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Application and validation of the lipofuscin method in the assessment of crustacean age


Lipofuscin, which is a fluorescent pigment detectable in crustacean neural tissue is correlated with age in laboratory populations of Homarus, Nephrops and Penaeus. In natural populations where age is unknown it is correlated with body size. In lipofuscin frequency distributions modes are present in some instances and may indicate the age structure of these populations. Lipofuscin accumulation is affected by temperature acting through its effect on metabolic rate. Some evidence has also be found that metabolic rate is affected by population density and size structure. Ontogenetic and sex specific differences occur in the levels of lipid peroxidation and antioxidant activity in Parapenaeus. These properties of crustacean neural tissue may affect the accumulation of lipofuscin and the possibility of using it as an index of age.
Measurement of lipofuscin in laboratory and natural populations

1. Lipofuscin can be detected and quantified in all species being studied. 2. In cultured populations of known age (H. americanus, H. gammarus, P. japonicus) there is a correlation between age and the percentage of the olfactory cell mass of the brain occupied by lipofuscin. The rate of accumulation is not linearly related to age but slows down in older animals (see O'Donovan and Tully 1996, Wahle et al 1996,). In P. japonicus females accumulate a significantly (1.5-2 fold) higher amount of lipofuscin than males 3. Application of a lipofuscin-age model for N. norvegicus to a population in the Irish Sea predicted a more restricted age distribution and higher mortality than currently predicted using standard fisheries length based assessment. 4. Lipofuscin frequency distributions for N. norvegicus in the Irish Sea do not show clear modes that could represent age groups. However, in juvenile cohorts of H. americanus modes are apparent in the small number of samples analysed to date.

Environmental control of growth, metabolic rate and lipofuscin

1. Differences in metabolic rate, as measured by citrate synthase activity, were detected in N. norvegicus in the Irish Sea, which may be related to population density or sediment type.
2. Density and temperature dependent growth and survival were demonstrated in juvenile H. americanus. Citrate synthase activity is being measured to indicate if this is mediated through metabolic rate differences
3. In the laboratory the relative size of upstream individuals of H. gammarus has been shown to affect growth rate and oxygen consumption of downstream target animals.
4. Both population density and structure may therefore play a role in controlling metabolic rate in decapods.

Ontogenetic changes in lipid biochemistry, lipid peroxidation and antioxidant activity in the crustacean brain

Age related changes in lipid profile, lipid peroxidation and lipid soluble fluorescent products (LSFPs) have been studied in detail in A. antennatus and P. Iongirostris captured from the wild, and in cultured P. japonicus. In P. Iongirostris substantial differences have been shown in the oxidation status of males and females. Vitamin E contents are higher in male brains than in females. Also peroxidation activity (expressed as malondialdehyde per gram of brain weight) and intensity of lipid soluble fluorescent products decreases with increasing size in males but not in females. These results may indicate a high protective effects of Vitamin E in males. These differences between male and female were not detected in P. japonicus fed on artificial diets.


Data produced to date indicates that lipofuscin can be quantified in all species concerned and that it is correlated with age, in species for which the age is known, or body size in species where the age in unknown. In laboratory experiments metabolic rate and growth is related to the size of neighbouring animals and in the field density dependent differences in metabolic rate and growth has been shown experimentally. Biochemical data indicate important differences in anti-oxidant activity in male and female crustaceans and in relation to age. These differences and different rates of accumulation of lipofuscin in males and females may indicate sex specific differences in metabolic rates, growth rates and susceptibility to oxidative stress.
Final interpretation of the data and evaluation of the lipofuscin method as an index of crustacean age will be included in the final project report to be issued early in 1999.


This project is entirely funded under the FAIR 4th framework programme


O' Donovan, V. & Tully, O., 1996. Lipofusion (age pigment) as an index of crustacean age: correlation with age, temperature and body size in cultured juvenile Homarus gammarus (L.). J. Exp. Mar. Biol. Ecol., Vol. 207, pp.1-14.

