A review on the “in vitro” culture of freshwater mussels (Unionoida)

Many Unionoida are considered to be extinct, endangered, or of special concern. These bivalves have complex life cycle stages that limit successful culture. In nature, the larvae (glochidia) of these bivalves must successfully parasitize a host (mainly fish) in order to metamorphose into juveniles. The two artificial methods used to obtain juvenile freshwater mussels in laboratory are either by induced attachment to host fish or by in vitro culture of glochidia. This article is focused on the in vitro method that represents a novel and alternative process to fish infestation, offering the ability to obtain larger numbers of juveniles without the need for host fishes and reducing the overall costs of propagation. In vitro culture requires a medium which fulfills the nutritional needs of each glochidia species and avoids microbial contamination. Recently, this methodology has presented excellent results with survival and transformation rates up to 94% using host fish plasma. High efficiencies on growth, and survival rates (84%) of juvenile freshwater bivalve Hyriopsis myersiana (Lea, 1856) up to 120 days were obtained when reared in adequate recirculating aquacultural systems using a very specific diet. More research is still needed to demonstrate successful propagation, mainly concerning the media nutritional composition to increase glochidia transformation and juvenile quality.     

Life history of an unusual marine fish: survival, growth and movement patterns of Hippocampus guttulatus Cuvier 1829

The life history of the long‐snouted seahorse Hippocampus guttulatus was characterized using mark‐recapture data collected within a focal study site and catch data from 53 additional sites in the Ria Formosa coastal lagoon, southern Portugal. Population structure in benthic habitats was characterized by high local densities (0·3–1·5 m^?2), equal sex ratios and few juveniles <70 mm. Adult H. guttulatus maintained small (19·9 ± 12·4 m²), strongly overlapping home ranges during multiple reproductive seasons. Recruited (benthic) juveniles exhibited significantly lower site fidelity than adults. A Ford‐Walford plot of standard length (L_S) at time t against L_S measured during the previous year from tagged juveniles and adults led to estimates of the von Bertalanffy parameters K = 0·571 and L_∞ = 197·6 mm. The growth rate of planktonic juveniles (inferred from previous studies), was greater than predicted by the von Bertalanffy model, providing evidence of an ontogenetic shift in growth trajectory. The instantaneous rate of natural mortality, M, ranged from 1·13 to 1·22 year^?1(annual survival rate = 29·4–32·2%). Sexes did not differ in movement, growth or survival patterns. On average, H. guttulatus measured 12·2 ± 0·8 mm at birth. Planktonic juveniles recruited to vegetated habitat at 96·0 ± 8·0 mm (0·25 years), had mature brood pouches (males only) at 109·4 mm (0·49 years), began maintaining home ranges and reproducing at 125–129 mm (0·85–0·94 years), and lived for 4·3–5·5 years. Early age at maturity, rapid growth rates, and short generation times suggested that H. guttulatus may recover rapidly when direct (e.g. exploitation) and indirect (e.g. by‐catch and habitat damage) effects of disturbance cease, but may be vulnerable to extended periods of poor recruitment.     

Morphological and morphometrical description of the glochidia of Westralunio carteri Iredale, 1934 (Bivalvia: Unionoida: Hyriidae)

Most freshwater mussel (Bivalvia: Unionoida) larvae (glochidia in Margaritiferidae, Hyriidae and Unionidae) are fish parasites. Knowledge of the larval morphology and the mechanism of release in freshwater mussels is useful in species systematics and ecology. Westralunio carteri is the only unionoid from south-western Australia. Little information is available on its biology and its glochidia have never been described. The aim of this study was to describe the glochidia of W. carteri and method of their release. Glochidia within vitelline membranes were embedded in mucus which extruded from exhalent siphons of females during spring/summer; they then hatched from vitelline membranes but remained tethered by a larval thread and began characteristically “winking”. Shells (n=120) were subtriangular, 308 μm long (±0.83 SE), 251 μm high (±0.73 SE) and had a hinge length of 212 μm (±0.78 SE). Larval teeth were singular with interlocking cusps and convex or concave basal protuberances on opposing valves.     

