IN VITRO CULTURE OF MACROBRACHIUM EGGS*

C. BALASUNDARAM & T. J. PANDIAN

School of Biological Sciences, Madurai Kamaraj University, Madurai-625 021, India

* Presented in the Second International Symposium on Invertebrate Reproduction held in Davis, California during August, 1979

Received February 13, 1980

Keywords: Macrobrachium nobilii; egg incubation in situ; in vitro incubation; artificial incubator

Abstract

Causes for the death of the eggs in the prawn Macrobrachium nobilii are: i) shedding of eggs by ovigerous female, and ii) infec- tion by epibionts: a Saprolegnial fungus, bacteria (gram negative) and protozoans (Vorticellids and Paramecium). A cause for the death of freshly hatched larvae of some decapods is the reduction in reserve yolk energy in the larvae hatched in the last few batches. To circumvent these disadvantages, an artificial incubator was designed, in which 70% of the J-day old eggs can successfully be incubated and hatched simultaneously. The isolted eggs are ir- rigated with filtered and aerated water over a diaphragm in the incubator; the water flushed from below through the diaphragm in the artificial incubator, sways and keeps the eggs continuously in a suspended motion, simulating the irrigation technique of the mother.

Introduction

For prawn culture freshwater prawns are often chosen, as they readily mature, mate and spawnincaptivity(Wickins, 1970). Of the several species, Macrobrachium nobilii was chosen, as it does not undertake spawning migration. High variability in the number of eggs inchbated by the females of different crustaceans under laboratory and field conditions has been reported (e.g. Paralithodes camt- schatica, Haynes, 1968; PandaIus jordani, Dahlstrom, 1968; Macrobrachium spp., Dugan et a!., 1975; Ligia oceanica, Pandian, I 972). This variability is due to the loss of eggs incurred during in situ incubation; such loss of eggs has been found to range between 20 and 80% in Palaemon serratus and about 30% in M. rosenbergii (Wickins & Beard, 1974). Castlow & Bookhout (1968) too observed that the berried Cancer magister ‘frequently’ tears off the eggs loosing them totally. Berried P. serratus is highly sensitive to water quality; at higher temperatures, the eggs

may be lost in toto due to a premature moult (Phillips, 1971). In species of Cancer and Homarus, Fisher (1976) and Nilson et al. (1975) reported that the incubated eggs

are infected by microbial epibionts, which kill them by occluding effective gas exchange.

‘The incubated eggs of Homarus spp. hatch in batches over an extended period of about 15 days (Pandian, 197oa, b; Ennis, 1973). Pandian (r97oa) noted that none of the available views on the hatching mechanisms of crustacean eggs is adequate to explain the batching effect; additional cost of metabolism of the embryos hatched on subsequent batches is significantly high (8% of reserve yolk). Pandian & Katre (I 972) showed that the larvae of Macrobrachium idae hatched in subsequent batches were weaker and swam to a beam of light slower than that hatched in the first batch; they considered that such larvae may not be able to

predate, or escape from predators successfully. To cir-

cumvent the egg loss incurred during in situ incubation and to synchronise hatching in vitro, the present work was undertaken in the freshwater prawn Macrobrachium nobilii.

Materials and methods

Healthy individuals of Macrobrachium nobilii collected from the river Cauvery (S. India) were reared in the ratio of I $ : 7 Q/aquarium (1: 70 x b : 40 x h : 35 cm; 80 1). They were fed ad libitum on minced beef, boiled bengal gram and goat liver. Females spawned within 12 hrs following

mating. The time, at which the female was berried, was counted as o hr of incubation. From these females, eggs of chosen age were removed and used for experiments.

Results

In situ incubation Eggs in an egg mass of M nobilii commence development

-at the same time. At any given time of incubation, all the

203

Hydrobiologia 77, ZOj-208 (1981). 0018-8I~%/%I/O~~~-OZO~%O1.20. 8 Dr. W. Junk b.v. Publishers, The Hague. Printed in the Netherlands.

