Volume 51

RESEARCH GRANT REPORT

Antioxidants in Molluscan Larvae

Rachel Przeslawski

Rach...@ga.gov.au

Stony Brook University, Department of Ecology & Evolution, Stony Brook NY 11794-5245, USA

Larvae are considered the most vulnerable life stage of most organisms, and their survival and fitness are thus critical to the success of future populations. Molluscan larvae can be negatively affected by various environmental stressors, including temperature, salinity, ultraviolet radiation (UVR), and pollutants, which may synergistically affect larvae. However, these effects may be sublethal and therefore difficult to detect in experimental periods covering only larval stages. Other measures of stress, such as antioxidant potential, may provide accurate indications of stress (Regoli 2000) but have yet to be tested on molluscan larvae.

UVR causes a range of negative effects in both terrestrial and aquatic ecosystems. In particular, UV-B (280-315nm) is strongly absorbed by nucleic acids and proteins, resulting in DNA damage and impairment of biological function. UVR exposure also triggers production of reactive oxygen species (ROS) which can react with other molecules, producing cytotoxic photo-products and deleteriously affecting biological processes (Vincent & Neale 2000). These effects may be exacerbated by other stressors, including temperature.

Antioxidants can limit damage done by ROS by detoxifying them or the resulting cytotoxic compounds. However, the antioxidant potential of molluscan larvae has not yet been investigated. Most antioxidant assays focus on one or few related enzymes, making overall protection afforded to an organism difficult to assess. Commercially available assays testing for total antioxidant potential are now widely available.  These assays are based on the reduction of Cu⁺⁺ to Cu⁺ by antioxidants in a supplied standard, and a colorimetric test and calibration curve from a uric acid standard are then used to quantify Cu⁺.

In this study, I conducted three experiments to test whether the antioxidant potential of hard clam larvae and its algal food varies with UV-induced stress, reflecting the use of antioxidants to protect against ROS and cytotoxic compounds. The hard clam (Mercenaria mercenaria) is one of the most historically important fisheries in the eastern United States, but unsustainable harvesting led to its collapse in the 1980s, negatively impacting not only the regional economy, but also community structure and ecosystem dynamics (McHugh 1991). Current research is heavily focused on ecosystem restoration and the reintroduction of the hard clam into this region but is hindered by lack of sufficient knowledge about larval responses to stressors.

In all experiments, larvae were fed Isochrysis galbana, an alga rich in long-chain PUFAs and triglycerols on which hard clam larvae have been shown to thrive (Brown et al. 1997, Przeslawski et al. 2008). Spectral treatments were achieved using cutoff filters under a mercury fluorescent lamp (10.6 +/- 1.1umol m^-2 sec^-1 at 250-400 nm) and a standard fluorescent light (2.6 +/- 0.4 umol m^-2 sec^-1at 400-800 nm). In the first experiment, six containers each with 100 four-day old veligers were exposed to either full spectrum or darkness for six hours at 22°C. Unfortunately no differences were detected between the larval samples and the blank samples, indicating that more larvae were needed in each sample to ensure the assay was sensitive enough to quantify antioxidant potential. In the second experiment, four-day old larvae were cultured under various densities (100, 250, 500, 1000, 10,000, 50,000 larvae per litre) and spectral treatments (full spectrum, UV-blocked, dark) for three days at 22°C and 14 h light: 10 h dark cycle. Again, no differences were detected among the larval samples. In the third experiment, Isochrysis galbana was cultured under a combination of spectral treatments (full spectrum, UV-blocked, dark) and exposure times (5 or 10 hours). Algal cells were sonicated and assayed for antioxidant potential (2 x 10⁷ cells per treatment). Contrary to predictions, algae in full spectrum treatments did not have a significantly lower antioxidant potential than UV-blocked and dark treatments (df = 2, F = 1.0937, p = 0.3662) (Figure 1). In addition, time of spectral exposure did not significantly affect antioxidant potential (df = 2, F = 1.0900, p = 0.3171).

 

Fig. 1: The effects of spectral treatments on antioxidant potential of Isochrysis galbana after various exposure times. n = 3. Error bars are standard error means.

 

No effects of UVR were detected in M. mercenaria larvae or its food Isochrysis galbanna, suggesting that for this assay, larval sample size should exceed 50,000 and I. galbanna sample size should exceed 2 x 10⁷ cells. Alternatively, M. mercenaria larvae or I. galbanna may simply not use antioxidants in response to levels of UVR used in this study. Results from this study indicate that the total antioxidant potential is not an appropriate gauge of stress in marine larvae or its food, at least at larval and cell densities used here. Further studies using higher numbers of larvae are highly recommended but were unfortunately unable to be conducted in the current study due to the difficulty of spawning hard clam adults and obtaining high densities of viable trochophores. Antioxidant assays have the potential to become a useful tool in larval ecology and malacology, and they may eventually help managers and researchers estimate tolerance of hard clam larvae to abiotic stressors, thereby improving success of fisheries management and restoration ecology. Nevertheless, results from the current study suggest that these assays may only be of use with species from which high densities of larvae can be reliably obtained.

Brown MR, Jeffrey SW, Volkman JK, Dunstan GA (1997) Nutritional properties of microalgae for mariculture. Aquaculture 151:315-331

McHugh JL (1991) The hard clam fishery past and present. In: Schubel JR, Bell TM, Catrter HH (eds) The Great South Bay. SUNY Press, New York, p 107

Przeslawski R, Bourdeau PE, Doall MH, Pan J, Perino L, Padilla DK (2008) The effects of a harmful alga on bivalve larval lipid stores. Harmful Algae doi:10.1016/j.hal.2008.04.003

Regoli F (2000) Total oxyradical scavenging capacity (TOSC) in polluted and translocated mussels: a predictive biomarker of oxidative stress. Aquatic Toxicology 50:351-361

Vincent WF, Neale P (2000) Mechanisms of damage to aquatic organisms. In: de Mora S, Demers S, Vernet M (eds) Effects of UV Radiation in the Marine Environment. Cambridge University Press, Cambridge, p 149-176

 

Fig. 2: Mercenaria mercenaria larvae seven days after fertilisation.
The small spheres visible inside the veliger are Isochrysis galbana.