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Glycogen is a primary metabolic reserve in bivalves and can be suitable for the evaluation of bivalve condition and health status, but the use of glycogen as a diagnostic tool in aquaculture and biomonitoring is still relatively rare. A tissue biopsy combined with a simplified phenol–sulfuric acid method was used in this study to evaluate the inter‐ and intraindividual variation in the glycogen concentrations among several tissues (foot, mantle, gills, adductor muscle) of the unionid bivalve, the duck mussel Anodonta anatina. This short report documents that individual bivalves differ in the spatial distribution of glycogen among tissues. Sampling of different types of tissues can cause distinct results in the evaluation of energetic reserves at the individual level. At the same time, spatial variability in glycogen content has the potential to provide a more detailed evaluation of physiological conditions based on tissue‐specific glycogen storage. The results obtained and the simplified methodology provide a new opportunity for researching the energetic reserves and health status of freshwater mussels in various applications.
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Variation in Glycogen Distribution among Freshwater Bivalve Tissues:
Simplied Protocol and Implications
Barbora Vod
a and Karel Douda*
Department of Zoology and Fisheries, Czech University of Life Sciences Prague, Kam
a 129, CZ-16500, Prague,
Czech Republic
Glycogen is a primary metabolic reserve in bivalves and can be
suitable for the evaluation of bivalve condition and health status,
but the use of glycogen as a diagnostic tool in aquaculture and
biomonitoring is still relatively rare. A tissue biopsy combined with
a simplied phenolsulfuric acid method was used in this study to
evaluate the inter- and intraindividual variation in the glycogen con-
centrations among several tissues (foot, mantle, gills, adductor mus-
cle) of the unionid bivalve, the duck mussel Anodonta anatina. This
short report documents that individual bivalves differ in the spatial
distribution of glycogen among tissues. Sampling of different types
of tissues can cause distinct results in the evaluation of energetic
reserves at the individual level. At the same time, spatial variability
in glycogen content has the potential to provide a more detailed
evaluation of physiological conditions based on tissue-specic glyco-
gen storage. The results obtained and the simplied methodology
provide a new opportunity for researching the energetic reserves and
health status of freshwater mussels in various applications.
Monitoring the energetic reserves of aquatic inverte-
brates is still rarely implemented, although invertebrates
are increasingly being used in the food production sector,
in conservation aquaculture, and for biomonitoring (Koop
et al. 2008). The evaluation of the condition of macroin-
vertebrates is often determined indirectly from growth and
mortality data, but more specic physiological markers
can be critical for the early identication of changes in
their health status (Fritts et al. 2015b).
This study addresses freshwater mussels, order Union-
ida, which are increasingly being propagated in aquaculture
facilities for conservation purposes, and there is a need to
employ reliable and noninvasive methods to assess their
energetic status (Fritts et al. 2015a). Despite the critical
conservation status of freshwater mussels, the determina-
tion of the physiological condition of this group is mostly
based on whole-body analyses performed by lethal methods
(Gustafson et al. 2005). These methods include the analysis
of the lipid and fatty acid composition in adult mussels
(Prato et al. 2010) and the analysis of lipids or glucose in
juveniles (Tankersley 2000; Sim-Smith and Jeffs 2011). In
contrast, nonlethal methods can be applied for a larger part
of a population to obtain more replications (Gustafson
et al. 2005) and for the evaluation of changes during long-
term studies. Nonlethal methods are particularly important
for the research of threatened and endangered species
(Naimo et al. 1998; Fritts et al. 2015a), where the sacrice
of individuals could put populations of such species in jeop-
ardy (Naimo et al. 1998; Haskell and Pan 2010).
Glycogen is the primary metabolic reserve in mussels
(Ke and Li 2013). Stored glycogen is a source of glucose,
which can be mobilized to tissues (Martinez-Pita et al.
2012). Glycogen level is suitable as a physiological marker
for the evaluation of the condition and health of bivalves
(McGoldrick et al. 2009; Sim-Smith and Jeffs 2011; Cor-
deiro et al. 2017). Glycogen quickly reacts to changes in
the environment (Fisher and Dimock 2006), and it is
connected to the nutritional condition, different types of
stress, stages of the life cycle, and sexual maturity
(de Zwaan and Zandee 1972; Dridi et al. 2007; Anacleto
et al. 2013; Cordeiro et al. 2017). Monitoring changes in
glycogen levels has been used in various studies dealing
with bivalves, for example, to monitor stress (Fearman
and Moltschaniwskyj 2010; Fritts et al. 2015b; Andrade
et al. 2017) under different conditions such as starvation
(Cordeiro et al. 2016) or transportation (Yusufzai et al.
