First Issue

Mail to the editor


[Editorial] [Basics for raising brine shrimp] [Bivalve larvae metamorphosis 1] [Bivalve larvae metamorphosis 2] [Bivalve larvae metamorphosis 3] [Cod Farming] [Concluding remarks of the Larvi'95 symposium] [Hatchery hygiene manual] [Indian shrimp farming hit by disease] [Laboratory studies on the culture of the brine shrimp Artemia using organic wastes] [Collection of wild shrimp seed in India] [Technical appraisal manual - shrimp and freshwater prawn hatchery] [Larvae and bacteria] [Larvae metamorphosis 1] [Larvae metamorphosis 2] [Water treatment in algae cultures 1] [Water treatment in algae cultures 2] [Water treatment in algae cultures 3] [Feeding rates of gilthead seabream (Sparus aurata), larvae on microcapsules] [Capacity of gilthead seabream, Sparus aurata L., larvae to break down dietary microcapsules] [Fluorometric determination of selectivity between live and inert food by Penaeus Japonicus larvae] [Variation of Ascorbic acid content in different live food organisms] [The effectiveness of tricaine, quinaldine sulfate and metomidate as anesthetics for larval fish] [Artemia culture in the Mekong delta of Vietnam] [Taura disease] [Hatchery developments in Honduras] [Molecular biology and genetics engineering of fish] [Crustacean reproduction structure and biosynthesis of lipoproteins and accumulation of lipids in the ovary] [The Samuel Lunenfeld Rotifer center] [A gamete and embryo bank for cultivated fish]

EDITORIAL TO THE FIRST ISSUE OF THE ILWG @NL


To all subscribers,

We're happy to send you the first issue of the Electronic Newsletter of the International Working Group on Larviculture of Fish and Shellfish. It's still suffering from some children's diseases, but in the near future we hope to send it in a more definitive form at least on a monthly basis. Our future email address, larvi@UGent.be is not operational yet, so I suggest that any comments/suggestions/requests are sent, for the time being, to gilbert.vanstappen@UGent.be.

As we are planning to get on WWW in the (near ?) future, we'd like to know if you have access to this means of electronical communication.

Please inform us.

Subscription to the IWGL can be made via the standard form, that can be obtained from the EAS (European Aquaculture Society), Coupure Rechts 168, B-9000 Gent, Belgium, tel. 32-9-2237722, fax 32-9-2237604 (email address not yet available), or from our lab.

Of course this @NL can hardly be viable without the contribution of its readers. Feel free to comment and to communicate.

Regards,

Gilbert Van Stappen


BASICS FOR RAISING BRINE SHRIMP


Kai Schumann is an engineer in a biotech company, and has made a FAQ for Artemia culture, with information he has gathered from various books, publications and his own experience. It contains very practical and useful guidelines for hatching and culturing Artemia. The text can be obtained from Kai Schumann, 4901 Mt. Gaywas Dr., San Diego, CA 92117, USA, email SCHUMANN_KAI@Lilly.com. He would appreciate very much receiving more information on the subject, as well as comments or additions to this FAQ.


BIVALVE LARVAE METAMORPHOSIS 1


By
Tom Lewis

In reply to Will Borgeson's comments (italics)

Most commercial and research hatcheries feed bivalve larvae live algae cells of species that have been shown to have good nutritive characteristics (tropical *Isochrysis,* *Chaetoceros* spp, etc.).

I think the most significant nutrition variable for larvae may well be what is in the egg. A typical pattern for crashing larvae is that they survive well to straight-hinge, then crash, apparently unable to feed on the *ad libitum* ration of live algae that is provided. Other times the crash happens later; around 10 days post-fert is not uncommon.

This 10-day crash often involves just a percentage of the larvae. Other times the major mortality is delayed until metamorphosis.

A group of researchers in Tasmania did some work about ten years ago which showed a positive correlation between bacterial levels in algal cultures (tropical *Isochrysis,* *Chaetoceros* spp, etc.) fed to Pacific oyster larvae (in a commercial hatchery) and crashes. (Lewis et al. 1988 Aquaculture 69: 241-251).

We found that algal cultures "seemed" to be safe until culturable bacterial counts exceeded ~2 x 10^6/mL, then any amount of said cultures fed to larvae (at any time during the larval cycle) resulted in mortalities within days.

Similar circumstances of bacteriologically -unsafe algal cultures could also explain the crashes mentioned by Will.

Tom Lewis
Department of Agricultural Science
University of Tasmania
GPO Box 252C
HOBART
Tasmania, Australia, 7001
Phone: (002) 20 2776
Fax: (002) 20 2642


BIVALVE LARVAE METAMORPHOSIS 2


By
Will Borgeson

Another interesting bit of info from a Tasmanian study found that oyster larvae cultured in .2 micron filtered water crashed, while controls grown in roughly filtered water thrived. Seems one can get things too clean, as well as too dirty. Apparently the few bacteria that came in with the zygotes found the clean water plus nutrient situation to be like a blank agar plate. I don't have that ref. in front of me now, but it may well have been Tom's work. In any case it was a good and fascinating paper.