Table 1. Mucrobrachium nobilii: Number of larvae hatched by a female on succes- sive days of incubation. Hatching commences normally after 11 days of incubation.

Length of prawn (mm)

Animals observed

00)

Day of incubation

12th 13th 14th 15th 16th 17th

32 2 0 152 0 70 5 2 34 3 250 0 25 0 75 0 36 5 60 75 0 12 1 0 38 2 0 0 320 215 10 20

Percentage 24 18 27 23 7 2

eggs are at the same developmental stage. Embryonic development is completed on the 12th day after berrying; hatching took place in irregular batches extending over a period of 4 f 2 days. Table I shows that about 24,18,27, 23,7 and 2% of the eggs were hatched on the 12,13,14,15, 16 and 17th day after incubation, respectively.

In vitro incubation

Synchronisation of hatching

To synchronise hatching under in vitro conditions, rz-day old egg masses were removed from berried females and incubated in standing water. In all egg masses, the periph- eral eggs successfully hatched but those in the centre, espe- cially in the larger egg masses, succumbed to death. Such egg mortality was found to be linearly related to the size of the egg mass (Table 2). This observation prompted us to reduce the size of the egg mass and ultimately individual eggs were isolated by careful teasing. In vitro incubation of isolated rz-day old eggs in standing water yielded 98% hatchability. Hatching lasted for a period of 6 hrs between the 3rd and 9th hr after in vitro incubation.

Table 2. Macrobrachium nobilii: Effect of standing water on hatchability and mortality of the 1 Zday old teased egg masses incubated in vitro. Each value is based on 6 experimental ob- servations.

Egg density Hatchability (No/mass) (%)

25* 5 93 + 6.1 45* 5 72 + 5.5 65~ 5 56 f 7.1

125 f 25 44 + 4.2

Mortality of em zoea (%) (%)

0 7 f 6.0 5 f 2.1 23 f 8.1

19 + 3.1 25 f 8.0 44 f 3.4 12 * 1.0

204

Influence of age of eggs

The second objective was to advance the age of developing eggs used for in vitro incubation, so as to decrease the chances of berried females loosing the eggs. Fertilized eggs, collected after extrusion but before attachment to the ovigerous setae of pleopods, were incubated under in vitro conditions; they died within 4 hrs in toto. Hence, arti- ficial incubation of the fertilized eggs collected prior to berrying may not be successful. The observations sum- marised in Table 3 reveal that for successful in vitro in- cubation, the eggs must be at least r-day old and a period of 3 days after berrying should elapse for the mother to withstand the strain of egg mass removal. Therefore, for all the in vitro experiments, 3-day old eggs were used.

Incubation in standing water

The 3-day old egg mass was incubated in 50 ml of disin- fected standing water. On the 3rd day of incubation, the egg mass began to deteriorate with the spoilage spreading from the centre to the periphery. Deficient supply of oxygen for the eggs in the centre, and/or microbial infec- tion were considered as the causative factors for the spoil- age of eggs. To increase oxygen supply, water was gently aerated. The turbulence created by even gently bubbling air swayed the entire egg mass, exposing the peripheral eggs to a fresh column of water, but failed to let water to percolate through the egg mass.

Microscopic examination of the spoiled eggs revealed prolific infection by gram negative bacteria,a Saprolegnial fungus (Fig. I) and ciliates. To suppress bacterial growth, potent antibiotic (5 ppm of Ampicillin, Streptopenicillin or Chloramphenicol) was incorporated into the incuba- tion medium; the egg mass was also disinfected by treating it with fungicides (0.003% Mercuric chloride), formalin (50 to IOOO ppm) and Sodium hypochlorite (0.003%) for

Table 3. fkfacrobrachium nobilii: Effects of egg mass removal on the mother animal and egg development. Each observation is based on 6 mother animals. Age of egg is counted from the time of berrying.