*Corresponding author:
Received June 8, 2018; accepted December 5, 2018
Journal of Aquatic Animal Health 31:107111, 2019
©2019 American Fisheries Society
ISSN: 0899-7659 print / 1548-8667 online
DOI: 10.1002/aah.10057
2010; Anacleto et al. 2013; Cordeiro et al. 2017), or in
ecotoxicological studies (Hazelton et al. 2014).
The biopsy of foot tissue followed by the phenolsulfu-
ric acid method of glycogen determination by Naimo
et al. (1998) is a nonlethal method for the analysis of this
physiological marker in freshwater mussels. Glycogen can
be determined by means of the glycogen biopsy method
developed by Naimo et al. (1998) and is a very promising
marker of the physiological condition in bivalves. How-
ever, it is not routinely applied in aquaculture and conser-
vation physiology, probably because of the high workload
and material consumption of this method and of the lim-
ited knowledge of the spatial and temporal dynamics of
glycogen reserves in freshwater bivalve tissues.
The aim of this study was to determine the distribution
of glycogen in soft-body tissues (foot, gills, mantle, and
adductor muscle) in a freshwater bivalve, the duck mussel
Anodonta anatina, to estimate the potential effect of glyco-
gen spatial distribution on the evaluation of the energetic
status of a bivalve. We also aimed to simplify the glycogen
analysis method (phenolsulfuric acid method) by Naimo
et al. (1998) to reduce the workload, costs, and material
consumption for easier amplication of the methodology.
Sample Collection
The duck mussel was used as a test organism because it
is a widespread European species (Lopes-Lima et al. 2017)
and has a potential use in biomonitoring (Falfushynska
et al. 2013). Six adult individuals (shell length, 82
98.5 mm) sampled in the Lu
znice River, Czech Republic
(49°18024N, 14°30014E), were transported in a 25-L tank
with aerated (through an airstone) river water to the labo-
ratory where the tissue sampling was performed on the
same day (November 6, 2017). Biopsy samples were taken
through gently opened valves (using a shell opening
device) from the outer edge of the foot (posterior part),
mantle (medial part), and gills (medial part). Dissecting
scissors were used to cut off three independent samples
close to each other from the foot, mantle, and gill tissue,
in that order, from each individual. Then, the individual
was sacriced by cutting the adductor muscles, and the
shell was opened completely. The tissue of the anterior
adductor muscle was sampled in the same way as that for
other tissues. The target weight of samples was 410 mg
(sample weight identied as suitable for nonlethal biopsy
of foot tissue in Unionidae and glycogen analysis protocol
by Naimo et al. 1998), which corresponded to a piece of
tissue approximately 6 9191.5 mm in size. There were
68 samples collected (four pairs of replicated samples were
merged due to the small sample weight), and the true
weight of the nal samples ranged from 2.17 to 12.68 mg
(mean weight, 7.36 mg) of wet tissue. Samples were stored
at 75°C and processed within 40 d.
Glycogen Analysis
The method by Naimo et al. (1998) was used with sev-
eral modications (details may be found online in the Sup-
porting Information section at the end of the article), and
the bias associated with glycogen determination was esti-
mated by recovery of known additions using matrix stan-
dards. The precision of the method was estimated from
triplicate analyses of all matrix standards (relative stan-
dard deviation [RSD]); the method detection limit (MDL)
was determined using 11 blank samples from three differ-
ent analysis sets according to the EPA (2016) guidelines.
Calibration standards.Glycogen calibration standards
were prepared by dissolving 40 mg of powdered oyster
glycogen (type II, Sigma-Aldrich, St. Louis, Missouri) in
20 mL of deionized water and then creating serial dilu-
tions of 2,000, 1,000, 500, 250, and 125 mg/L immediately
before analyses.
Internal standards.The in-house reference material
was prepared according Naimo et al. (1998) from the
homogenized foot tissue of six duck mussels sampled on
July 24, 2017, from the same location as the study samples.
Two milliliters of 30% KOH (Penta, Prague, Czech Repub-
lic) was added per gram of tissue to a 3-mL cryovial (Sim-
port, Beloeil, Quebec), heated for 20 min in a water bath at
100°C (RTC Basic, IKA, Wilmington, North Carolina),
then vortexed for 10 s (MS2 Minishaker, IKA), and stored
in a freezer at 75°C. The in-house reference material was
thawed at room temperature and vortexed before use during
the whole study (four analytical days).