Unfortunately we don't have a pathology group set up to do the plating work to routinely test our algae for bacteria. I pasteurize the water that will be used for the algae, and rarely have an algae crash; have had none at all this year. But that doesn't mean there aren't bacteria in there. I do tend to tweak thru algae cultures rapidly, they aren't hanging around a long time for bacteria to proliferate. But I would like to be examining this. Still, our bivalve larval survivorship seems closely linked with the gonad cycle of the parents, especially the mothers.

My gut feeling is that bivalve larvae can handle having some bacteria around, as long as there are enough competing microbes to keep each other in balance. There has been much work that indicates that there is a distinct seasonality to oyster bivalve success. The gametes need to be optimal to get optimal survival...weak larvae can't feed well, and probably have compromised immune systems, making it difficult for them to deal with bacteria that may well be ubiquitous.

I'll check out Tom's paper mentioned above when I get a chance, but I'm wondering how long it usually takes to get up to that number of bacterial colonies...


BIVALVE LARVAE METAMORPHOSIS 3


By
Tom Lewis

Further to Will's latest message:

No it wasn't my paper, but by the same group:
Garland et al (1986) Aust J Mar Freshwater Res 37 pp 713-720

Our work was done using algae cultured on a semi-continuous basis. i.e. 10-50% of the algae culture was drained fom the algal culture bags, and the bags refilled with fresh culture medium. Is this similar to your work Will?

The hatchery in which we did this work was pastueriseng algae water when I first arrived. The pasteurised culture medium routinely had bacterial levels around 10^4 by the time the water had cooled enough for inocculation. Yuk!!

We quickly moved to 0.2um filtering for similar reasons to those Will expressed earlier. i.e. it's best to provide some "natural" bacteria in the culture medium to prevent opportunisitic contaminants getting an unimpeded chance to grow in the cultures. In this case the 0.2um filtration gear gave us ~50 bacteria/100ml at the time of inocculation.

When using the original and (admittedly) poorly designed pasteurisation facility, algal cultures were achieving bacterial counts in the order of 10^6 to 10^7 within 3-5 days of inocculation, with most (>90%) cultures exceeding 2 x 10^6 bact/ml during their harvest life.

Once the 0.2um filtration system was correctly installed, most algal cultures reached around 10^5 to 10^6 bact/ml within 4-6 days and rarely (<10%) exceeded 2 x 10^6 bact/ml during their harvest life.

Obviously, the level of larval survivorship will be determined by a combination of many factors, including genetics, water quality and bacteria in feed algae, but I thought I'd drop in my few cents worth to this discussion.


COD FARMING


By
Rodney Melvin Penney

This is in response to your request for information on cod farming. I am a graduate student in the Master of Aquaculture program at Simon Fraser University, Burnaby, B.C. I have domee a fairly extensive literature search on cod aquaculture myself and here are some references that may be of interest to you.

I am also interested in the development of new species for aquaculture and would like to get involved in cod aquaculture in the future. I would like to discuss this further in the future. I hope this information was helpful to you.


CONCLUDING REMARKS OF THE LARVI'95 LARVICULTURE CONFERENCE HELD IN GENT, BELGIUM, SEPTEMBER 3-7, 1995


Impressions of the meeting presented at the closing ceremony on behalf of the academic community by Dr B R Howell (Fisheries Laboratory, Ministry of Agriculture, Fisheries & Food, Conwy, UK)

It's an awesome task to attempt to summarise such a multifaceted meeting in just a few minutes but in doing so I must emphasise that I am the front man for the combined efforts of Prof Tom Lam (Department of Zoology, University of Singapore) and myself. I also acknowledge the helpful comments of other participants.

We were asked to highlight advances made since Larvi '91 and to identify, where appropriate, perceived deficiencies in our coverage of the subject area. With regard to advances, I think it has to be said that at this stage in the development of the field of aquaculture quantum leaps forward can not be expected, rather we are seeking a steady step-wise erosion of the problems that confront us.

The first two days of the meeting were dedicated to the subjects which most concern culturists, namely egg quality and the availability and quality of food. With regard to the former we learned there has been some progress in relation to our ability to identify practical criteria for recognising poor quality eggs as well as in understanding the importance of hormones in controlling over-ripening and subsequent embryonic and larval development.

Evidence was presented that mollusc investigators are ahead of the rest of us in understanding parental influences on egg and larval development. We also heard that cryopreservation techniques for fish and mollusc gametes are at or near the point of commercial exploitation.

