1 NOVEMBER 1999
Sent: October 21, 1999
From: Jerome Davis <jeromed@iafrica.com>
To: <crust-l@VIMS.EDU>
QUESTION:
In order to do in vitro studies of Scylla eggs, I need to separate the egg mass into several portions and to separate the eggs so that they can be incubated in artificial incubators. I have two questions regarding this.
1) Can anybody tell me what the physical mechanism is that is produced by the female to attach the eggs to one another and to the female?
2) Is it possible to dissolve these links chemically or separate the
eggs from one another by any other means?
3) Has anybody had experience incubating the eggs of decapods in this
manner? I have seen some work on Macrobrachium where the egg mass is incubated as a whole in glass funnels, but this will not be suitable for Scylla.
Jerome Davis
Research Officer
Department of Ichthyology & Fisheries Science
Rhodes University/Liberty Life Crab Aquaculture Project
Mtunzini Prawn Farm
Box 98
Mtunzini
3867 South Africa
Tel: (0353) 402 494
Fax : 402 495
E-mail : jeromed@iafrica.com
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COMMENTS 1:
I did in vitro incubating of Scylla eggs many years ago in order to
determine the effects of temperature (5 to 35 C) on the development of the crab embryos. To my knowledge, it was Costlow and Bookhout who first reported success incubating crab eggs in vitro with a recipical agitator in the 60's. However, if you are lack of facilities, you can do it with a very simple 'still water' incubation method that I used for Scylla eggs. You simply put appropriate amount of separated eggs into containers and cover them. If no serious contamination occurs, normal larvae will hatch out of it. Any kind of containers would be fine for the purpose, but the containers and seawater need to be sterilized beforehand. The hatching rates were generally low (though could be high in some cases) but if your experiment is not so much depending on hatching rates, it should be OK. The biggest headache for either
'agitating' or 'still' method is separating of eggs, it is tedious but can
be done with a fine needle and patience. What I would suggest is that you better use the 'miscarried eggs', i.e. those eggs failed to attach to the abdomen of female crabs at spawning, for the purpose. It would save whole lots of your time for separating eggs and also provide you with eggs that are healthy (eggs sometimes are damaged during the manual separation) and at the beginning stage of embryo development.
Regarding to mechanisms that eggs attach to the female crab, I have only limited knowledge about it, which is also not up-to-date. I think general consensus is that first female crab dig a shallow hollow on the sediment under their abdomen and then lay eggs onto it. At this stage, the eggs are separate. The female then beats her abdomen continuously and vigorously on the eggs. During the process, the setae on the pleopods pierce through outside membranes of the double layered membranes of the eggs and then form the egg stems that attach them to crab pleopods; the fluid between the two membranes is also believed helping the eggs to adhere to the setae of the female pleopods.
Chaoshu Zeng
Seikai National Fisheries Research Institute
49 Kokubu-machi, Nagasaki-shi
Nagasaki 850-0951
JAPAN
Tel: +81-95-821-4494 or +81-95-824-9335
Fax: +81-95-833-2695
E-mail: cszeng@snf.affrc.go.jp or chaoshuzeng@hotmail.com
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COMMENTS 2:
I would counsel against the use of the "miscarried" eggs as they will have a higher prevalence of bacterial and fungal infections and more physical damage from exposure. With Cancer spp. I've used sharp scissors and fine forceps to remove setae from the pleopods. Individual setae with 20-150 eggs work well for hatching (or symbiont) studies in small petri plates. I used a 1% bleach wash to clean the external surfaces of the eggs, then transferred through several water changes (to get rid of excess yolk from broken eggs) into pen-strep seawater. Water changes were done every 1-3 days. Dick Lee has used an amphotericin pen-strep commercial mix for blue crab eggs and we've had some success with it at 0.25% amphotericin. One of the key elements is that the female crab has to be handled relatively
gently. We've lost several batches of eggs from heat stress during
capture of the female.
There are references for batch culture of brachyuran embryos/larvae but I don't have them.
Jeffrey Shields
Assistant Professor
Virginia Institute of Marine Science
Gloucester Point, VA 23062, USA
Tel (804) 684-7128
Fax (804) 684-7186
E-mail: jeff@vims.edu
http://www.vims.edu/~jeff/
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COMMENTS 3:
I agreed that the 'miscarried' eggs are prone to bacterial and fungal infections if they are left for long after having been extruded. I did not
state clearly in my last mail that what I suggested is that those eggs extruded not long ago from the female crab and therefore, they are relatively clean. I used 'miscarried' eggs right after being extruded (I observed the spawning process) and compared them with those removed from the pleopods. The result indicated that eggs from pleopods generally have higher contaminant rates, probably because setae from female pleopods carried microbiotics. Eggs damaged during the separating process may also promote the growth of microbiotics. Another phenomenon I observed is that if a few eggs in a removed batch got infected, it very quickly spread to the whole batch while for individual 'miscarried' eggs, the situation was better because they were separated. Of course, the situation could vary depending on the method of incubation. I did not use antibiotics during the incubation and partial water exchange was only performed a few times during the whole embryo development. The water exchange was also done very gently to reduce the chance of spreading of the microbiotics colonies.
Chaoshu Zeng