Spiked calibration standards.Spiked samples were pre-
pared the same way as the aqueous calibration standards,
but they were spiked by adding 10 μL of the in-house refer-
ence material.
Digestion and extraction of glycogen.Glycogen from
all standards and samples was digested and extracted from
tissues. Thirty percent KOH was added to the samples in
3-mL cryovials in a volume of 100 μL to the tissue sam-
ples, blank solution, and in-house reference material sam-
ples. The volume of 30% KOH added to the spiked
samples and aqueous calibration standards was 280 μL.
Then, cryovials were boiled in a water bath for 20 min for
homogenization. In the next step, 96% ethyl alcohol
(Penta) was added to the solution, and cryovials were
placed in a boiling water bath for 15 min. The volume of
ethyl alcohol was 1.5 times more than the added KOH to
prevent the precipitation of other polysaccharides (Naimo
et al. 1998). The solutions were diluted with deionized
water to the same total solution volume; after homoge-
nization, 2,660 μL of solution were removed by pipette
from the samples, and 390 μL of deionized water were
added to the solutions to reach the optimal ratio of water
108 VOD
volume in the analyzed samples (see the detailed step-by-
step protocol of the modied methodology 113 in the
Supporting Information).
Quantication of glycogen.Quantication of glycogen
was based on spectrophotometry. Forty microliters of 80%
phenol (Carl ROTH, Karlsruhe, Baden-W
Germany) and 2,180 μL of 96% sulfuric acid (Penta) were
added to the sample solutions to gain coloration. A 250-μL
aliquot of the solution of each sample was pipetted into a
96-well microplate (Anicrin S.R.L., Scorz
e, Venice, Italy),
and the absorbance of the samples was determined by
spectrophotometry (see the detailed step-by-step protocol
of the modied methodology 1419 in the Supplement
available in the online version of this article).
The content of glycogen in samples was determined by
using the calibration slope, which was calculated from the
absorbance of triplicate samples of the aqueous calibration
standard of used concentrations. The calibration slope was
estimated individually for every analytical set with linear
regression models. The mean recovery of glycogen in
spiked samples, the mean CV (100SD/mean) (SD) for
each concentration of aqueous calibration standards, and
the mean percentage difference between slopes were calcu-
lated according to Naimo et al. (1998) from three repli-
cated samples on four analysis dates.
Data Analysis
A two-way ANOVA was used to examine the effects of
tissue (foot, mantle, gills, adductor muscle) and the mussel
individual (AF) on the glycogen content. The interaction
term of explanatory variables was included in the analysis.
A post hoc Tukeys honestly signicantly different (HSD)
test was used to identify the signicant differences between
pairs of tissues. Before statistical analyses, data were
assessed for the homogeneity of variance and normality
using Levenes test and a KolmogorovSmirnov test,
respectively. Glycogen data were transformed by means of
a natural-log transformation before analyses. The data
analysis was performed using the R software (R Develop-
ment Core Team 2017).
The glycogen concentrations in duck mussels ranged
from 5.6 mg to 60.1 mg/g wet tissue, with a mean concen-
tration of 20.7 mg/g (SD, 10.7). The mean glycogen con-
tent in the biopsied samples was 0.15 mg (SD, 0.07), and
MDL was established to be 0.049 mg of glycogen in the
whole analyzed sample (0.0075 mg of glycogen in the nal
solution for spectrophotometry).
The slopes of the calibration standard and spiked cali-
bration standard regression lines were not signicantly dif-
ferent (ANOVA: P>0.05, with the interaction term of
concentration level by spiking) in all analytical sets,
validating the use of the calibration standards line to pre-
dict the glycogen content. The aqueous calibration standard
curves had a mean slope of 258.28 (range, 213.44283.44),
mean intercept of 0.1973 (range, 0.18600.2113), and mean
of 0.9769 (range, 0.94030.9928). The curves of the
spiked samples had a mean slope of 261.55 (range, 209.35
290.57), mean intercept of 0.2889 (range, 0.26010.3058),
and mean R
of 0.9490 (range, 0.92750.9669). The mean
percentage difference between slopes was 1.258%.
The mean recovery of glycogen in spiked samples was
85% (SD, 13) for the concentration of 125 mg/L of the
aqueous calibration standard, 82% (SD, 27) for a concen-
tration of 500 mg/L, 109% (SD, 20) for a concentration of
1,000 mg/L and 83% (SD, 25) for a concentration of
2,000 mg/L. The CVs of these concentration samples were
7.7% (SD, 2.7), 10.5% (SD, 3.4), 7.4% (SD, 4.5) and 7.9%
(SD, 1.6), respectively.