High stocking density during larviculture and effect of size and diet on production of juvenile Lophiosilurus alexandri Steindachner, 1876 (Siluriformes: Pseudopimelodidae)

Different stocking densities were investigated in larviculture and feeding of Lophiosilurus alexandri, as well as analyses of the effects on juveniles of two size‐classes and two different commercial formulated diets. The first experiment was two‐phased: (a) larvae stocked at densities of 60, 120, 180, 240, and 300 larvae L⁻¹ fed with Artemia nauplii and reared for 15 days; (b) in phase 2, densities of 5, 10, 15, 30, and 40 juveniles L⁻¹ were evaluated during feed training (20 days). Mean water temperature in both phases was 28°C. In the first phase of experiment 1, the different stocking densities did not affect fish growth or survival. In phase 2, growth was similar in all densities; however, survival was lower at higher densities. The increased density provided a rise in biomass and number of individuals produced in both phases. In the second experiment, two size‐classes of feed‐trained juveniles (30.22 ± 1.84 and 34.66 ± 2.41 mm) were given pellets of two different diameters (1.2 and 2.6 mm) for 20 days. The largest juveniles fed the 1.2 mm inert diet had higher final weights and lengths. Larviculture and feed training of L. alexandri can thus be performed successfully at high stocking densities of 300 larvae L⁻¹ during the first 15 days of feeding, and at densities of up to 40 juveniles L⁻¹ during the 20 days of feed training, respectively.     

Ecological and morphological characteristics of the endoparasitoids of larval Acronicta rumicis (Lepidoptera: Noctuidae)

The Hymenopterans Glyptapanteles liparidis, Microplitis sp. and Diadegma sp. were found to be larval parasitoids and koinobionts of Acronicta rumicis (Lepidoptera: Noctuidae). Mesochorus semirufus is believed to be a new unreported hyperparasitoid of G. liparidis, which, along with M. semirufus, is a gregarious parasitoid. In contrast, the parasitoids Microplitis sp. and Diadegma sp. are solitary. All of the hymenopteran parasitoids are multivoltine insects that emerge from A. rumicis more than once. Compcilura concinnata, Euexorista sp. and Exorista sp. of the Diptera were found to be larval–pupal parasitoids, solitary parasitoids and koinobionts. These three species are univoltine, and emerge only once from A. rumicis. Morphological and life cycle data were collected for G. liparidis, and for the parasitoids of that species found in this study. The major and minor axes of an egg of G. liparidis were 0.10 and 0.02 mm, respectively, while the mean clutch size of G. liparidis was 67.71 ± 39.36 individuals. The body length of female and male G. liparidis were 2.25 ± 0.06 and 2.21 ± 0.12 mm, respectively, and the longevity of an adult was 2.93 ± 0.96 days. Among the parasitoids, the mean body length of an adult Microplitis sp. was 3.5 mm and adults lived for an average of 8.13 ± 3.54 days. The adult Diadegma sp. was larger (mean body length 6.5 mm) but lived for a shorter interval (3.33 ± 1.32 days). The body lengths of female and male M. semirufus were 3.16 ± 0.11 and 3.10 ± 0.23 mm, respectively, greater than the body lengths of female and male G. liparidis. The body lengths of adult C. concinnata, Euexorista sp. and Exorista sp. were 9.5, 9.53 and 8.68 mm, respectively. All of their pupae were dark brown.     

The critical thermal limits for the bullhead, Cottus gobio, from three populations in north-west England

