LÁGMÖRKUN Á DÁNARTÍÐNI FYRIR HUMAR Í GEYMSLU TIL ÚTFLUTNINGAS Á LIFANDI MARKAÐ
(Improved Survival Of Lobsters Stored For Live Export)
Progress Report
Heather Philp MSc
Feb 2011
Contents
Summary 3
Report Introduction 3
Task 1 – Define the relationship between nutritional parameters and concentration of circulating protein 4
Introduction 4
Methods & materials 4
Results 6
Haemolymph Protein 7
Lobster weight 9
Hepatopancreas proximate composition 12
Discussion 14
Conclusion & Future Direction 15
Task 2 - Determine maximum storage time based on nutritional status 15
Introduction 15
Methods & materials 15
Results 16
Discussion 16
Future direction 16
Task 3 - Determine effects of nutritional status on transport survival rates 16
Introduction 16
Methods & materials 17
Results 17
Discussion 17
Future direction 17
Report Conclusions 18
References 18
Summary
All three of the experimental work packages have been started with WP1 being completed and WP2&3 are being halfway through. The preliminary results indicate that the strongest motivator of changes in haemolymph protein concentration is the moult cycle. This is very interesting since it is the first time such a relationship has been found in clawed lobsters and one of the few studies to determine this in crustaceans. Further, it appears that mobilisation of energy reserves during starvation begins with carbohydrate, then lipid and finally protein. This is also a new discovery for both this species and clawed lobsters in general. Samples are still being processed and when this has been completed a comprehensive analysis of the data will be carried out in order to complete WP4, a protocol for management of stored lobster stock in order to maximise survival. To date, survival of experimental animals up to four months has been good
Report Introduction
The aim of this project is to investigate the effect of extended storage on lobsters destined for live export. Previous work determined that for lobsters to be maintained in good health past two weeks, a closed system that re-circulated filtered water was necessary. However, during the course of the experiments, it was also found that up to one third of animals stored longer than three weeks died despite showing no obvious evidence of disease (unpublished data). It was theorised that nutritional status could be the reason for this high level of mortality.
In the American lobster industry, some companies test the blood protein level of animals upon intake as a measure of their physiological condition. Typically, a hand-held refractometer is used: a small drop of haemolymph is removed with a needle & syringe and placed upon the optical surface. When the instrument is directed towards a source of light, a system of lenses and prisms within cause the light to refract and a shadow line is cast upon the reticle where a scale allows a reading to be taken. Whilst mildly invasive, this non-lethal approach has facilitated improved management of stock and reduced mortalities during storage and transport.
Little research has been directed towards this aspect of crustacean health and condition in Nephrops although there is a rapidly expanding live industry for the species. The main reason for this is that to date, animals are usually sold and transported within a few weeks of capture. However, in Iceland access to the fishery is very weather-dependant with boats potentially being prevented from fishing for weeks at a time, especially in winter. This risks a situation where producers are only able to supply the market during the summer months when prices are depressed and miss the opportunity to sell during the winter when product value can be three times this.
We intend to evaluate the use of haemolymph refractometry as a means to assess the condition of lobsters. Further, we will explore the effect of extended storage on live lobsters in terms of mortality, taste and capacity to survive the stress of transport. The first part of the project focuses on identifying the relationship between haemolymph protein and other measures of condition including hepatopancreas stores of lipid, protein and glycogen, muscle protein and haemocyanin. Following this, a series of experiments will be carried out where animals stored for progressively longer periods of time are subjected to simulated transport covering a range of times.
The project has been divided into four tasks of which the first three involve data collection and experimentation. The final task comprises report writing and the publication of results. This mid-project report summarises the developments in the research so far. The task titles have been used as chapter headings under which a full description of the work carried out and findings made is given.
Task 1 – Define the relationship between nutritional parameters and concentration of circulating protein
Introduction
The life history of crustaceans is more complex than most vertebrates because the hard exoskeleton must regularly be shed and replaced with a new larger one; around these moments of ecdysis, foraging stops completely for extended periods of time. Further, the environment that many marine crustaceans inhabit is subject to great variation in food availability. Consequently, within the natural environment animals such as lobsters are exposed to, and are physiologically tolerant of, periods of starvation. This feature has been exploited by processors who catch, store and transport the animals, all without feeding. During two to three weeks storage period typical for Nephrops, a very small weight loss occurs which is measurable in grams and unlikely to affect the sale price. However, the mobilisation of reserves is a finite process after which the animal may either die or be sufficiently weakened as to not survive the stress of transportation to the market.
Several species have been subjected to controlled starvation in order to elucidate the mechanisms of reserve mobilisation. Interestingly, a variety of responses have been observed which are not only species-specific but developmental stage-specific. For example, Spiny lobster larvae were found to catabolise more lipids than carbohydrates and proteins in stages II, IV and VI than other stages (Ritar et al., 2003). Early work on Nephrops by Dall (1981) focused on lipid storage and metabolism where it was found that the hepatopancreas formed the main storage site. Lipids levels did not decrease in 5 weeks of storage, indicating that another source of energy was being utilised preferentially. Baden et al (1994) found that hepatopancreas glycogen reduced to 3% of the original value in Nephrops starved for 7 months. Finally, a recent study by Mente (2010) compared protein metabolism and free amino acid accumulation between two different diets in cultured Nephrops.
