Evaluation of a nonlethal technique for hemolymph collection in Elliptio complanata, a freshwater bivalve (Mollusca: Unionidae)

Hemolymph, the circulatory fluid of bivalves, transports nutrients, respiratory gases, enzymes, metabolic wastes, and toxicants throughout the body. Hemolymph can provide information pertinent to health assessment of animals or populations, but is not commonly used in freshwater bivalves partly because of the lack of tested, practical techniques for its nonlethal collection. The objective of this study was to evaluate the effect of hemolymph collection on the growth and survival of Elliptio complanata, a freshwater bivalve (Unionidae). We describe a simple technique for the collection of hemolymph from the anterior adductor muscle sinus of E. complanata. To evaluate the effect of hemolymph sampling on mussel survival and growth, 30 mussels sampled using the technique and 30 unsampled controls were followed for 3 mo post collection. Nine animals were sampled 3 times over 7 mo to monitor effects of repeated sampling. No negative impacts on survival or growth were observed in either the singly or repeatedly sampled animals. We also compared the composition of hemolymph collected from the adductor muscle sinus with that collected from the ventricle of the heart. Calcium levels and cell count of hemolymph obtained from the adductor sinus and ventricle were significantly different. There was no significant difference between collection sites for magnesium, phosphorus, ammonia, protein, sodium, potassium, or chloride. We conclude that collection of hemolymph from the adductor sinus is safe for sampled E. complanata and should be explored as a relatively non-invasive, and potentially useful, approach to the evaluation of freshwater mussel health.     

Locomotion in Elliptio complanata (Mollusca: Unionidae): a reproductive function?

1. Temporal variation in the spatial aggregation of the freshwater bivalve Elliptio complanata was studied at a sandy site in an oligotrophic lake over three years.
2. Epibenthic populations varied in aggregation over the season bringing animals closer together during spawning. The complex link between movements of mussels and aggregation dynamics suggested a functional reproductive role for horizontal locomotion of unionid mussels in lentic systems.
3. The rate of locomotion did not differ systematically among males, females or hermaphrodites, and was independent of gravidity, whether compared during spawning, after spawning or throughout the ice‐free season.
4. In spite of the high reproductive output of mussels and the energetic cost of locomotion, no relationship was found between the rate of movement of spawning gravid mussels and reproductive output.     

Length‐specific growth rates in freshwater mussels (Bivalvia: Unionidae): extreme longevity or generalized growth cessation?

1. North American lacustrine freshwater mussels (Bivalvia: Unionidae) are one of the world's most imperilled groups of organisms. Knowledge of their age structure and longevity is needed for the understanding and management of mussels. Current methods for age estimation in freshwater mussels are insufficient and may have resulted in an erroneous view of the ages of lacustrine freshwater mussels.
2. We collected growth data through mark-recapture in Minnesota and Rhode Island, U.S.A., examining four lentic populations of three of the most common species of freshwater mussels, Elliptio complanata , Lampsilis siliquoidea , and Pyganodon grandis . Using an inversion of the von Bertalanffy growth equation, we estimated age at length from length-specific growth relationships.
3. In some populations, lacustrine mussels may be much older than previously predicted. Ages predicted from actual growth rates suggest that individuals in some populations frequently reach ages in excess of a century, placing unionid mussels among the Earth's longest-lived animals. Alternatively, if growth has only recently slowed in these populations, generalized growth cessation may be occurring over a broad distributional range of some common North American lacustrine mussels.     

Substratum patch selection in the lacustrine mussels Elliptio complanata and Pyganodon grandis grandis

1. Sediment selection was investigated under controlled conditions in two common lake-dwelling species of freshwater mussels (Bivalvia: Unionidae), Elliptio complanata and Pyganodon grandis grandis .
2. Sediment choice was determined in six independent experiments under controlled conditions by distributing mussels randomly or evenly in tanks containing patches of sand and mud, and following their movement among sediment patches in experiments lasting between 30 and 45 days.
3. In all experiments, both species were found most frequently in muddy sediment patches. Movement toward muddy patches occurred rapidly: an average of nearly 80% of Pyganodon grandis grandis were found in mud after 30 days. Elliptio complanata moved rapidly to patches of mud at the start of experiments, but occupation of muddy sediments appeared to decrease after about 30 days.
4. Our results contrast with many field studies that suggest populations of lake-dwelling freshwater mussels infrequently inhabit mud and silt. We therefore postulate that large-scale mussel distribution in lakes is influenced most strongly by factors other than sediment composition.     