Sheehy, M.R.J. 1989. Crustacean brain lipofuscin: an examination of the morphological pigment in the freshwater crayElsh, Cherax cuspidatus. J. Crust. Biol., Vol. 9, pp. 387-391.

Sheehy, M.R.J. 1990. Individual variation in and the effect of rearing temperature and body size on the concentration of fluorescent morphological lipofuscin in the brains of freshwater crayfish, Cherax cuspidatus (Crustacea: Parastacidae). Comp. Biochem. Physiol., Vol. 96a, pp. 281-286.

Sheehy, M.R.J. Greenwood, J.G. & Fielder, D.R. 1995. Lipofuscin as a record of rate of living in an aquatic poikilotherm. J. Gerontol., Vol. 50B, pp. 327-336.

Tully, O., Fletcher, D., O'Donovan, V, Howie, D.I.D. and Jones, D.A. (1995). Use of the age pigrnent lipofuscin as an indicator of age in Nephrops and Homarus. Final Report of Project FAR MA 3 651, DGXIV-C-2, Rue de al Loi 200, B1049, Brussels.

Wahle, R.A. Tully, O. & O'Donovan, V., 1996. Lipofuscin as an indicator of age in crustaceans: analysis of the pigrnent in the Amerian lobster Homarus americanus Milne Edwards. Mar. Ecol. Prog. Ser., Vol. 138, pp. 117-123

Current management of exploited European crustacean populations is hampered because of the general lack of information on population age structure. Previous work on Cherax spp (Sheehy 1989, 1990, Sheehy et al 1995), Homarus (Wahle et al 1996, O'Donovan and Tully, 1996) and Nephrops (Tully et al 1995) has shown that the amount of the pigment lipofuscin, which can be observed in histological sections of brain tissue, is correlated with physiological age under experimental conditions. The first objective of the project is to assess if this pigment is sufficiently related to chronological age to be used in estimation of age structure of natural populations. The technique is being applied to various natural populations of Nephrops norvegicus, Homarus americanus, Parapenaeus longirostris and Aristeus antennatus and to Penaeus japonicus and Homarus gammarus of known age which are cultured under controlled conditions.
Various factors controlling metabolic rate in crustaceans may affect the rate of deposition of the pigment as it appears to be formed as a result of oxidative stress and in particular lipid peroxidation in the brain. Although there is a general correlation between lipofuscin and age there can be important and unexplained differences in pigment content between individuals of the same age maintained in similar environmental conditions (Tully et al 1995). The second objective of the project is, therefore, to identify internal and external factors controlling the rate of lipofuscin deposition in crustacean neural tissue through their effects on organism metabolic rate and antioxidant activity.


Brains are dissected out and fixed in 10% formal saline and embedded in paraffin wax or plastic. Sections of the brain are cut, air dried, cleared in xylene and mounted in DPX. The tissue is viewed under epi-fluorescence (x 20 or x 40) using a B2A (450 - 490 nm) excitation filter. Lipofuscin granules are visible as bright yellow fluorescing granules. Images of the olfactory cell mass of the brain are captured via an integrating CCD camera and viewed in pseudocolour. Image analysis software is then used to process the image and to quantify the amount of lipofuscin in the tissue sections. The % area of tissue occupied by lipofuscin, the number of granules per unit area of tissue and the average size of the granules is estimated.
Two approaches are followed in assessment of the pigment as an index of age in natural populations. Lipofuscin-age-temperature models developed in laboratory populations of known age are used to predict the age of wild individuals based on their lipofuscin content and secondly lipofuscin frequency distributions are constructed and analysed using methods developed for extracting age information from body size distributions.
Various laboratory and field based experiments are conducted to assess the role of temperature and population density on organism metabolic rate and lipofuscin accumulation.
Characterisation of ontogenetic changes in the lipid biochemistry, lipid peroxidation and antioxidant enzyme activity in the crustacean brain is an integral part of the process of assessment of the lipofuscin technique as an index of age in decapod crustaceans in this project.

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East Theatre, Trinity College , College Green
2 Dublin

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