Age of egg (hr) developing eggs

Observations on mother animals

Fertilized eggs before berrying

0

3

12

24

48

12

100% mortality within 4 hrs of incubation

100% mortality within 6 hrs of incubation

Normal

Succumbed to the strain of egg removal immediately or later

Normal development; 10% abnormal in shape

Normal development; no abnormality in egg shape

Too sensitive to handle; when eggs are force-relieved, some died

Too sensitive to handle; often abandon egg mass with appendages

3,

Successfully withstand the stress of egg mass removal

30 sec. prior to incubation. Despite these treatments, dete- rioration of the eggs in the centre could not be prevented. Similar results were obtained, when such chemically treat- ed egg masses were incubated with or without aeration.

The fact that the spoilage of eggs commenced always from the core of the egg mass suggested that deficiency of oxygen is perhaps the real cause for the spoilage and not the microbial infection; for, if the spoilage were due to microbial infection, eggs from all parts of the egg mass- perhaps the peripheral eggs, which are more exposed than the central ones-should suffer mortality; mortality would commence from the exterior to the interior of the egg mass (see also Nilson et al., 1975; Fisher & Wickham, 1976).

The fact that 98% of the isolated, Iz-day old eggs suc- cessfully hatched prompted us again to provide maximum surface area of contact between the egg and the medium. Hence, 3-day old egg mass was isolated into individual eggs before incubation. To ward off infection, the isolated eggs were dip-treated in formalin (300 ppm). To biologists interested in artificial culture of fish eggs, such dip-treat- ment is not a new and is, in fact, preferably adopted in salmon hatcheries (Bardach et. al., 1972).

An increase in density of isolated eggs in the incubation

chamber containing standing water necessarily decreases the surface area of contact between individual eggs and the incubation medium. To study the influence of egg density on hatchability 50,40,30,20 or IO eggs were incubated in a flask with the surface area of IO cm’. Maximum hatchabi- lity (70 * 8.2%) was observed at a density of IO eggs/flask, affording I cm*/egg (Table 4). Under identical conditions, formalin treatment failed to improve hatchability. Provid- ing sufficient oxygen by increasing the area of contact of the eggs with the medium was therefore felt critically more important.

Egg membrane of aquatic crustaceans represents an important structure through which gaseous and ionic ex- changes are effected (Pandian, 197oa, b). Therefore, any device used for mass culture of eggs should have provi- sion to keep the outer surface of the egg membrane free from epibionts. Increasing egg density reduces the area of contact of the egg with the medium. Mass rearing of eggs in standing water will demand an enormous surface area in the flask, since an egg requires a minimum of I cm* to ensure successful incubation and hatching. The conditions afforded by the standing water in a conical flask were therefore considered disadvantageous for reasons given below:

205

Fig. I. Developing egg of Macrobrachium nobilii infested by the epibiont belonging to Saprologniales. Note the ramifying hypae of the fungus forming a mat on the surface of the egg.

I. Since part of the egg rests on the lower surface of the conical flask, the egg is not fully exposed to the medium, and

2. Since water remains static, replenishment of oxygen is minimal. The incubator designed to incubate the eggs comprises

of I. Tank, 2. Sand filter, 3. Incubation chamber, and 4. Collection chamber(Fig. 2). The incubation chamber con- sists of an inverted conical flask (capacity IOO ml) with an opening at the top, through which the eggs can be intro- duced; the eggs rest on a diaphragm made of bolting silk (mesh size: 137 p) fixed at ‘/j height of the flask. There is an inlet at the bottom of the flask and an outlet at the shoulder level. The sand filter unit consists of a jar con-

Table 4. Macrobrachium nobilii: Effect of egg density on the hatchability of the 3-&y old formalin treated (300 ppm) and untreated eggs incubated in standing disinfected water.