Biomass and annual production of the freshwater mussel Elliptio complanata in an oligotrophic softwater lake

SUMMARY. An extensive survey of Mirror Lake, New Hampshire, was carried out by divers with SCUBA to assess the importance of the freshwater mussel Elliptio complanata in this softwater lake ecosystem. Density (0.032 adults m⁻²), biomass (52 mg m⁻² as dry organic matter) and annual production (6.4 mg m⁻² as dry organic matter) of the mussel population are low when compared with results from other studies, corresponding with the general observation that mussels are scarce in soft, oligotrophic waters. We reject the traditional view that the low mussel density is a result of low calcium concentrations in Mirror Lake, and propose that mussel populations may be regulated by a scarcity of appropriate fish hosts in unproductive lakes. Elliptio complanata is probably not important in the metabolism or biochemistry of the Mirror Lake ecosystem.     

Evaluation of estrogenic effects of municipal effluents to the freshwater mussel Elliptio complanata

Municipal effluents are an important source of estrogens to the aquatic environment. The purpose of this study was to examine the estrogenicity of municipal effluents to the indigenous freshwater mussel, Elliptio complanata. First, estradiol-binding sites in gonad homogenates were characterized to determine the binding affinity and specificity of estrogens. Mussels were exposed to increasing concentrations of a municipal effluent for 96 h at 15 degrees C. In another experiment, mussels were placed in cages and submerged for 62 days at 1.5 km upstream and 5 km downstream of a municipal effluent plume in the St. Lawrence River. Mussels were harvested for assessment of vitellogenin-like proteins in the hemolymph and determination of total lipid, carbohydrate and protein in the gonad. The presence of specific estrogen-binding sites was found in both male and female gonads. Binding of estradiol to cytosol proteins reached saturation, yielding a dissociation constant of 0.4 nM. Vitellogenin (Vg) levels increased significantly in both the hemolymph and the gonad after exposure to the effluent. Moreover, females appeared to be more sensitive than males to producing Vg. Mussels exposed in situ to contaminated surface waters had higher levels of Vg at the downstream site, again, females had higher levels of Vg than did males. On the other hand, lipid and sugar levels in male gonads were significantly increased at the downstream site. Moreover, mussels at the downstream site had decreased shell growth length and increased total and soft tissue weights. We conclude that municipal effluents contain bio-available xenoestrogens at levels sufficient to elicit effects in freshwater mussels.     

Internal shell annuli yield inaccurate growth estimates in the freshwater mussels Elliptio complanata and Lampsilis radiata

1. The objective of this study was to compare, in freshwater bivalves, growth rates inferred from the spacing of internal annuli to those obtained directly by measuring annual changes in shell length.
2. The unionid mussels studied were Elliptio complanata and Lampsilis radiata from a pond in the north-eastern U.S.A. Age was inferred from internal lines of 157 E. complanata and twenty-five L. radiata . Actual length change was determined from comparisons of annual remeasurements of marked E. complanata ( n = 520) in 1992–95 and L. radiata ( n = 81) in 1993–95.
3. High retrieval rates of painted mussels demonstrated the efficacy of the marking method.
4. Annual length changes determined by remeasurement were significantly lower than annual length changes predicted by length-at-age data from internal annuli. Should this be a common occurrence, past estimates of annual growth based on annuli are probably too large, and unionid mussels may be much older than previously assumed.     