The current study represents the first dedicated investigation into the metabolism of storage reserves in the species Nephrops norvegicus. Further, the aim is to determine the relationship between the nutritional parameters and the concentration of haemolymph protein. This approach was first utilised by Stewart in 1967 who proposed that lobster haemolymph protein levels directly relate to their diet. A decade later, Leavitt & Bayer (1977) used a hand-held refractometer to measure protein in lobster haemolymph in the field. Since then, the practice has been almost universally adopted by the American lobster industry as a means to quickly and easily measure vitality (Ozbay & Riley, 2002).
Methods & materials
The initial months of the project were spent preparing the facilities and acquiring the necessary equipment. The vessel intended for use, the Hafro boat Fredrik Jesson, required a series of modifications including the addition of a new winch arm to enable the lobster traps to be hauled from the side rather than the back. The filtration system which cleans and circulates the water in the onshore storage tanks needed replacement parts from the UK to ensure its effective operation for the duration of the project.
During September and October 2010, several fishing trips were undertaken in which male lobsters with a carapace length between 50 and 65 mm were selected for use in the study (Figure 1). 180 animals were randomly chosen to be used in Task 1 and arbitrarily allocated to one of 9 groups of 20 (8 weeks storage + one control group). The carapace length of each individual was recorded along with weight, moult stage and any other noticeable characteristics (for example shell hardness, claw damage). A haemolymph sample of 1ml was drawn and the protein level measured by refractometer (Figure 2). The remaining haemolymph (approximately 0.8ml) was frozen in a labelled eppendorf for later haemocyanin analysis. The lobster was placed into a pre-marked ‘tube’ in 40-space crate which when full was placed to the storage tank.
Figures 1-3: Catching the lobsters (top), using the refractometer to measure haemolymph protein (centre) and the crates in which the lobsters are stored for the duration of the experiment (bottom).
Starting at a storage time of 0 Weeks, one group per week were removed and a full spectrum of analyses performed on each individual. The weight of the animal was recorded and a large sample (2-3ml) of haemolymph drawn. A small drop was placed on the optical surface of the refractometer and the protein level recorded. The remainder was divided between two eppendorfs for subsequent haemocyanin and total protein analysis. The lobster head was separated from the body and the exposed hepatopancreas removed and weighed after being blotted dry. Both the hepatopancreas and tail were placed in pre-marked ziplock bags and frozen to be analysed later.
In the laboratory, the hepatopancreas was divided into three parts for lipid, protein and carbohydrate analysis. All sections were dried to a constant weight and the water content calculated. The lipid extraction was performed using the petroleum ether method; briefly, ether was passed through the sample using distillation equipment which moved only the lipid portion from the sample. Protein analysis followed the Kjeldahl method which uses the amount of reduced nitrogen liberated from the sample by heating with sulphuric acid to calculate the protein content of the sample. Glycogen concentration within the sample was measured using anthrone reagent which turns from yellow to blue-green when heated in the presence of sugars. The colour change was measured spectrophotometrically and compared against a calibration curve prepared using glucose as a standard.
Haemocyanin was measured using the method of Baden et al (2003): the defrosted sample was oxygenated by the addition of distilled water and the absorbance measured at 335nm by spectrophotometer. The extinction coefficient of Nickerson & Holte (1971) was used to calculate the concentration of haemocyanin. Disposable cuvettes with a 1cm pathlength and 1.5ml maximum capacity were used throughout the study. Haemocyanin concentration 1 (measured upon intake) has been analysed but Haemocyanin 2 (taken when animal was sacrificed) is still outstanding. Analysis of the lipid and protein content was completed as far as Week 7 (from 8 weeks) whilst carbohydrate testing was completed as far as Week 2.
Results
Haemolymph Protein
Haemolymph protein concentration was highly variable ranging between 3.4 and 16 g/dl with no discernable pattern across the size classes measured (Figures 4 & 5). However, the histogram indicated that whilst most of the data followed an approximately normal distribution, a separate peak at very low protein levels (less than 5 g/dl) was present. When moult cycle stage was introduced as an independent variable, a strong correlation was detected (Figure 6). The difference between the haemolymph concentration upon intake and at the point of sacrifice increased as storage time increased (Figure 7), which was statistically significant between weeks 4 & 5, and 5 & all subsequent weeks ( χ28= 109.362; p<0.005). In the first week of storage, reduction in concentration was found predominantly in the lobsters with the highest intake concentration, i.e. those at the most advanced stage in the moult cycle (Figure 8). However, as the experiment progressed the reduction was found across all other intake concentrations too and this pattern was not detected again.
Figure 4. Histogram of haemolymph protein concentration measured upon intake
Figure 5. Haemolymph protein concentration plotted against carapace length
Figure 6. Protein concentration grouped by moult stage
Figure 7. Difference between intake and final haemolymph protein concentration
Figure 8. Difference between final and initial protein concentration against intake protein concentration.
Lobster weight
During the first week, a small amount of weight was lost from most of the lobsters (Figure 9). Interestingly, during the second week of storage many animals were found to have actually gained weight relative to their intake measurement (Figure 10). However, by the third week a decrease was observed and although it appeared that the weight loss was steadily decreasing over the remainder of the experiment, this was found to be due to normal variation. It is important to note that although the weight loss from Week 3 onwards relative to the previous weeks is statistically significant (χ28 = 112.8; p < 0.005), it is only a matter of grams and thus unlikely to affect the market value.
The relative contribution from the hepatopancreas as calculated by the hepatosomatic index (HSI) decreased during the course of the experiment. During the first week of storage the relative weight was maintained but the following week it was found to decrease significantly (F (1,155) = 38.3; p<0.0005) and again in the final week (F (1,155) = 38.3; p<0.0005).
Figure 9. Change in weight between intake and final measurements during the experiment
Figure 10. The range of differences at each storage time during the experiment