1. The objective was to determine the thermal limits for feeding and survival in the bullhead, Cottus gobio, using juveniles (total length 20–30 mm, live weight 0.5–1.5 g) from one population and adults (50–70 mm, 3.5–5.5 g) from three populations. 2. Fish were acclimated to constant temperatures (3, 7, 10, 15, 20, 25 or 27 °C) and the temperature was then changed at a rate of 1 °C /30 min to determine the critical limits for feeding, survival over 7 days (incipient lethal temperature), or survival for 10 min or less (ultimate lethal temperature). The rate of 1 °C/30 min was the optimum value from preliminary experiments, using nine rates from 0.5 °C/48 h to 18 °C h^?1. As values for adults were not significantly different between populations, they were pooled to provide arithmetic means (with 95% CL) for the thermal limits at each acclimation temperature. 3. Feeding limits increased with acclimation temperature to upper and lower mean values (± 95% CL) of 26.5 ± 0.16 °C and 4.2 ± 0.20 °C for adults, 26.6 ± 0.59 °C and 5.0 ± 0.55 °C for juveniles. Incipient lethal levels defined a tolerance zone within which fish survive indefinitely; upper limits increased with acclimation temperature to a plateau of 27.6 ± 0.22 °C for adults and 27.5 ± 0.47 °C for juveniles, lower limits increased from near 0 °C to 2.5 ± 0.31 °C for adults and 2.7 ± 0.47 °C for juveniles. Ultimate lethal levels increased with acclimation temperature to a plateau of 32.5 ± 0.24 °C for adults and 32.6 ± 0.46 °C for juveniles, whilst the lower limits increased from near 0 to 0.9 ± 0.29 °C. Upper feeding, incipient and ultimate lethal values were significantly lower for juveniles than those for adults at acclimation temperatures < 20, < 20 and < 15 °C, respectively. 4. The thermal tolerance of bullheads was slightly lower than that of stone loach, similar to that of juvenile Atlantic salmon and higher than that of brown trout; the thermal limits for feeding were much wider than those for salmon or trout.     

Seasonal dynamics and emergence pattern of a tropical earthworm,Drawida calebi (Oligochaeta)

Cocoon morphology (shape, size, colour and ornamentation), hatching, and seasonal dynamics of cocoon and juveniles of D. calebi have been studied in an Indian pasture site. The average live weight of cocoon is 15.17 mg, length/diameter 0.94, and moisture content 72.5 g%. Just-hatched juveniles weigh on an average 10.4 mg, and are 10.5–17.5 mm in length. The incubation period lasts 56.33 ± 2 days during November—December in laboratory cultures (25°C, and moisture 15 g%). A maximum of 64.0 live cocoons/m² with a live weight of 0.971 g/m² were deposited during October—November 1977, but very few during summer months. A surface-living species, D. calebi shows single peak emergence pattern in December—January. It is estimated that 78 cocoons (live and dead)/m² (780,000/hectare), were produced in the field by D. calebi population in a year (1976–77). Thus the total cocoon production is 11.83 kg/hectare/year. It is estimated that 3 cocoons/individual/year are produced by D. calebi in field conditions.     

The critical thermal limits for the stone loach, Noemacheilus barbatulus, from three populations in north-west England

1. The chief objective was to determine the upper and lower thermal limits for feeding and survival in the stone loach, Noemacheilus barbatulus, using juveniles (total length 30–45 mm, live weight 0.25–0.80 g) from one population and adults (total length 77–100 mm, live weight 3.6–7.9 g) from three populations. 2. Fish were acclimatized to constant temperatures of 3, 7, 10, 15, 20, 25 and 27°C; then the temperature was changed at a rate of 1°C/30min to determine the critical limits for feeding, survival over 7 days (incipient lethal temperature), or survival for 10 min or less (ultimate lethal temperature). The rate of 1°C/30min was the optimum value from preliminary experiments, using nine rates from 0.5°C/48h to 18°Ch^?1. As values for adults were not significantly different between populations, they were pooled to provide arithmetic means (with 95% CL) for the thermal limits at each acclimation temperature. 3. Feeding limits increased with acclimation temperature to upper and lower mean values of 28.0 ± 0.15°C and 5.1 ± 0.55°C for adults, 25.0 ± 0.54°C and 6.1 ± 0.92°C for juveniles. Incipient lethal levels defined a tolerance zone within which stone loach survive for a considerable time; upper limits increased with acclimation temperature to reach a maximum plateau of 29.1 ± 0.18°C for adults and 29.0 ± 0.40°C for juveniles; lower limits also increased from near 0°C to 3.0 ± 0.40°C for adults and juveniles. Upper limits for the ultimate lethal level increased with acclimation temperature to a maximum plateau of 33.5°C for adults (95% CL ± 0.19) and juveniles (95% CL ± 0.40), whilst the lower limits increased from near 0°C to 2.5 ± 0.30°C. At acclimation temperatures below 20°C, upper incipient and ultimate lethal values were significantly lower for juveniles than those for adults. 4. The thermal tolerance of stone loach was higher than that of juvenile Atlantic salmon or brown trout, one or both of these species often being dominant in streams with stone loach.     