Seasonal variation in vertical and horizontal movement of the freshwater bivalve Elliptio complanata (Mollusca: Unionidae)

1. Vertical and horizontal movement were studied in the freshwater bivalve Elliptio complanata at a sandy site in an oligotrophic lake over 3 years. Mussel movement did not vary significantly between day and night. On average, between 2 and 8% of 527 mussels moved each month during the ice-free season and the distance travelled by moving mussels averaged 0.6 cm day^–1.
2. Mussels were endobenthic during the winter, emerged from the sandy substrate in mid-May, peaked in sediment surface abundance in July, and descended into the sediments for the winter in September–October. Vertical displacement of mussels was closely correlated with water temperature although daylength may play a role. Mussels apparently move very little beneath the sediment during the winter.
3. The number of mussels moving horizontally at any given time was linearly correlated with daylength, but the distance travelled during a sampling period was related to daylength in a non-linear fashion. Greatest horizontal displacement of epibenthic mussels was found during spring and early summer, coincident with spawning in E. complanata     

Seasonal variation in vertical and horizontal movement of the freshwater bivalve Elliptio complanata (Mollusca: Unionidae)

1. Vertical and horizontal movement were studied in the freshwater bivalve Elliptio complanata at a sandy site in an oligotrophic lake over 3 years. Mussel movement did not vary significantly between day and night. On average, between 2 and 8% of 527 mussels moved each month during the ice-free season and the distance travelled by moving mussels averaged 0.6 cm day^–1.
2. Mussels were endobenthic during the winter, emerged from the sandy substrate in mid-May, peaked in sediment surface abundance in July, and descended into the sediments for the winter in September–October. Vertical displacement of mussels was closely correlated with water temperature although daylength may play a role. Mussels apparently move very little beneath the sediment during the winter.
3. The number of mussels moving horizontally at any given time was linearly correlated with daylength, but the distance travelled during a sampling period was related to daylength in a non-linear fashion. Greatest horizontal displacement of epibenthic mussels was found during spring and early summer, coincident with spawning in E. complanata     

Internal shell annuli yield inaccurate growth estimates in the freshwater mussels Elliptio complanata and Lampsilis radiata

1. The objective of this study was to compare, in freshwater bivalves, growth rates inferred from the spacing of internal annuli to those obtained directly by measuring annual changes in shell length.
2. The unionid mussels studied were Elliptio complanata and Lampsilis radiata from a pond in the north-eastern U.S.A. Age was inferred from internal lines of 157 E. complanata and twenty-five L. radiata . Actual length change was determined from comparisons of annual remeasurements of marked E. complanata ( n = 520) in 1992–95 and L. radiata ( n = 81) in 1993–95.
3. High retrieval rates of painted mussels demonstrated the efficacy of the marking method.
4. Annual length changes determined by remeasurement were significantly lower than annual length changes predicted by length-at-age data from internal annuli. Should this be a common occurrence, past estimates of annual growth based on annuli are probably too large, and unionid mussels may be much older than previously assumed.     

Pathophysiologic alterations in Biomphalaria glabrata infected with Angiostrongylus costaricensis

Hemolymph glucose, alkaline phosphatase, lactic dehydrogenase, and creatine phosphokinase in Biomphalaria glabrata infected with Angiostrongylus costaricensis were significantly higher on day 27 postinfection (PI) than in uninfected snails. Hemolymph total calcium from infected snails was less on days 6, 12, and 27 PI than that from controls. Total hemolymph protein was similar for controls and infected animals during the entire study. Throughout the study the mean number of amoebocytes/mm³ hemolymph from infected snails was significantly less than that for controls. Mean total wet weights of digestive gland and foot muscle from infected and uninfected snails was similar throughout the study. Mean μg glycogen/mg wet weight of digestive gland from infected snails was significantly greater on days 24, 27, and 28 PI than that from controls. Mean μg glycogen/mg wet weight of foot muscle from infected snails was significantly reduced between days 12 and 28 PI from that of uninfected snails. It is suggested that hemolymph glucose and digestive gland glycogen in infected snails are augmented by glycogen breakdown in the foot muscle of parasitized animals. Elevations in hemolymph enzymes are due to tissue destruction by larvae emerging from the foot muscle of infected snails. Parasite-induced derangements in shell metabolism underlie observed changes in hemolymph calcium in infected snails.     