Egg density (eggs/l0 cm’)

10 20 30 40 50

Hatchability (%)

Treated Untreated

70.0 f 8.2 82.0 f 7 56.3 f 12.0 58.8 f 10 46.6 f 11.5 51.5 f 12 34.6 + 9.0 40.6 f 5 16.0 + 4.0 18.0 + 7

taining graded sand, through which the incubation me- dium is filtered and the suspended impurities and air bub- bles are trapped. The sand filter is replaced once in three days. Filtered tapwater mixed with I ppm malachite green enters into the incubation chamber through a polythene tube via the sand filter at a rate of 3.5 Vhr, flushes the eggs on the diaphragm and flows out through the collection chamber. The collection chamber consists of two plastic buckets of identical size (height 18 cm) fitted one into the other. The inner bucket consists of 6 holes (diameter 0.5 cm) made at equal distance in the mid level and pasted with the bolting silk to prevent escape of larvae. The outer bucket consists of a polythene tube fitted at the shoulder level through which water flows out to the drain. When hatching commences, the apparatus is covered with a black paper except at the outlet through which a beam of light is passed in. The larvae, attracted by the light come out of the incubation chamber, flow through the outlet and get collected in the inner bucket of the collection chamber.

Incubation of egg mass

With a view to expose a larger surface area in each egg, the egg mass was teased and eggs were isolated. However, teasing and isolation of eggs from an egg mass involved mechanical injury to the eggs. This was still accomplished so that the 12-day old eggs are hatched synchronously.

206

Fig. z. The newly designed incubator for successful incubation and synchronised hatching of j-day old eggs of Mucrobrachium nobilii.

Experiments were made to incubate egg masses containing 200-300 eggs in the newly developed apparatus maintain- ing a flow rate of 3.5 I water/hr. Hatching commenced 3-4 hrs after incubation and it was completed between the 4th and 10th hour of incubation, recalling the observations on the incubation of isolated rz-day old eggs in standing water. Evidentlywaterflowthroughthe hatcherpercolated through egg masses providing sufficient oxygen to indivi- dual eggs.

the experiments. Hatchability progressively decreased from 98% in the rz-day old eggs to 70% in the 3-day old eggs (Table. 5). Attempts are being made to increase the flow rate so that hatchability of 3-day old eggs could also be enhanced.

Discussion

Incubation of isolated eggs

To study the influence of age of teased eggs on hatchability 3,6,9 and r2-day old eggs were incubated in the apparatus maintaining a flow rate of 3.5 1 water/hr; the density of eggs was maintained between 2 and 3.6 thousand eggs in all

Only a few publications concerning artificial hatching of crustacean eggs are available. Sandoz & Rogers (1944)

were, perhaps, the first to artificially hatch the eggs of the crab Cancer magister; the eggs were incubated at a density of 50 eggs/cm*. In 1953, Costlow & Bookhout were able to increase the egg density to I IO eggs/cm2 by affording bet- ter aeration keeping the incubating set up on a variable

Table 5. Macrobrachium nobilii: Effect of age of developing eggs on hatchability in the incubator

Age of egg Observations Incubated eggs Hatchability (day) (No) (No) (%)

3 7 3061 * 220 70 + 12.6 6 9 2068 f 714 83 f 8.7 9 11 3586 f 456 90 f 4.7

12 8 2768 + 278 98 f 2.0

speed shaker. Lewis (1975) made an in-depth study on the modifying role of ecological factors like photoperiod, aeration and temperature on artificial incubation of the barnacle Pollicipes polymerus. Incubating the eggs of the shrimp Palaemon serratus, Phillips (1971) considered clean running water and adequate aeration are the most impor- tant criteria in designing an egg incubator. Though Phillips successfully hatched the eggs of P.serratus, the hatchabili- ty of the eggs was not consistent, perhaps, due to the con- tamination of the eggs with the bacterium Leucothrix sp. For the first time, consistent success has been achieved in artificially hatching the eggs of the prawn M. nobilii at a synchronised time in the apparatusdescribedin the present work.