The effects of different cycles of starvation and re‐feeding on growth and body composition in rainbow trout (Oncorhynchus mykiss,Walbaum, 1792)

This study was designed to investigate the effects of starvation and re‐feeding cycles on the growth performance and body chemical composition of Oncorhynchus mykiss juveniles. A total of 360 juveniles with initial mean weights (IW) of 8.46 ± 0.07 g (n = 360) were stocked into 400‐L tanks in triplicate for each group, with 30 juveniles per tanks. The control group received regular feed, as is the common practice. The three other groups were periodically starved: 1 day starvation followed by 6 days re‐feeding (S1), 2 days starvation followed by 5 days re‐feeding (S2) and 3 days starvation followed by 4 days re‐feeding (S3). The experiment lasted for 10 weeks, over the course of which the water flow rate was 4 L min^?1 and the water quality parameters determined as: temperature 14.4 ± 1.1°C, oxygen 8.2 ± 0.4 mg L^?1 and pH 7.5 ± 0.2. At the end of the study, S1 had the best growth performance (final weight, specific growth rate, average daily growth) of all test groups (P < 0.05). The lowest daily feed intake (DFI) and growth performance parameters were observed in S3 (P < 0.05), while protein efficiency ratio (PER), net protein utilization (NPU) and lipid efficiency ratio (LER) were higher in the S3 fish group than in the other groups (P < 0.05). Whole body protein and lipid contents were highest in S1 fish. The hepatosomatic index (HSI) and viscerosomatic index (VSI) were significantly different among groups (P < 0.05). Feed conversion ratio (FCR) was significantly lower in starvation groups S1, S2 and S3 than in the control (P < 0.05). Compensation coefficient (CC) values were higher than 1 in all starvation groups. The concluding indicate that rainbow trout exposed to 1 and 2 days of starvation in week cycles could achieve over compensation compared to the control. Additionally, partial growth compensation and improved feed utilization could be achieved in a starvation group within 3 days in a week, by beginning with the juvenile size over a 10‐week experimental period.     

Feasibility of early outplanting of sexually propagated Acropora verweyi for coral reef restoration demonstrated in the Philippines

Over the last 20 years, coral sexual propagation techniques for reef restoration have been steadily developed and improved. However, these techniques involve considerable time and costs to grow coral propagules. There is a need to examine the optimal size of juvenile corals for outplantation. Here, we outplanted sexually propagated small (3–5 mm diameter) and large (10–15 mm diameter) Acropora verweyi corals at 4 months after fertilization at two sites in northwestern Philippines, and compared their survival and radial growth rate after a year. A. verweyi coral juveniles (n = 240) exhibited an overall mean survival of 29.5% and growth rate of 11.12 ± 6.2 mm/year (mean ± SD). Large colonies had a significantly higher growth rate than smaller colonies. Although survivorship of large juveniles was significantly better than that of the smaller ones at one site, it did not differ significantly at the other. Each 4‐month‐old coral cost US$1.52 to produce, while the cost of each of the outplanted juveniles (n = 240) was about US$2.67, whereas the cost of each survivor about a year after outplantation was US$11.47. Results suggest that A. verweyi reared in ex situ nurseries for only 4 months can survive reasonably well when outplanted onto coral reefs.     

Growth and morphological development of laboratory-reared larvae and juveniles of the Laotioan indigenous cyprinid Hypsibarbus malcolmi

The morphological development, including the pigmentation, body proportions, fins, and survival rate for 30 days after hatching, of laboratory-reared larval and juvenile Hypsibarbus malcolmi is described. Body lengths (BL) of larvae and juveniles were 2.0 ± 0.2 (mean ± SD) mm at 1 h after hatching (day 0) and 9.2 ± 0.6 mm on day 16, reaching 12.1 ± 0.9 mm on day 30. Yolk volume decreased linearly, with the yolk being completely absorbed by day 3 in all preflexion larvae (all specimens >3.2 mm BL). Feeding was observed on day 2 in fish which had rapidly undergone complete yolk absorption following mouth and anus opening on day 1, and on day 3 in all remaining fish. Myomere numbers were 20–21 + 11–12 = 31–33, although they were not clearly visible in juveniles. Melanophores were few on the body during days 0–2, but increased with growth and covered the entire upper dorsal body surface during the juvenile stage. Body proportions tended to become constant in juveniles. Notochord flexion began in larvae >5.2 mm BL on day 8, and was completed in larvae >8.4 mm BL on day 14. Specimens with full fin ray complements were initially observed on day 22 (10.4 mm BL in juveniles). All specimens >11.5 mm BL had attained the juvenile stage. A high survival rate of 92.7% was estimated on day 30.     

