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Eggs and hatchlings of the freshwater crayfish,
ž
/
marron Cherax tenuimanus , can be successfully
incubated artificially
Mark Henryon
a,), Ian W. Purvis
ba
Danish Institute of Agricultural Sciences, Department of Breeding and Genetics, Research Centre Foulum, P.O. Box 50, 8830 Tjele, Denmark
b
CSIRO DiÕision of Animal Production, Pastoral Research Laboratory, Armidale NSW 2350, Australia
Accepted 6 September 1999
Abstract
The objective of this study was to test whether marron eggs and hatchlings can be incubated artificially with high levels of survival. Marron eggs were collected from 30 gravid females. The eggs from each female were divided into two replicate groups, and each replicate group was incubated separately within an artificial incubator until the eggs had hatched and developed into independent juveniles. Eighty-nine percent of the eggs hatched and developed into independent juveniles, while the variation in survival between replicate female groups was not significant. These results demonstrate that the artificial incubator was a suitable replacement for the maternal care provided by the female marron after spawning, and that a uniform environment existed within the incubator. The success of the artificial incubation technique enables experiments to be carried out on eggs and hatchlings independent of the females, under a common and controlled environment, and with many separate treatment groups.q2000 Elsevier Science B.V. All rights reserved.
Keywords: Cherax tenuimanus; Marron; Crayfish; Eggs; Hatchlings; Incubation
1. Introduction
Artificial incubation of crayfish eggs and hatchlings involves the collection of fertilised eggs from gravid females and the incubation of these eggs in an artificial incubator where they hatch and develop into independent juveniles. In principle, the
)Corresponding author. Tel.:q45-89-99-1220; fax:q45-89-99-1300; e-mail: [email protected]
0044-8486r00r$ - see front matterq2000 Elsevier Science B.V. All rights reserved.
Ž .
technique is relatively simple as all endogenous contributions to the eggs and hatchlings
Ži.e., yolk and cytoplasmic material. are made during oogenesis and fertilisation
ŽAnderson, 1982 , and the artificial incubator need only to replace the maternal care.
Ž
provided by the females after spawning i.e., aeration, protection from predators, and .
removal of dead eggs and hatchlings . The eggs and hatchlings of the freshwater
w Ž .x
crayfish, marron Cherax tenuimanus Decapoda: Parastacidae , appear to be particu-larly receptive to artificial incubation. Specifically, marron eggs and hatchlings have a simple development, void of the problems that have restricted attempts to artificially incubate the eggs and hatchlings of other crayfish species. Many of these attempts have
Ž . Ž
been with Astacidea species and have resulted in poor survival 0–70% e.g., Mason,
1977; Rhodes, 1981; Carral et al., 1988; Carral et al., 1992; Matthews and Reynolds, .
1995; Perez et al., 1998; Perez et al., 1999 . This was presumably because the eggs of
´
´
Ž .
these species have a long development 7–9 months , including a period of winter diapause. The long development has the potential to increase the risk of infection and mechanical damage during artificial incubation, while development during the period of winter diapause requires a specific thermal regime, conditions for which have been difficult to reproduce artificially. By comparison, the eggs and hatchlings of marron
Ž .
have a short development 3–4 months that lacks a period of diapause. Other than favourable water temperatures, their development does not require specific environmen-tal conditions, suggesting that they can be artificially incubated with high levels of survival.
In this study, marron eggs were collected from gravid females and incubated in an artificial incubator developed for marron eggs and hatchlings. The objective was to test whether marron eggs and hatchlings can be incubated artificially with high levels of survival.
2. Materials and methods
2.1. Marron eggs
Marron eggs were derived from matings between broodstock captured from a location
Ž X Y X Y
.
on the Deep River 34849 30 S 116834 36 E , matings between broodstock captured
Ž X X Y
.
from a location on the Donnelly River 34820 S 115850 06 E , and crossbred matings
Ž
between broodstock from both of these river locations i.e., the eggs were either
purebred stock from the Deep or Donnelly Rivers, or Deep=Donnelly River crossbred
.
stock . The Deep and Donnelly Rivers are two major river systems in the south-west of
Ž .
Australia. The broodstock were captured from the rivers in autumn April and then maintained in an experimental pond, located 50 km north-east of Perth, Australia, until
Ž .
they had bred during the following spring breeding season October .