Rapid evolution of body fluid regulation following independent invasions into freshwater habitats

Colonizations from marine to freshwater environments constitute among the most dramatic evolutionary transitions in the history of life. Colonizing dilute environments poses great challenges for acquiring essential ions against steep concentration gradients. This study explored the evolution of body fluid regulation following freshwater invasions by the copepod Eurytemora affinis. The goals of this study were to determine (1) whether invasions from saline to freshwater habitats were accompanied by evolutionary shifts in body fluid regulation (hemolymph osmolality) and (2) whether parallel shifts occurred during independent invasions. We measured hemolymph osmolality for ancestral saline and freshwater invading populations reared across a range of common-garden salinities (0.2-25 PSU). Our results revealed the evolution of increased hemolymph osmolality (by 16-31%) at lower salinities in freshwater populations of E. affinis relative to their saline ancestors. Moreover, we observed the same evolutionary shifts across two independent freshwater invasions. Such increases in hemolymph osmolality are consistent with evidence of increased ion uptake in freshwater populations at low salinity, found in a previous study, and are likely to entail increased energetic costs upon invading freshwater habitats. Our findings are consistent with the evolution of increased physiological regulation accompanying transitions into stressful environments.     

Temporal and spatial variability in stable isotope compositions of a freshwater mussel: implications for biomonitoring and ecological studies

Stable isotopes can be used to elucidate ecological relationships in community and trophic studies. Findings are calibrated against baselines, e.g. from a producer or primary consumer, assumed to act as a reference to the isotopic context created by spatio-temporal attributes such as geography, climate, nutrient, and energy sources. The ability of an organism to accurately represent a community base depends on how, and over what time-scale, it assimilates ambient materials. Freshwater mussels have served as references for trophic studies of freshwater communities and as indicators of change in nutrient pollution load or source. Their suitability as reference animals has not yet been fully explored, however. We conducted a series of studies examining the suitability of freshwater mussels as isotopic baselines, using their ability to reflect variation in ambient nutrient loads as a case scenario. (1) We analyzed bivalve foot tissue δ¹⁵N and δ¹³C from 22 stream reaches in the Piedmont region of North Carolina, USA to show that compositions varied substantially among locations. Site mean bivalve δ¹³C values correlated with site ambient particulate organic matter (POM) δ¹³C values, and site mean bivalve δ¹⁵N values correlated with site ambient water dissolved δ¹⁵N-NO₃ values. (2) Similarity of results among sample types demonstrated that the minimally invasive hemolymph sample is a suitable substitute for foot tissue in δ¹⁵N analyses, and that small sample sizes generate means representative of a larger population. Both findings can help minimize the impact of sampling on imperiled freshwater mussel populations. (3) In a bivalve transplantation study we showed that hemolymph δ¹⁵N compositions responded to a shift in ambient dissolved δ¹⁵N-NO₃, although slowly. The tissue turnover time for bivalve hemolymph was 113 days. We conclude that bivalves serve best as biomonitors of chronic, rather than acute, fluctuations in stream nutrient loads, and provide initial evidence of their suitability as time-integrated isotopic baselines for community studies.     

Ionic and acid-base consequences of exposure to increased salinity in the zebra mussel, Dreissena polymorpha