Improved survival and growth of silver therapon Leiopotherapon plumbeus early juveniles through co-feeding with Artemia and commercial feeds

This study examined the effects of co-feeding Artemia and commercial feeds on survival, growth and fatty acid composition of silver therapon Leiopotherapon plumbeus early juveniles. Triplicate groups of 36 days post hatch (DPH) early juveniles (17.09 ± 1.69 mm; 0.07 ± 0.02 g) were stocked in nine glass aquaria at 25 individuals per aquarium and reared for 60 days on three feeding regimes: (A) Artemia + powdered commercial tilapia feed (35% crude protein (CP)); (B) Artemia + powdered commercial prawn feed (38% CP); and (C) Artemia nauplii only as the control group. Early juveniles co-fed Artemia and commercial feeds had significantly higher survival (97%) than those fed Artemia alone (86%). Except for the condition factors that were similar to the control group, higher mean total length (30.2 ± 1.3 mm and 27.6 ± 1.2 mm), body weight (401 ± 64 mg and 339 ± 46 mg), length- (SGRL; 0.95 ± 0.07%/day and 0.80 ± 0.07%/day) and weight-specific growth rates (SGRW; 2.85 ± 0.27%/day and 2.58 ± 0.22%/day) were also observed in the co-feeding groups, independent of protein, fat and other nutrient levels in commercial feeds. Higher levels of long-chain polyunsaturated fatty acids were reflected in early juveniles co-fed Artemia and commercial feeds than those fed exclusively on Artemia contributing, in part, to the higher growth and survival observed in the co-feeding groups. Together, these results suggest that co-feeding strategy showed best results in terms of growth and survival, and that commercial feed with 35% protein and 6% crude fat levels may be beneficial in supplementing live feed with essential nutrients to optimize production of silver therapon fry during nursery culture.     

Effect of temperature on oocyte development of Oreochromis karongae (Trewavas, 1941)

Oreochromis karongae (Trewavas, 1941) is one of the indigenous Tilapia species exhibiting favourable traits for aquaculture in Malawi. However, commercial fingerling production is still a problem. An experiment was carried out to investigate the effect of raising ambient temperature to 27°C on oocyte development of the fish. Female O. karongae were reared under two temperature regimes: at room (20.3 ± 0.8°C) and at raised (26.5 ± 0.5°C) temperature for 90 days. Changes in gonadosomatic index (GSI) and oocyte developmental stages were determined every 45 days. Fish samples from the pond (22.5 ± 3.4°C) from which experimental fish were collected were used for comparison. Results showed that raising temperature to 26.5 ± 0.5°C significantly enhanced oocyte development. Higher GSIs (P ≤ 0.05) were obtained after 45 and 90 days in fish cultured at elevated temperature (0.82 ± 0.66 and 1.13 ± 0.47%, respectively) than at room temperature (0.06 ± 0.03 and 0.37 ± 0.05 %, respectively). GSI of fish samples from the pond were not different from that of fish from room temperature. After 45 days, relative frequency of mature oocytes was higher (P ≤ 0.05) in fish from raised temperature (60.42 ± 3.63%) than in fish from room (1.76 ± 0.84%) and pond temperature (2.43 ± 1.38%). After 90 days, the frequency of mature oocytes in fish from raised temperature was not different from pond fish (8.68 ± 2.40 and 10.99 ± 3.41%, respectively). Fish from room temperature had a low (P ≤ 0.05) frequency of mature oocytes (3.12 ± 2.03%). The results suggest that O. karongae has the potential to spawn throughout the year when the temperature is manipulated.     