2.2. Experimental design
Eggs were collected from 30 gravid females. The eggs from each female were divided into two replicate groups of equal number and each replicate group was allocated to a separate ‘incubation cell’ within the artificial incubator. The eggs were maintained in the incubator until they had hatched and developed into independent juveniles. Each replicate group contained between 25–50 eggs. The water in the incubator was maintained at 20"0.58C. From a survival perspective, 208C was within
Ž .
the optimal temperature range Henryon, 1995 .
When collected from the females, the eggs were between the 0% and 65% stages of
Ž .
egg development, as described for C. destructor Decapoda: Parastacidae by Sandeman
Ž .
and Sandeman 1991 . Stage of development varied between female groups, but was synchronised within female groups.
2.3. Artificial incubator
The artificial incubator was maintained at the Faculty of Agriculture, University of Western Australia, Perth. It consisted of cylindrical incubation cells that were
sub-Ž . Ž .
merged vertically in a 48-l bath of water 80=50=12 cm Fig. 1a . The eggs were
incubated in the cells, and held in suspension by a continuous flow of water that flowed up from the bottom of each cell. When the eggs in each cell hatched, a piece of nylon
Ž .
cloth 10=2 cm was provided as support for the hatchlings.
Each incubation cell was constructed using the barrel of a ‘50-ml’ plastic syringe
Ž w . Ž .
with a catheter tip Terumo , Australia Fig. 1b,c . The barrel had a total volume of 75
Ž .
ml. A piece of polythene mesh 1=0.8 mm aperture was placed across the internal
diameter of the catheter tip, and across the open end of the barrel, to confine the eggs and hatchlings to the cell. The mesh placed across the internal diameter of the catheter tip was held in position by a piece of hollow plastic tube, while the mesh placed across the open end of the barrel was held in position by a hollow rubber stopper. A plastic ring, the size of the internal diameter of the barrel, was placed at the internal base of the barrel to prevent the eggs from becoming trapped around the base.
The incubation cells were positioned within the incubator along a closed network of
Ž .
12 mm diameter polythene pipe that lay at the bottom of the water bath Fig. 1b . Each cell was fastened to the pipe with its catheter tip inserted into one of a series of holes made along the pipe network. In turn, the network of pipe was connected to a submerged pump, which continuously cycled water from the bath, through the pipe network, into the cells, and back into the water bath. However, a portion of the water from the pump was by-passed through a biological filter before returning to the water
Ž
bath. The biological filter consisted of a plastic container 20 cm diameter=25 cm
.
height that was filled with four 5-cm layers of substrate. The bottom layer was fine
Ž . Ž .
sand 20–60mm diameter , which was covered by a layer of coarse sand 0.1–1 mm ,
Ž . Ž .
Throughout the incubation period, the water in the incubator was saturated with
Ž .
oxygen, had a very low level of ammonia nitrogen less than 0.1 ppm , and a pH between 6.0 and 7.8.
2.4. SurÕiÕal measurements
Survival for the whole development period was assessed for each replicate female group as the proportion of eggs that hatched and developed into independent juveniles.
Ž . Ž .
Fig. 1. Artificial incubator used to incubate marron eggs and hatchlings. a Artificial incubator. b Incubation
Ž .
Ž .
Fig. 1 continued .
Survival during the egg development phase was measured as the proportion of eggs that
Ž .
hatched, whilst survival during the two stages of hatchling development Stages I and II were measured as the proportion of hatchlings alive at the end of each stage relative to the number of hatchlings alive at the beginning of the stage.
Survival for the whole development period, and the individual phases of develop-ment, are presented as weighted means of the survivals for each replicate female group. The weights were the number of eggs or hatchlings in each replicate group.
Variation in survival between replicate groups within female groups was assessed
w Ž .
using the PROBIT procedure from the SAS System SAS Institute, 1988 . Survival
was assumed to be a discrete characteristic with an underlying liability, such that
Ž . Ž . Ž .
SisF l , where S was either the survival for i the whole development period, iii i Ž .
during the egg development phase, or iii during the two stages of hatchling develop-ment. The variable, l , was the underlying liability of S , logistically distributed withi i
mean zero and variancep2r3. The models fitted included the fixed effects of female
group and replicate group nested within female group.