Dreissena polymorpha, an invasive freshwater bivalve, displays physiological characteristics that reflect its ancestry in brackish water, yet it has limited ability to withstand modest increases in salinity. We examined changes in hemolymph ion concentrations and acid-base variables in mussels transferred to and incubated in 10% artificial seawater (ASW) for 7 days and then returned to pondwater (PW) for a further 7 days. Hemolymph was sampled (10 animals per sample period) every 4 h for the first 24-h incubation and at 72 h and 168 h for both the transfer to 10% ASW and the transfer back to PW. The initial response to transfer to 10% ASW was a rapid attainment of an apparent isoosmotic steady state, with most hemolymph ion concentrations rising and attaining steady state within 12 h. Hemolymph magnesium rose more slowly, and hemolymph calcium declined despite an increase in its concentration in the bathing medium. Hemolymph pH rose significantly during the first 24 h, from 7.96 to 8.25, as a result of increases in bicarbonate; pH subsequently returned to normal through increases in PCO2. When animals were returned to PW after 7 days' incubation in ASW, the response of the major hemolymph ions was largely the reverse of that effected by the transfer to ASW. Hemolymph pH was not altered significantly until after 72 h in PW, when declines in bicarbonate lowered the pH to 7.73. Strong ion difference (SID) was related significantly to hemolymph pH. Hemolymph calcium and magnesium showed a reciprocal relationship throughout both transfer and incubation. Solubility interactions between sulfate and calcium and magnesium may be important in determining calcium availability in solution. The Na/K ratio in hemolymph was maintained within relatively narrow bounds throughout the procedure and may contribute to the mussels' ability to volume-regulate during an osmotic challenge. Overall, the responses of D. polymorpha to modest changes in salinity were largely the result of passive processes.     

Mechanisms involved in parasitic castration: in vitro effects of the trematode Prosorhynchus squamatus on the gametogenesis and the nutrient storage metabolism of the marine bivalve mollusc Mytilus edulis

The mechanisms involved in the parasitic castration of the marine mussel Mytilus edulis by the trematode parasite Prosorhynchus squamatus Odhner, 1905, have been investigated in vitro with two bioassays employing dissociated host tissues. There is no conclusive evidence that P. squamatus affects the secretion of two host neuroendocrine factors, viz., gonial mitosis-stimulating factor and glycogen mobilization hormone, involved in the gametogenesis/nutrient storage cycles of the mussel. In contrast, extracts of P. squamatus sporocysts and cercariae significantly stimulated glycogen mobilization in host glycogen cells and strongly inhibited host gonial mitosis. A gonial mitosis-inhibiting factor (GMIF) was found in the hemolymph of parasitized mussels. The existence of an endogenous GMIF in mantle tissue of uninfected mussels has been demonstrated. This factor appeared to be secreted into the hemolymph during the period of sexual maturity. Whether the parasite acts directly on the host gonia, or by provoking the liberation of this endogenous GMIF, has yet to be ascertained. It would appear, however, that the parasite acts directly on host glycogen cells.     

Effects of pharmaceutical products and municipal wastewaters on temperature-dependent mitochondrial electron transport activity in Elliptio complanata mussels

The advent of global warming has given rise to the issue on how temperature impacts the susceptibility of ectothermic organisms to pollution. The purpose of this study was to examine the effects of pharmaceutical products and domestic wastewaters on temperature-dependent mitochondrial electron transport activity in the freshwater mussel Elliptio complanata. Mitochondria from mussels were freshly prepared and exposed to increasing concentrations of various pharmaceutical products known to be found in municipal effluents for 30 min at 4, 12 and 20 degrees C. Electron transport activity as well as lipid peroxidation and DNA strand breaks were determined in the mitochondria. Next, mussels were placed in the aeration lagoons of two municipal wastewater treatment plants for 30 days. Mitochondrial electron transport (MET), temperature-dependent MET (MET(T)) and lipid peroxidation in gonad were then determined. The results show that all products were able to increase MET activity, but at two different ranges of threshold concentration. Certain pharmaceutical products (i.e., ibuprofen, cotinine, fluoxetine, coprostanol and trimethoprim) increased MET(T) at a lower threshold concentration than observed at 20 degrees C. Products of lesser potency in reducing lipid peroxidation were those that produced more DNA strand breaks in mitochondria. Both MET and MET(T) were significantly increased in mussels exposed to aeration lagoon effluents. Lipid peroxidation was also increased in the gonad and was significantly correlated with MET and MET(T) activities. The data indicate that pharmaceutical products and municipal effluents increase respiration rates in isolated mitochondria, such that interaction with temperature could enhance the susceptibility of mitochondrial energy production and oxidative stress in environments contaminated by domestic wastewater.     