Eimeria ladronensis n. sp. and E. albigulae (Apicomplexa: Eimeriidae) from the Woodrat,Neotoma albigula (Rodentia: Cricetidae)1

Of 50 white-throated woodrats (Neotoma albigula) collected from Socorro Co., New Mexico, 21 (42%) had eimerian oocysts in their feces when examined. Of the 21 Neotoma found positive for Eimeria, 19 (90%) harbored a single eimerian species at time of examination. Eimeria albigulae Levine, Ivens & Kruidenier, 1957, was found in 18 (86%), and E. ladronensis n. sp. was found in five (24%) infected woodrats. Sporulated oocysts of E. ladronensis are ellipsoidal, 19–25 × 13–15 (21.4 ± 1.3 × 14.1 ± 1.1) μm, have a smooth wall and one or two polar granules, but lack a micropyle and an oocyst residuum. Sporocysts are tapered at one end, 7–10 × 6–7 (8.5 ± 0.7 × 6.5 ± 0.3) μm, and have a Stieda body and sporocyst residuum, but no substieda body. Prepatent periods for E. albigulae and E. ladronensis n. sp. are 5–6 and 8–9 days, respectively; patent periods are 7–18 and approximately 11 days, respectively.     

Characteristics of Sinanodonta woodiana (Lea, 1834) Populations in Fish Ponds (Upper Silesia,Southern Poland) in Relation to Environmental Factors Abstract

We analyzed the biomass, density and age structure of S. woodiana populations in relation to different environmental factors at new sites of its occurrence in southern Poland. The highest recorded biomass was 4,413 g m^–2. Mussel density was 19 individuals m^–2. The age structure was formed by juveniles as well as by specimens more than 8 years. The rapid expansion of S. woodiana in European countries is believed to be the result of accidental introduction of fishes bearing glochidia of this species. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Response of juvenile tench Tinca tinca (L.) to the anaesthetic 2-phenoxyethanol

The response of tench Tinca tinca aged 40–171 days post‐hatch (22–49 mm TL) to the anaesthetic 2‐phenoxyethanol was studied at 25°C. The lowest effective concentration of 2‐phenoxyethanol increased with age, while the highest safe concentration decreased. The fish aged 40 days post‐hatch required a significantly (P ≤ 0.05) shorter time to become anaesthetized than did older fish. The recovery time after 15 min of exposure to 2‐phenoxyethanol at 0.45 g dm^?3 was significantly shorter in the 40‐day‐old fish than in older fish. In juveniles of the same age, induction time or recovery time did not depend on their size or condition (Fulton's coefficient). At 25°C, 2‐phenoxyethanol at 0.5 g dm^?3 may be used to efficiently and safely anaesthetize T. tinca juveniles.     

Using stable isotope ratios as a tracer of feeding adaptation in released Japanese flounder Paralichthys olivaceus

Release‐recapture experiments were conducted to examine temporal changes of the carbon and nitrogen stable isotope (δ¹³C and δ¹⁵N) ratios in the muscle tissue of artificially produced Japanese flounder Paralichthys olivaceus, juveniles. About 9000 juveniles (mean ± s .d . 43·3 ± 5·2 mm in standard length and 1·07 ± 0·37 g, n = 15) were released in each of three coastal areas: Chojagasaki, Arasaki and Jogashima with different geographical conditions, along Sagami Bay, Pacific coast of central Japan. Recapture efforts were made on 4, 11, 18, 40 and 55 days after the release. The stable isotope ratios, RNA:DNA ratio, stomach content mass (per body mass M_sc) and condition factor (K) of recaptured individuals were measured. The mean ± s .d . δ¹³C and δ¹⁵N values (n = 15) were ?18·3 ± 0·2‰ and 12·2 ± 0·2‰, respectively at the release. Wild Japanese flounder juveniles were captured only in Chojagasaki, and the δ¹³C and δ¹⁵N values (n = 6) were ?14·0 ± 0·4‰ and 13·2 ± 0·7‰, respectively; these values were considered to represent the wild diet. Nutritional conditions of the released and recaptured juveniles as determined by the RNA : DNA ratio, M_SC and K were indicated to be the best in Chojagasaki, in which the stable isotope ratios gradually shifted towards and reached the wild values within 40 days. This result along with stomach content analyses suggested that the released juveniles had acquired a wild feeding habit. In Arasaki and Jogashima, nutritional conditions of the recaptured juveniles were poorer, with no clear changes in the stable isotope ratios. Greatly varied stable isotope ratio values were observed in the juveniles recaptured in Chojagasaki 11 days after the release, ranging from the release levels to the wild levels. The extent of changes in the stable isotope ratios had a positive correlation to the RNA : DNA ratio and K of these juveniles (r = 0·87, n = 10 and r = 0·83, n = 18, respectively). The analyses of stable isotope ratios coupled with nutritional condition were considered to be an effective tool to examine post‐release feeding adaptation of Japanese flounder juveniles.     