2.5. DeÕelopment time of the eggs and hatchlings
independent juveniles. Mean development time was also assessed for the egg
develop-Ž .
ment phase, and the two stages of hatchling development Stages I and II . However, because the eggs were between the 0% and 65% stages of development when collected from the females, the length of time assessed for the egg development phase and, in turn, the whole development period does not represent the time from spawning.
The length of the whole development period and the individual phases of develop-ment are presented as weighted means of the developdevelop-ment times for each replicate female group. The weights were the number of eggs or hatchlings in each replicate group.
3. Results
3.1. SurÕiÕal of the eggs and hatchlings
Eighty-nine percent of the eggs collected from the females and incubated artificially,
Ž .
hatched and developed into independent juveniles Table 1 . Survival during the egg
Ž .
development phase 91.5% was lower than survival during the two stages of hatchling development, where almost all of the eggs that hatched, developed into independent juveniles.
Variation in survival between replicate groups within female groups was not signifi-Ž cant. This was the case for survival during the individual phases of development i.e.,
. during the egg development phase, and the two stages of hatchling development and for the whole development period.
3.2. DeÕelopment time of the eggs and hatchlings
The eggs took 42 days to hatch and develop into independent juveniles at the 208C
water temperature. Eggs collected from the females at the 0% stage of development took 59 days to hatch and develop into independent juveniles, while those collected at the 65% stage took 36 days. The time spent in the egg development phase was 18 days, and ranged between 35 and 12 days for eggs collected from the females at the 0% and 65% stages of development. The hatchlings spent 7 and 17 days as Stage I and II hatchlings.
Table 1
Ž .
Survival % of marron eggs and hatchlings incubated artificially. Survival during the egg development phase was the proportion of eggs that hatched. Survivals during the two stages of hatchling development were the proportion of hatchlings alive at the end of each stage relative to the number of hatchlings alive at the beginning of the stage. Survival for the whole development period was the proportion of eggs that hatched and developed into independent juveniles
Development period Survival
Egg development phase 91.5
Hatchlings development — Stage I 98.5
Hatchlings development — Stage II 99.1
4. Discussion
This study established that marron eggs and hatchlings can be incubated artificially with high levels of survival. Eighty-nine percent of the eggs collected from gravid females and incubated artificially, hatched and developed into independent juveniles. This result indicates that the artificial incubator was a suitable replacement for the maternal care provided by the female marron after spawning.
Previous attempts to artificially incubate crayfish eggs and hatchlings were carried out on Astacidea species, and resulted in much lower survivals than those observed in this study on marron. As considered in Section 1, this was presumably because it is more difficult to provide suitable conditions for the eggs and hatchlings of the Astacidea species due to their development, which is much longer than that of marron and includes a period of winter diapause. It is unlikely that the artificial incubators used to incubate the eggs and hatchlings of the Astacidea species were a major reason for the lower survival. Many of these incubators were, in principle, similar to the artificial incubator used in this study to incubate the eggs and hatchlings of the marron. That was, to hold the eggs in suspension by a continuous flow of water flowed up from beneath the eggs. The lower survival of the marron during the egg development phase, compared to during the stages of hatchling development, was also observed in the Astacidea species
ŽStrempel, 1973; Mason, 1977; Rhodes, 1981; Koksal, 1988; Carral et al., 1992 . The
¨
.lower survival of the eggs of the marron was not surprising considering that the
Ž .
processes associated with early egg development e.g., cell differentiation are sensitive
Ž .
to environmental changes c.f. Schmidt-Neilson, 1970 , and environmental changes would have been encountered during egg collection and transfer to the incubator. However, it was possible that the incubation technique of holding the eggs in suspension also contributed to the reduced survival of the eggs by increasing the risk of mechanical damage. By comparison, the hatchlings were not held in suspension, but were supported by a piece of nylon cloth provided in each of the incubation cells.
The lack of variation in survival between replicate female groups indicates that Ž
uniform environmental conditions i.e., water temperature, water quality, and water .
flow existed between the incubation cells within the incubator. This result has particular implications for research studies carried out on marron eggs and hatchlings using the
Ž
technique of artificial incubation. In such studies, experimental groups e.g., genetic .
groups andror treatments of eggs and hatchlings need not be replicated across large
numbers of cells.
