’ rarity and ongoing decline. However, it is also possible that the endangered mussel is a novel host for the bitterling, which is expanding its range throughout Europe.     

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.     

The function of multiple ejaculations in bitterling

In some taxa, males perform multiple ejaculations, which may function in sperm competition or in maintaining a baseline density of spermatozoa in the female reproductive tract to ensure fertilization, a process that has been termed ‘topping up’. We investigated the function of multiple ejaculations in two species of bitterling, the European bitterling (Rhodeus amarus) and Chinese rose bitterling (Rhodeus ocellatus). Bitterling oviposit in living freshwater mussels, with fertilization taking place within the mussel gill cavity. Thus, although fertilization is external, the mussel is analogous to the female reproductive tract in an internally fertilizing species. We measured the frequency of ejaculations and mussel inspections by individual males of two bitterling species in 28 replicated mesocosms and examined focal male responses to rival ejaculations and the presence of females in spawning condition. We used a model of ejaculatory behaviour to simulate the temporal abundance of spermatozoa in mussels. Male R. amarus exhibited high rates of ejaculation and inspection of the siphons of mussels and increased their ejaculation rate in response to the presence of females in spawning condition. Rhodeus ocellatus showed lower overall rates of ejaculation, but significantly elevated ejaculation rate in response to rival ejaculations. The ejaculatory strategy of R. amarus is one that maintains a minimum level of spermatozoa in mussels, which is elevated when the probability of oviposition increases. In contrast, R. ocellatus engages more directly in sperm competition with rivals. We discuss these results in the context of the function of multiple ejaculations and male mating tactics.     

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.     

The zebra mussel (<Emphasis Type="Italic">Dreissena polymorpha</Emphasis>) impacts European bitterling (<Emphasis Type="Italic">Rhodeus amarus</Emphasis>) load in a host freshwater mussel (<Emphasis Type="Italic">Unio pictorum</Emphasis>)

The European bitterling, Rhodeus amarus, is a non-indigenous fish species in British fresh waters. It lays its eggs in unionid mussels which themselves are vulnerable to fouling by the non-indigenous zebra mussel, Dreissena polymorpha. Observations from an unmanipulated natural system showed that only 27% of zebra mussel-fouled Unio pictorum hosted bitterling, while 47% of unfouled U. pictorum hosted bitterling. We conducted a field experiment in the River Great Ouse catchment, Cambridgeshire, England in May–June 2007 and 2008 to quantify the impact of zebra mussels on bitterling load in host mussels. Zebra mussel-fouled unionids were significantly less likely to host bitterling than unfouled unionids. The number of unionids hosting bitterling did not differ significantly whether the zebra mussels fouling the unionid were alive or dead. Bitterling appeared to discriminate against zebra mussel-fouled unionids less as the 2007 breeding season advanced, potentially because preferred unfouled unionids had a higher bitterling load, and were therefore relatively lower quality hosts than at the start of the breeding season.