Molecular characteristics and expression of calmodulin cDNA from the freshwater pearl mussel, Hyriopsis schlegelii

Calmodulin (CaM) is a multifunctional intracellular calcium ion receptor protein that participates in a range of cellular processes, including calcium metabolism in mussels. To investigate the role of CaM in freshwater mollusk shell calcium metabolism, the full-length CaM cDNA was isolated from the freshwater pearl mussel, Hyriopsis schlegelii (referred to as hsCaM) using SMART RACE technology. The full-length hsCaM was 855 bp in size, containing a 70-bp 5'-untranslated sequence, a 447-bp open reading frame, a 309-bp 3'-untranslated sequence, and a 26-nucleotide long poly(A) tail. The hsCaM mRNA expression in different mussel tissues was examined using real-time PCR. The hsCaM mRNA was found to be ubiquitously expressed, but far more abundant in the gill, foot, and mantle than in the posterior adductor muscle. Real-time PCR was also used to determine hsCaM mRNA expression levels in mantle tissues of H. schlegelii at different ages. No significant differences between one-, two-, and three-year-old mussels were detected, but expression increased in four-year-old mussels and then decreased in five-year-old mussels. CaM appears to be involved in calcium regulation of the mantle in four-year-old mussels, which may secrete more mother of pearl during pearl culture.     

Declining freshwater mussel diversity in the middle and lower reaches of the Xin River Basin: Threat and conservation

Freshwater mussels provide important functions and services for aquatic ecosystems, but populations of many species have been extirpated. Information on biodiversity plays an important role in the conservation and management of freshwater mussels. The Xin River Basin is a biodiversity hotspot for freshwater mussels in China, with more than 43 species known, but populations of which are decreasing. Here, we quantify the diversity of freshwater mussels in the middle and lower reaches of the Xin River Basin and study the correlation of habitat characteristics and freshwater mussel diversity. Compared to the historical period, the number of species, density, and biomass of freshwater mussels decreased 33%, 83%, and 82% in the current period, respectively. Fifty two percent of recorded species were empty shells, and 14 native freshwater mussels were not found in the study area. Four species are currently listed as vulnerable species using IUCN criteria and their global status. The assemblage structure of freshwater mussels exhibits significant spatial differences, and there was a correlation with substrate and physicochemical parameters. The main tributary of the Xin River with higher freshwater mussel diversity should be established as one large protected area because the nestedness component was the main pattern of beta diversity. These results indicated freshwater mussel diversity was declining rapidly, which can help focus conservation effort for freshwater mussel biodiversity.     

Physiological and metabolic variations in Carcinus mediterraneus (Crustacea: Decapoda) parasitized by Thelohania maenadis (Microspora: Microsporida): An ecophysiopathological approach

This paper examines the influence of Thelohania maenadis (Protozoa: Microsporida), a muscle microsporidian, on certain features of the metabolism of Carcinus mediterraneus (Crustacea: Decapoda). Comparison of biochemical parameters reveals that only hemolymph total protein and glucose levels are reduced significantly by this parasitic infection. Muscular effort results in a diminution of muscular glycogen and hemolymph glucose levels, while hepatopancreatic glycogen remains unaffected; lactate accumulates in the muscle tissue. Experimental variations in the environmental water temperature and salinity affect protein levels and metabolic indicators of carbohydrate metabolism. Proteinemia varies with temperature and salinity, but parasitic infection accentuates these variations. Glucose and lactate concentrations in the hemolymph are increased in healthy specimens after muscular effort, under conditions of increased temperature or hypoosmotic shock; microsporidiosis provokes the reverse of these tendencies, on a less pronounced scale. Hyperosmotic shock at high temperature leads to a slight reduction in glucose and lactate concentrations. The paper concludes with a discussion of the physiopathological significance of the results obtained as an expression of the effects of parasitism infestation.