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
Protein (final) - Protein (intake)
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
5.0
7.0
9.0
11.0
13.0
15.0
Haemolymph protein concentration (g/dl)
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
Figure 11. HSI measured at various storage times
Hepatopancreas proximate composition
During the eight weeks of storage, the hepatopancreas tissue dissected from individual lobsters was
assessed for water content and protein & lipid concentration by dry weight. The water content was
consistent throughout the experiment until week 7 (Figure 12) when it increased significantly (F(1,124)
= 8.82; p<0.005). By comparison, the concentration of protein in dry tissue increased progressively
over the duration of storage (Figure 13). However, only the final week of analysis (Week 7) was
found to be significantly different from tissue sampled upon lobster intake (F (1,131) =10.77; p<0.005).
After increasing in concentration over the first three weeks of storage, the lipid component of the
tissue began to reduce, significantly so at week 7 (F(1,118) =7.602; p<0.05).
Figure 12. Water content of dissected hepatopancreas tissue at various storage times
Figure 13. Protein concentration in dry hepatopancreas tissue at various storage times
Figure 14. Lipid concentration in dry hepatopancreas tissue at various storage times
Discussion
The samples from this work package have been processed as far as possible within the time
available. However, the carbohydrate content of the hepatopancreas and the proximate
composition of all tissues from the final week have to be completed. Nevertheless, preliminary data
analysis has revealed some extremely exciting results. In particular, the relationship between the
concentration of circulating protein in the haemolymph and the moult cycle is a major breakthrough.
The inherent variability in protein measurements from wild-caught lobsters of various species has
long been recognised. In American lobsters, it was suggested almost four decades ago that nutritive
status could be inferred from the level of protein. However, in only a handful of studies since then
have the authors pursued an understanding of the underlying physiology. Generally, it is assumed
that inter-individual differences in nutritive status and thus protein concentration can be explained
by the challenging and highly stochastic environment in which members of the same population live.
The work from this project has revealed that in actual fact moving through the moult cycle is
predominant motivator behind changes in haemolymph protein. This in intuitive: following a single
ecdysis, a lobster’s energy reserves have been depleted but it gradually replaces them, moving from
post- to inter-moult. This phase is associated with the accumulation of new reserves and when this
reaches a maximum for the particular shell size, it becomes necessary to move to pre-moult in
preparation for the next ecdysis. Thus, a lobster’s nutritive state is most likely to be closely linked to
the position in the moult cycle.
One of the other important discoveries is that although many of the lobsters in the experiment were
found to be at an advanced stage in the moult cycle (i.e. in pre-moult), only one actually moulted
whilst in captivity. The decrease in protein concentration over the duration of the experiment
suggests that under starvation conditions, the depletion of accumulated reserves causes the
advancement of moulting to halt. This is very important because of animals are to be stored for
several months it is very likely that without this mechanisms, moulting related mortality would
become a considerable problem to producers.
The results of the proximate composition analysis suggest that mechanisms for reserve utilisation
initially involve carbohydrate mobilisation. During the course of the experiment, there was an
overall weight loss (although small relative to the weight of the animal) and a concurrent reduction
in HSI, i.e. the relative contribution by the hepatopancreas to the overall weight decreased. For the
first half of the storage period, both lipid and protein showed an increase in concentration per gram
of dry weight hepatopancreas tissue. From this, we can infer that these components were not the
causative factors behind the progressive weight loss from the organ. It is therefore likely that stored
glycogen is the primary energy source during the first phase of starvation; this will be confirmed
when the carbohydrate analyses have been completed.
This is very interesting since previous research has found that carbohydrates are a well-utilised
resource in shrimps but poorly digested by lobsters (Verri et al, 2001). However, many studies have
focused on dietary requirements for cultured animals and research dedicated to the effects of
starvation in lobsters is scarce. It is very likely that lobsters also use carbohydrate pathways when
mobilising reserves but absorb carbohydrates poorly from feedstuffs. Studies such as ours which
focus on the animals’ physiology under this time of stress can be expected to reveal considerable
new knowledge on the mechanisms of starvation.