In vitro culture of glochidia of the threatened freshwater mussel Unio crassus Philipsson 1788 – the dilution problem

We applied in vitro techniques in culturing glochidia of the thick-shelled river mussel Unio crassus, seriously threatened European species. Glochidia were freshly isolated from a gravid female. The sterile phase of the cultures was terminated at different time points to assess the optimal length of this phase. We imitated the process of juvenile excision from a fish host by diluting the culture with water at regular time intervals. The metamorphosed juveniles that survived until the end of the experiment and started growing their shells were observed for 24–27 days from the start of the culture in samples diluted for the first time between days 13 and 17. Long-lasting cultures usually became infected and died, whereas in those that were terminated too early, glochidia were unable to develop further in clean water. The transfer of juveniles from an artificial medium to pure water should be done gradually, through a series of dilutions, so that the larvae have the opportunity to feed on the diluted medium after metamorphosis. Only individuals with an active foot capable of operating outside the shell were ready to inhabit water and forage on solid food from the external environment.     

Mineral composition variation in the shells of freshwater mussel Anodonta woodiana at different growth stages

The levels of mineral element Na, Mg, K, Ca, Cr, Mn, Fe, Co, Ni, Cu, Zn, Se, Mo, Al, As, Ag, Cd, and Tl were quantified in the whole shells of the freshwater bivalve Anodonta woodiana at three different growth stages (i.e. J1 juveniles of 1 month old, J2 juveniles of 3.5 months old, and adults of 36 months old). The concentrations of Na and Al were different between different growth stages (p < 0.05). The highest Na concentrations (2715 ± 86 μg/g dry weight) were found in J2 juveniles. The highest Al concentrations (303.9 ± 5.95 μg/g dry weight) were found in J1 juveniles. Manganese concentrations (517.0 ± 47.98 μg/g dry weight) were significantly higher in J2 juveniles than in J1 juveniles (432.3 ± 9.87 μg/g dry weight) (p < 0.05). Copper concentrations (27.32 ± 0.15 μg/g dry weight) were significantly higher in J1 juveniles than in J2 juveniles (26.21 ± 0.86 μg/g dry weight) and adults (24.74 ± 1.43 μg/g dry weight) (p < 0.05). Burdens of Na, Ca, Mn, Fe, Co, Cu, Mo, Ag, and Tl were positively correlated with the shell length (p < 0.05). These findings can possibly contribute to an understanding of elemental requirements for shell growth and, hence, facilitate improvement of survival and growth rates during artificial mussel culture.     

Spatial and temporal variability in filament length of a toxic cyanobacterium (Anabaena affinis)

1. This study examines the distribution of Anabaena affinis filament lengths under natural conditions as a function of depth and season, and in the laboratory as a function of growth phase. Because Anabaena affinis is only toxic when consumed, both its filament length and position in the water column are important determinants of its potential impact on zooplankton populations. 2. Star Lake (Norwich, Vermont, U.S.A.), a natural, eutrophic pond, remained thermally stratified throughout the Anabaena bloom. Filament number and length differed significantly with both sampling date and water depth. Most filaments occurred at 0.5 m, particularly at the height of the bloom. Throughout the entire water column average filament length decreased from approximately 0.53 mm in May to 0.14 mm in July. The shortest filaments occurred at the 2.5 m depth. Filament length distributions (combined for all depths) for 29 May, 12 June and 3 July, corresponding to the beginning, middle and end of the bloom, respectively, differed significantly among the three dates. These patterns most likely reflect variable growth conditions, both during the season and in the water column. 3. In the laboratory, Anabaena filament length was affected by medium composition and growth phase. Filaments were significantly longer when grown in MBL than in ASM medium. Also, the average length of Anabaena filaments grown in MBL changed significantly as cultures aged; by day 13 filament length (2.01 ± 0.38 mm, mean ± SD) was twice that on day 0 (0.97 ± 0.71 mm). As cell concentration continued to increase, mean filament length gradually decreased.