The glycogen concentration signicantly differed among
tissues (two-way ANOVA: F
3, 44
=6.9, P<0.001; Figure 1).
The mean glycogen concentration was 18.7 mg/g (SD, 4.9)
in the foot tissue, 20.1 mg/g (SD, 16.1) in the mantle,
20.2 mg/g (SD, 8.6) in the adductor mussel, and 26.1 mg/g
(SD, 9.0) in the gills (Figure 1; Table S1 available in the
Supplement). Subsequent post hoc comparison tests revealed
higher glycogen content in gills than in all other tissues (all
There was a signicant effect of individual mussels
(two-way ANOVA: F
5, 44
=22.0, P<0.001; Figure 1). A
signicant interaction term between tissue and specimen
(two-way ANOVA: F
15, 44
=3.3, P<0.01) demonstrated
that individual mussels differed in the spatial distribution
of glycogen among tissues.
Foot Gills Mantle Adductor
Glycogen (mg/g)
FIGURE 1. Glycogen content in duck mussel tissues sampled in the
znice River, Czech Republic. The mean (black line), median (white
line), interquartile range, minimummaximum (without values >1.59
interquartile range) are displayed by box plots. Values measured for
individual mussels (six individuals, two or three samples per tissue) are
indicated by different symbols.
A simplied methodology for glycogen analysis was
demonstrated to be precise and reliable for the identica-
tion of differences in the glycogen level between types of
tissue in the duck mussel as well as in the body distribution
of glycogen between individuals. The results conrm previ-
ous ndings (de Zwaan and Zandee 1972; Naimo and
Monroe 1999) that there is a signicant difference in glyco-
gen levels in different types of tissues of unionid bivalves
and that the glycogen content in the mantle is the least
stable, while the most stable glycogen is in foot tissue (Fig-
ure 1). Furthermore, this study demonstrated that (despite
relatively high variability even within the same anatomical
structure) individual mussels differed signicantly in the
body distribution of the glycogen content. A tissue-specic
glycogen evaluation can provide more detailed data for the
monitoring the health and condition of mussels and can
provide new valuable information for future sampling,
where more than one type of tissue for the glycogen analy-
sis can be quantied. These ndings also provide a new
view of the evaluation of results from eld studies where
only one tissue was sampled. Although the duck mussels
were not sampled nonlethally in this study, previous
ndings corroborate that both foot and mantle tissue can
be biopsied nondestructively and without increasing
mortality in a long-term perspective (Berg et al. 1995;
Naimo et al. 1998). However, the effect of gill or adductor
mussel biopsy (or simultaneous extraction of more samples
per individual) on the survival rate of unionid mussels
needs to be tested.
The modied method is more economical by reducing
the analytical supplies, specically the sulfuric acid volume
by 56.4%, and by using only one 3-mL cryovial per sam-
ple instead of transferring the solution among three types
of laboratory test tubes. The analysis is easily performed
by one person without assistance, and it allows an analysis
of up to 60 samples (tissue samples plus calibration of
standard solutions) in one analytical set. These improve-
ments will signicantly reduce amount of hazardous waste
that is produced by this analysis.
Further studies are needed to clarify the connection
between the glycogen level and its distribution in the body,
considering changes that occur annually (de Zwaan and Zan-
dee 1972), as well as other factors. Bivalves are exposed to
annual cycles of food availability which highly inuences
their glycogen reserves (Albentosa et al. 2007; Cordeiro et al.
2016). Glycogen storage and utilization are also closely con-
nected to the annual reproductive cycle (Lemaire et al. 2006)
because glycogen reserves can be converted into lipids during
the mating season for gamete development (Martinez-Pita
et al. 2012; Ke and Li 2013; Irisarri et al. 2015). Glycogen is
catabolized to add glucose to hemolymph when the glucose
level declines or when it is mobilized by the inuence of some
stress factor (Fritts et al. 2015a). The glycogen concentrations
in bivalve soft tissues are largely used for monitoring the
impacts of stress under different conditions (Anacleto et al.