Conclusion & Future Direction
Preliminary analysis showed that the mechanisms of starvation control follow a path similar to
shrimp species such as Peneus vannemei rather than that of more closely related lobster species.
However, we feel that this is because this element of crustacean physiology is somewhat poorly
understood and our study will make a valuable contribution to the field. Further, the discovery that
almost all of the variability in haemolymph protein concentration can be explained by moult cycle
position is both novel and likely to impact on the field of crustacean research very heavily. The most
significant finding for the lobster industry is that storing the lobsters appears to have the unexpected
effect of arresting moult cycle progression, a feature that is essential for extended storage.
The next stage of this work package is to complete the both the sample processing and data analyses
in order to fully understand the relationship between the nutritional parameters and haemolymph
protein. It is intended that this be undertaken in the next four weeks and the results publicised in a
scientific journal. The full interpretation of the information is more complicated than initially
thought, mostly due to the paucity of studies focussing on either protein fluctuations throughout the
moult or the mechanism of reserve mobilisation in starved lobsters. To aid this, the results will be
presented at the four-yearly International Lobster Biology Conference being held in Bergen in June
with advice being sought from the other attendees.
Task 2 - Determine maximum storage time based on nutritional status
Introduction
The aim of this work package is to perform an extended storage trial over a period of nine months in
order to monitor the capacity for this species to survive prolonged starvation. Anecdotal evidence
suggests that Nephrops can be maintained without food for several months (Fisheries Research
Services, UK) however no research has been formally undertaken to verify this. This experiment will
fully explore the capacity of Icelandic lobsters to survive storage based on their condition upon
intake.
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. 180 animals were
randomly chosen to be used in Task 2 and arbitrarily allocated to one of 9 groups of 20 (9 month
storage groups). 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. 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.
Starting at a storage time of one month, one group per week was 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. Samples
have been taken from animals stored for months 1-4 and the remaining animals are still in the
storage tanks. All samples have been marked and frozen at -20oC until they can be processed.
Results
No results are available yet.
Discussion
Future direction
Over the next several months, the experiment will be completed by storing lobsters for up to nine
months. The full spectrum of analyses will be performed on the samples and the data analysed.
Task 3 - Determine effects of nutritional status on transport survival rates
Introduction
The aim of this work package is to perform a series of simulated transport experiments on animals
that have been stored for up to nine months. The intention is to determine whether animals that
have survived prolonged starvation also have the capacity to survive the rigour of aerial transport.
In Iceland it is necessary to dispatch live lobsters by air which means that they must be emersed for
a minimum of 12 hours. In animals stored for three weeks, this was possible without decreasing
survival but no information exists on the effects of emersion on animals stored for longer.
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. 540 animals were
randomly chosen to be used in Task 2 and arbitrarily allocated to one of 9 groups of 60 (9 monthly
storage groups). 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. 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.
Starting at a storage time of one month, one group per month was removed and subjected to
simulated transport. Briefly, they were packed into polystyrene boxes following industry protocol
and placed to a temperature controlled room. At times of 24, 36 and 48 hours one set of twenty
animals was removed and assessed for vigour. The extent of idiopathic muscle necrosis (a pathology
affecting animals exposed to air for extended periods of time) was recorded using a previously
developed index. Blood parameters including protein, haemocyanin, pH, lactate and ammonia were
measured.
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. Samples
have been taken from animals stored for months 1-4 and the remaining animals are still in the
storage tanks. All samples have been marked and frozen at -20oC until they can be processed.
Results
No results are available yet.
Discussion
Future direction
Over the next several months, the experiment will be completed by storing lobsters for up to nine
months. The full spectrum of analyses will be performed on the samples and the data analysed.
Report Conclusions
All three of the experimental tasks are underway: the sampling phase of Task 1 has been completed
and the final laboratory work is being undertaken, Tasks 2 & 3 are halfway through with all
experimental animals from the latter half of the experiment still in the tanks and samples from those
from the first half in cold storage until they can be processed in the lab. The preliminary results are
extremely interesting and likely to benefit both the industry and the scientific community. The
project was slightly delayed at the start whilst preparations were being made in the holding facility
however everything is progressing rapidly now and the timescale outlined in the original application
is still correct.
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