Acknowledgements

Financial support by the International Foundation for Science, Stockholm (Research Grant Agreement No. R- 107) is gratefully acknowledged.

References

Bardach, J. E. Ryther, J. H. & McLarney W. D. 1972. Aquacul- ture: The farming and husbandry of freshwater and marine organisms. Wiley-Interscience, New York. 868 pp.

Costlow, J. D., Jr. & Bookhout, C. G. 1968. A method for developing the brachyuran eggs in vitro. Limnol. Oceanogr. 5: 212-225.

Dahlstrom, W. A. 1968. Synopsis of biological data on the ocean shrimp Pandalus jordani Rathbun. F.A.O., World Scientific Conference on the biology and culture of shrimp and prawns. Pr: BCSP/67/SS/9.

Dugan, C. C., Hagood, W. R. & Frakes, A. T. 1975. Develop- ment of spawning and mass larval rearing techniques for brack- ish-water shrimps of the genus Macrobrachium (Decapoda: Palaemonidae). Florida Department of Natural Resources No. 12. pp. 1-28.

Ennis, G. P. 1973. Endogenous rhythmicityassociated with larval hatching in the logster Homarus gammarus J. mar. biol. Ass. U.K., 53: 531-538.

Fisher, W. S. 1976. Relationships of epibiotic fouling and mor- talities of eggs of the Dungeness crab (Cancer magister) J. Fish. Res. Bd. Can. 33: 2849-2853.

Fisher, W. S. & Wickham, D. E. 1976. Mortalities and epibiotic fouling of eggs from wild populations of the Dungeness crab (Cancer magister). U.S. Fish. Wildl. Serv. Fish. Bull. 74: 201- 207.

Haynes, E. B. I 968. Relation of fecundity and egg length, carapace length in the king crab Paralithodes camtschatica. Proc. natl. Shell Fish. Ass. 50: 60-63.

Lewis, C. A. 1975. Some observations on factors affecting em- bryonic and larval growth of Pollicipes polymerus (Cirripedia: Lepadomorpha) in vitro. Mar. Biol. 32: 127-139.

Nilson, E. H., Fisher, W. S. & Shleser, R. A 1975. Filamentous infestations observed on eggs and larvae of cultured crusta- ceans. Proc. 6th Ann. Workshop World Maricult. Sot., Seattle, Wash.

Pandian, T. J. I 97oa. Ecophysiological studies on the developing eggs and embryos of the European lobster Homarus gammarus. Mar. Biol. 5, 153-167.

Pandian, T. J. r97ob. Yolk utilisation and hatching time in the Canadian lobster Homarus americanus. Mar. Biol. 7,249-254.

Pandian, T. J. 1972. Egg incubation and yolk utilisation in the isopod Ligia oceanica. Proc. Indian natl. Sci.Acad. 38, 430- 441.

Pandian, T. J. & Katre, S. 1972. Effect of hatching time and larval mortality and survival of the prawn Macrobrachium idae. Mar. Biol. 13: 330-337.

Phillips. G. 1971. Incubation of the eggs of the English prawn Palaemon serratus. J. mar. biol. Ass. U.K., 51: 43-48.

Sandoz, M. & Rogers, R. 1944. The effect of environmental factors on hatching, moulting and survival of zoea larvae of the blue crab Callinectes sapidus Rathbun. Ecology, 25: 216- 228.

Wickins, J. F. 1970. Prawn biologyandculture. In Oceanography and Marine Biology. An annual review. Ed. H. Barnes, Aber- deen University Press 14; 435-507.

Wickins, J. F. &Beard, T. W. 1974. Observations on the breeding and growth of the giant freshwater prawn Macrobrachium rosenbergii (de Man) in the laboratory. Aquaculture, 3: 159- 174.

208