2013; Cordeiro et al. 2016, 2017) and in ecotoxicologal stud-
ies (Hazelton et al. 2014), because the glycogen level
decreases long before changes in growth and survival rates
are known (Sim-Smith and Jeffs 2011). The simplication of
the methodology used in this study allows this method to be
used for routine applications and highlights the importance
of tissue-specic analyses for understanding mussel energetic
metabolism. In particular, it would be promising to focus on
the link between specic types of tissues and the condition of
the individual and its environment during different periods of
the year and with respect to environmental conditions. This
can help determine the reasons for the variation in the distri-
bution of glycogen in the body so that glycogen evaluations
can be put to practical use. Regarding the pilot character of
this study, further data are needed to establish which organ
and at which life stage and gender of the mussels is the best
for nonlethal biomonitoring.
In summary, using the simplied nonlethal glycogen
determination method, this study documented that indi-
vidual freshwater duck mussels differ in their spatial distri-
bution of glycogen among tissues. The results obtained
and the simplied methodology provide new opportunities
for the research of energetic reserves and the health status
of freshwater mussels in various aquaculture and conser-
vation applications.
This study was supported by the Czech Science Foun-
dation (19-05510S) and European Regional Development
Fund (Project No. CZ.02.1.01/0.0/0.0/16_019/0000845).
There is no conict of interest declared in this article.
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Additional supplemental material may be found online
in the Supporting Information section at the end of the
... Mussel hemolymph, foot, and mantle samples can be collected without causing significant mortality ( Naimo et al. 1998;Gustafson et al. 2005a, b;Fritts et al. 2015b, Bartsch et al. 2017). Refinement of these methods is needed to provide guidance on the sample type and volume or mass required for specific assays (e.g., Vodáková and Douda 2019) and the amount of tissue that can be sampled nonlethally based on individual body mass. ...
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INTRODUCTION The imperiled status of freshwater mussels (order Unionida) is well documented (Williams et al. 1993; Lydeard et al. 2004; Strayer et al. 2004; Régnier et al. 2009). The most commonly cited contributors to mussel declines are habitat destruction or alteration, pollution and poor water quality, impoundment, and invasive species (Strayer et al. 2004; Dudgeon et al. 2007; Downing et al. 2010; Haag and Williams 2014). However, these factors do not explain the declines and large-scale die-offs of mussels in otherwise healthy, unimpounded streams across a wide geographic region. The significant decline of mussels that occurred from the 1970s to 1990s has been described as “enigmatic” with characteristics suggesting a virulent and widespread factor specific to mussels (Haag and Williams 2014; Haag 2019). One topic missing from most publications related to mussel conservation is organismal health and disease. The role of the microbiota and pathogens in mussel health has been understudied, and, as a result, their role in mussel declines is unknown. No clinical signs or biomarkers have been established to distinguish a healthy mussel from one that is of compromised health or dying. Although the suggestion that mussel mortality and declines could be pathogen related has not been widely considered among freshwater biologists, the effects of epizootics on other aquatic invertebrates are well documented. For example, fungal, bacterial, and viral diseases (Edgerton et al. 2002; Jiravanichpaisal et al. 2009; Longshaw 2011; Bower 2012) have adversely affected crayfish populations worldwide. Numerous diseases have significantly impacted marine bivalves, including ostreid herpesvirus and the protozoan disease bonamiasis in oysters (Ostrea spp.; Zanella et al. 2017). More recently, a Densovirus (Parvoviridae) has been associated with sea star wasting disease, the cause of extensive mortality among populations of 20 asteroid species in the Pacific Northwest (Hewson et al. 2014). In 26 contrast, reports of pathogens in freshwater mussels are limited to those responsible for explosive epidemics in the Triangle-shell Pearl Mussel (Hyriopsis cumingii, family Unionidae) (see Zhong et al. 2016) in China. It seems unlikely that other freshwater mussel species are unaffected by comparable infectious agents. We discuss the state of knowledge on freshwater mussel health assessment and disease and outline a strategy for advancing and expanding that knowledge. First, we provide an overview of research efforts on mussel health and disease in the past 30 years. We use “disease” throughout the article to refer to any impairment that interferes with or modifies normal function, including responses to environmental factors such as food availability, toxicants, and climate; infectious agents; inherent or congenital defects; or combinations of those factors (Wobeser 1981). Definitions of terms related to health assessment and disease used in the article are provided in Appendix A. Second, we discuss the growing need for a focused effort on health and disease research in mussels and describe the application and benefits of a holistic approach. We discuss existing approaches for monitoring health in other faunal groups and highlight their application to mussel conservation. Finally, we discuss research and resources needed to advance the state of knowledge of mussel health.