The length of the egg development phase and, in turn, the whole development period for the eggs and hatchlings could not be assessed accurately because the eggs were collected from the females after spawning. However, given the stage of development when the eggs were collected from the females and the time taken for the eggs to hatch, the egg development phase is estimated to take 35 days when the eggs are incubated in
water maintained at 208C. Furthermore, with a time of 7 and 17 days for the two stages
Ž .
of hatchling development Stages I and II , it is estimated to take 59 days for the eggs to hatch and develop into independent juveniles.
potential advantages for marron production by increasing egg and hatchling survival, and reducing the space and food required to maintain gravid females, its main benefit is to research studies. The technique enables experiments to be carried out on the eggs and hatchlings independent of the females, under a common and controlled environment, and with many separate treatment groups.
Acknowledgements
This study was funded by Newmex Exploration, and Bequest Funds of the Faculty of Agriculture, The University of Western Australia, Australia.
References
Ž .
Anderson, D.T., 1982. Embryology. In: Bliss, D.E. Ed. , The Biology of Crustacea, Vol. 2. Embryology, Morphology, and Genetics. Academic Press, New York, pp. 1–41.
Carral, J.M., Celada, J.D., Gaudioso, V., Temino, C., Fernandez, R., 1988. Artificial incubation improvement˜ ´
Ž .
of crayfish eggs Pacifastacus leniusculus Dana under low temperatures during embryonic development. Freshwater Crayfish 7, 239–250.
Carral, J.M., Celada, J.D., Gonzalez, J., Gaudioso, V.R., Fernandez, R., Lopez-Baisson, C., 1992. Artificial´ ´ ´ ´
Ž .
incubation of crayfish eggs Pacifastacus leniusculus Dana from early stages of embryonic development. Aquaculture 104, 261–269.
Henryon, M., 1995. Genetic variation in wild marron can be used to develop an improved commercial strain. PhD Thesis, University of Western Australia, Perth, Australia, 128 pp.
Ž .
Koksal, G., 1988. Astacus leptodactylus in Europe. In: Holdich, D.M., Lowery, R.S. Eds. , Freshwater¨
Crayfish. Biology, Management and Exploitation. Croom Helm, London, pp. 365–400.
Ž Ž ..
Mason, J.C., 1977. Artificial incubation of crayfish eggs Pacifastacus leniusculus Dana . Freshwater Crayfish 3, 119–132.
Matthews, M., Reynolds, J.D., 1995. In vitro culture of crayfish eggs using a recirculating airlift incubator. Freshwater Crayfish 8, 300–306.
Perez, J.R., Carral, J.M., Celada, J.D., Munoz, C., Saez-Royuela, M., Antolın, J.I., 1998. Effects of stripping´ ˜ ´ ´
time on the success of the artificial incubation of white-clawed crayfish, Austropotamobius pallipes
ŽLereboullet , eggs. Aquaculture Research 29, 389–395..
Perez, J.R., Carral, J.M., Celada, J.D., Munoz, C., Saez-Royuela, M., Antolın, J.I., 1999. The possibilities for´ ˜ ´ ´
Ž .
artificial incubation of white-clawed crayfish Austropotamobius pallipes Lereboullet , eggs: comparison
between maternal and artificial incubation. Aquaculture 170, 29–35.
Ž .
Rhodes, C.P., 1981. Artificial incubation of the eggs of the crayfish Austropotamobius pallipes Lereboullet . Aquaculture 25, 129–140.
Sandeman, R., Sandeman, D., 1991. Stages in the development of the embryo of the fresh-water crayfish
Cherax destructor. Roux’s Arch. Dev. Biol. 200, 27–37.
SAS Institute, 1988. SASw
Technical Report P-179, Additional SASrSTATeProcedures, Release 6.03, SAS Institute, Cary, NC, 255 pp.
Schmidt-Neilson, K., 1970. Animal Physiology, 3rd edn. Prentice Hall, NJ, 145 pp.
Strempel, K., 1973. Edelkrebserbrutung in zuger-glasern und anfutterung der krebsbrut. Freshwater Crayfish 1,¨ ¨ ¨