Technical Report
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Los trabajos de 2019 corroboran la distribución pasada de Margaritifera auricularia en el tramo burgalés del Ebro, en un tramo comprendido aguas abajo del embalse de Puentelarrá, al encontrarse nuevas conchas tanto aguas arriba de Miranda de Ebro como aguas abajo. En esta ocasión además de la búsqueda de posibles animales y sus restos mediante técnicas de localización visual directa en zonas vadeables (27 parcelas) se han prospectado las zonas más profundas mediante buceo profesional (10 puntos) y videograbación (14 puntos) en una amplia red de puntos que incluyó todo el recorrido del Ebro en la provincia de Burgos. Se ha comprobado la ausencia en la actualidad de animales vivos a través de la utilización del eDNA (14 puntos) como técnica de alta sensibilidad. La recuperación a largo plazo de la especie en el Ebro burgalés pasará por la consolidación de las condiciones ambientales, la adaptación de los caudales a ritmos más naturales, la supervivencia de buenas poblaciones de blenios y la reducción del impacto de las especies exóticas en las comunidades bentónicas. Es necesario conocer más en detalle las necesidades vitales de M. auricularia y S. fluviatilis para caracterizar el área potencial de distribución, y además prevenir la colonización de los fondos por Corbicula fluminea.
Many proteins are known to be phosphorylated, affecting important regulatory factors of muscle quality in the aquatic animals. The striated and smooth adductor muscles of Yesso scallop Patinopecten yessoensis were used to investigate muscle texture and identify phosphoproteins by histological methods and phosphoproteomic analysis. Our present study reveals that muscle fiber density is in relation to meat texture of the striated and smooth adductor muscles. The phosphoproteomic analysis has identified 764 down-regulated and 569 up-regulated phosphosites on 743 phosphoproteins in the smooth muscle compared to the striated part. The identification of unique phosphorylation sites in glycolytic enzymes may increase the activity of glycolytic enzymes and the rate of glycolysis in the striated adductor muscle. The present findings will provide new evidences on the role of muscle structure and protein phosphorylation in scallop muscle quality and thus help to develop strategies for improving meat quality of scallop products.
1. Translocation is used to conserve mussels, yet there remains a debate about its merit owing to poor understating of its effects on transported mussels. 2. This study evaluated survivorship, body condition, and total glycogen and lipids for one common and widely distributed species (Cyclonaias pustulosa), two rare species (Cyclonaias petrina; Lampsilis bracteata), and one species complex (Fusconaia sp.-Fusconaia chunii and Fusconaia flava) from the East Fork of the Trinity River and the Llano River of Texas. 3. Survivorship estimates for C. pustulosa and Fusconaia sp. using the Kaplan-Meier estimator were high in the East Fork. Body condition, glycogen, and total lipids varied for C. pustulosa and Fusconaia sp., which may have indicated a short-term impact. For the Llano, survivorship of C. petrina and L. bracteata was high for the resident treatments but significantly reduced for the translocation treatments. 4. The decline in survivorship for C. petrina was mirrored by decreases in the body condition, which may indicate inability to acclimate to novel environments. For L. bracteata, declines in survivorship were due to predation by Procyon lotor, racoon. A large flood of 3,766 m 3 s −1 at the end of the study eliminated both study sites. 5. The findings of this study show that translocating mussels can be successful; however , sublethal effects and mortality may still occur. These effects are rooted in species-specific differences, which is not unexpected because mussel species vary in how they cope with environmental change based on their life-history traits. However, these traits are rarely considered when translocating mussels. 6. To complicate matters, most mussel species have yet to be evaluated on how they respond to translocation, and for species where such information is available, adults are the primary focus. Addressing these knowledge gaps is critical for determining the appropriateness of translocation and improving its efficacy.
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Limnoperna fortunei (Dunker 1857) is a freshwater mussel with physiological tolerance to different environmental conditions, which may explain its success as an invasive species. The role of abiotic factors in its establishment, abundance and projections of risk of further spread into several areas has been studied. These mussels may respond to multiple environmental stressors, such as temperature, through physiological mechanisms, behavioral responses, mortality or some combination of these. The aim of this study was to investigate the behavioral responses (valve closing), glycogen concentrations and mortality of L. fortunei under four different temperatures (5°C, 10°C, 20°C and 30° C) during a chronic test (30 days). Two-way analysis of variance (ANOVA) was used to compare glycogen concentrations across days of the experiment and at the different temperatures. Differences in valve-closing behavior and mortality among temperatures were tested using repeated-measures ANOVA. We observed that most of the mussels maintained at 5°C closed their valves (74.7 ± 15.3%), indicating that they remain inactive at low temperatures. The glycogen levels significantly differed among the temperatures tested. These differences occurred mainly due to the high glycogen values observed in mussels exposed to 10°C. Stability in glycogen concentrations was observed within each particular temperature. The cumulative mortality was higher at extreme temperatures (5°C and 30°C). The ideal temperature for laboratory maintenance and tests is approximately 20°C. Our data also show that L. fortunei can survive and maintain their energy reserves (glycogen) for several days at 5°C, an important feature related to its invasion success.
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Successful animal rearing under laboratory conditions for commercial processes or laboratory experiments is a complex chain that includes several stressors (e.g., sampling and transport) and incurs, as a consequence, the reduction of natural animal conditions, economic losses and inconsistent and unreliable biological results. Since the invasion of the bivalve Limnoperna fortunei (Dunker, 1857) in South America, several studies have been performed to help control and manage this fouling pest in industrial plants that use raw water. Relatively little attention has been given to the laboratory rearing procedure of L. fortunei, its condition when exposed to a stressor or its acclimation into laboratory conditions. Considering this issue, the aims of this study are to (i) investigate L. fortunei physiological responses when submitted to the depuration process and subsequent air transport (without water/dry condition) at two temperatures, based on glycogen concentrations, and (ii) monitor the glycogen concentrations in different groups when maintained for 28 days under laboratory conditions. Based on the obtained results, depuration did not affect either of the groups when they were submitted to approximately eight hours of transport. The variation in glycogen concentration among the specimens that were obtained from the field under depurated and non-depurated conditions was significant only in the first week of laboratory growth for the non-depurated group and in the second week for the depurated group. In addition, the tested temperature did not affect either of the groups that were submitted to transport. The glycogen concentrations were similar to those of the specimens that were obtained from the field in third week, which suggests that the specimens acclimated to laboratory conditions during this period of time. Thus, the results indicate that the air transport and acclimation time can be successfully incorporated into experimental studies of L. fortunei. Finally, the tolerance of L. fortunei specimens to the stressor tested herein can help us understand the invasive capacity of this mussel during the establishment process.
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p>The success of Limnoperna fortunei as an invasive species is related to its physiological plasticity that allows them to endure adverse environmental conditions. Starvation tolerance is considered to be an important trait associated with bivalve invasiveness. In natural ecosystems, food resources can vary during the year, exposing mussels to variable periods of starvation or limited food availability. Thus, mussels have developed physiological strategies to tolerate and survive fluctuations in food availability. Glycogen concentration has been used in different monitoring studies as an indicator of the nutritional condition of bivalves. The aim of this study was to investigate the physiological responses of L. fortunei based on the glycogen concentrations of specimens under four treatments, comprising different combinations of feeding and starvation, during 125 days. The experiment was carried out in two phases. In the phase I, mussels were divided in two treatments: starvation (S) and feeding (F). After 100 days, tissue samples were collected to quantify glycogen concentrations and, each phase I group was divided in two subgroups: starvation (S) and feeding (F), resulting in four treatments. In the phase II, that lasted 25 days, starvation specimens (S) from phase I were allowed to feed (starvation-feeding treatment , or S-F), or continued to undergo starvation (starvation-starvation treatment , or S-S) and the feeding specimens (F) continued feeding (feeding-feeding group, or F-F), or were subjected to starvation (feeding-starvation treatment , or F-S). Behavior (valve-closing) and mortality were recorded in 24 h intervals. After the 25 days (phase II) all specimens were killed, and their soft tissue was removed to quantify glycogen concentrations. The glycogen concentration of the S-F treatment was lower than that of the F-S treatment, which was initially allowed to feed (phase I) and then subjected to starvation (phase II). Stability in the glycogen concentrations was observed when the phase II feeding conditions were maintained during the experiments, as observed in the S-S (continued starvation) and F-F (continued feeding) treatments. Based on our glycogen concentrations results, the golden mussel shows a higher tolerance to starvation (125 days) than has previously been published, which suggests that its tolerance strongly influences its invasive behavior. </p
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Freshwater mussels of the Order Unionida provide important ecosystem functions and services, yet many of their populations are in decline. We comprehensively review the status of the 16 currently recognized species in Europe, collating for the first time their life-history traits, distribution, conservation status, habitat preferences, and main threats in order to suggest future management actions. In northern, central, and eastern Europe, a relatively homogeneous species composition is found in most basins. In southern Europe, despite the lower species richness, spatially restricted species make these basins a high conservation priority. Information on freshwatermussels in Europe is unevenly distributed with considerable differences in data quality and quantity among countries and species. To make conservation more effective in the future, we suggest greater international cooperation using standardized protocols and methods to monitor and manage European freshwater mussel diversity. Such an approach will not only help conserve this vulnerable group but also, through the protection of these important organisms, will offer wider benefits to freshwater ecosystems.
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The development of effective nonlethal biomonitoring techniques is imperative for the preservation of imperiled freshwater mussel populations. Changes in hemolymph chemistry profiles and tissue glycogen are potential biomarkers for non-lethally monitoring stress in mussels. We sampled three species in the Flint River Basin over two years to evaluate how these hemolymph and tissue biomarkers responded to environmental changes. We used hierarchical linear models to evaluate the relationships between variation in the biomarkers and environmental factors, and found that the responses of the hemolymph and tissue parameters were strongly related to stream discharge. Shifts in alanine aminotransferase and glycogen showed the largest relations with discharge at the time of sampling, while magnesium levels were most explained by the discharge for five d prior to sampling. Aspartate aminotransferase, bicarbonate, and calcium showed the strongest relations with average discharge for 15 d prior to sampling. The modeling results indicated that biomarker responses varied substantially between individuals of different size, sex, and species, and illustrated the value of hierarchical modeling techniques to account for the inherent complexity of aquatic ecosystems.
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Freshwater mussel populations are highly susceptible to environmental alterations due to their diminished numbers and primarily sessile behaviors; non-lethal biomonitoring programs are needed to evaluate the health of populations prior to mass-mortality events. Our objectives were to determine 1) which biochemical parameters in freshwater mussel hemolymph could be consistently quantified, 2) how hemolymph parameters and tissue glycogen respond to a thermal stress gradient (25, 30, and 35°C) and 3) the effects of tissue and hemolymph extraction on long-term growth and survival of smaller and larger-bodied mussel species. Glucose exhibited elevated expression in both species with increasing water temperature. Two transaminase enzymes had elevated expression in the 30°C treatment. The effects of hemolymph extraction and tissue biopsies were evaluated with a large-bodied species, Elliptio crassidens, and a smaller species, Villosa vibex. Individuals were monitored for 820 to 945 days after one of four treatments: hemolymph extraction, tissue biopsy, tissue and hemolymph extraction, and control. Hemolymph extraction and tissue biopsy adversely affected survival of V. vibex, suggesting that these extraction methods may add some risk of reduced survival to smaller-bodied species. Survival of E. crassidens was not impaired by any of the treatments, supporting the use of these techniques in non-lethal biomonitoring programs for larger-bodied mussel species.
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Mantle biopsy is a means of obtaining tissue samples for genetic, physiological, and contaminant studies of bivalves; but the effects of this biopsy on survival have not been determined. We describe a simple technique for obtaining such samples from unionacean bivalves and how we compared survival among biopsied and control organisms in field experiments. Survival was not significantly different between treatment and control groups. Power estimates for these results were between 0.42 and 0.73. Results were similar among species and among habitats. Mantle biopsy is a technique that allows genetic, biochemical, and contaminant studies of mussel populations when destruction of individuals should be avoided.
This volume represents the marriage of two separate symposia and one workshop paper, all involving freshwater mollusks. The first part of this book represents the proceedings of the Conservation, Captive Care, and Propagation of Freshwater Mussels Symposium held in Columbus, Ohio in March, 1998. The second part of the book represents the proceedings of the First Freshwater Mollusk Conservation Society Symposium held in Chattanooga, Tennessee in March, 1999. The final paper is a heretofore unpublished manuscript from a U.S. Geological Survey Biological Resources Division and Water Resources Division workshop held in Atlanta, Georgia in March, 1997. The workshop was entitled Freshwater Mollusks as Indicators of Water Quality.
Larvae of Utterbackia imbecillis normally undergo metamorphosis to the juvenile while attached to the gills or fins of a host fish; however, metamorphosis can also be induced in the laboratory in a modified cell culture medium. This study examined juveniles resulting from each of these rearing techniques to determine their relative physiological conditions. Juveniles reared in vitro grew more slowly and had higher mortality rates than did their fish-reared counterparts. Animals reared on their host fish accumulated triglycerides, cholesterol, glycogen, and protein during the parasitic metamorphic period. In contrast, animals reared in vitro showed an increase in the levels of triglycerides, but did not accumulate cholesterol, glycogen, or protein. These results suggest that fish-reared juvenile individuals of U. imbecillis are in more robust physiological condition than their in vitro-reared counterparts.