With regard to nutrition studies it is clear that our thoughts are still dominated by lipids. The HUFA story does seem to have become the soap opera of larvae culture - a never-ending tale with a plot that thickens only very slowly! Much of current work is repetitious or, to put it more kindly, confirmatory, though some increase in our understanding of requirements was evident. Since Larvi '91 the focus of attention has moved from a consideration of total or individual HUFAs to a greater appreciation of the importance of ratios, not only between DHA and EPA but also with arachidonic acid and possibly with saturates. In addition, the protective role of phospholipids has also been appreciated. It does seem we are moving towards a better understanding of how respective pathways may be interlinked, but in general the physiological and biochemical mechanisms are still unclear and important fundamental questions remain unanswered. These include:

While this part of the nutrition story gradually unfolds, other aspects of nutrition are perhaps being neglected. For example, the role of lipids vis-a-vis free amino acids and monosaccharides was barely mentioned, though there were some valuable contributions on the importance of vitamin C for both broodstock fish and larval fish and crustacea.

The session on the availability of food stressed the importance of reducing our dependence on Artemia in the light of the current world shortage of cysts, and in this respect it was encouraging to hear that some progress toward the development of microdiets both as a partial or total replacement for live foods has been made. It would, however, be premature to throw out our Artemia hatching apparatus!

Subsequent sessions on culture techniques and new species gave us some encouraging snapshots of impressive commercial developments and new species that will be coming on line, some sooner and others clearly later. The scale and diversity of recent developments in Taiwan was particularly impressive. Perhaps our enthusiasm for new species was a little dampened by the view expressed by Prof Anderson (University of Rhode Island) at an Economics Workshop that ultimately product diversity will be accomplished not by increasing the number of species but by devising a variety of ways of presenting a relatively small number of species to the market.

It was perhaps appropriate that microbiology and disease was left to the final session since disease is the last thing that a culturist wants to be reminded about! The importance of the subject, however, is clearly evident as were the various contributions that probionts can make through competitive exclusion of pathogens, stimulation of the immune system and the provision of nutrients as part of the intestinal flora. Subsequent contributions elaborated on alternative ways of stimulating the non-specific immune system using substances such as glucans and mannuronic acid. This is clearly a most important research field that will undoubtedly contribute much in the future.

Finally, an appeal not to neglect other areas of importance to the development of culture techniques which have not been prominent at this meeting. These would include studies of the behaviour and environmental requirements of target species as well as perhaps the greater use of genetic and microbiological techniques.

In summary, given that communication is the essence of science, I would say that this has been a most successful conference. We should all be grateful to Patrick Sorgeloos, his organising committee and all those who have provided us with this opportunity to exchange our information and views in such pleasant surroundings. The organisation has been impeccable and I do thank and congratulate you for your efforts on our behalf.

B. R. Howell
T. Lam


Larvi'95 - Concluding Remarks - Private Sector Participants


prepared by Richard Prickett (Marine Farm Technology, Ltd., UK) and Gidon Minkoff (Israel Salt Co Ltd., Israel)

On behalf of Gidon and myself, firstly I would like to thank Patrick and the committee for inviting us to give our opinions on behalf of the private sector and to add our congratulations to those already voiced by Bari on the usual high standard of organisation and presentation shown at this conference. Not many of us start a days work with a classical music interlude, more usually it is to the sounds of telephones and transistor radios down on the farm. Many thanks and much appreciated.

We have tried as much as possible even the last few days to collate the feelings and opinions of our colleagues and I hope that my comments will reflect most of their views as well as our own.

Concerning the quality of the posters and presentations and without singling out any single piece of work we were all impressed and informed on presentations reviewing several relevant topics. In particular:

Also we fish farmers tend to pick up odd ideas from lectures which are often not part of the main presentation. One example of this was the use of water jets instead of air for surface cleaners in larval tanks given in a paper on flatfish farming in Ecuador. I remember looking at the audience and seeing everyone who was working in a marine fish hatchery making a note in their books!

On the slightly critical side, we would have to add that some of the work presented seemed to be repetitive and "old hat" and I wonder if some researchers carry out literature searches before say 1985 when there was a lot of good ground work done for example on larval rearing using green water techniques notably by people like Barry and Ed Hoode.

On the positive side we would like to make some suggestions as to the future organisation of the conference but we realise we are a minority and these are merely ideas to be bounced around.

Finally we have already received some feed-back on future research and development projects from colleagues in the private sector. These cover such areas as:

We hope these views are seen as being constructive and will lead to further discussions in the future on a mutually beneficial basis.

Thank you for your attention.


HATCHERY HYGIENE MANUAL


By
Tom Lewis

Dear all,

Following the recent discussion on laval metamorphosis/genetics/bacteria etc I've had a number of requests for copies of our, and I emphasise **non-peer-reviewed and maybe somewhat dated**, Manual of Hygiene in Shellfish Hatcheries 45pp, (1986). I don't want to sound like I'm pushing this manual, but it seems as though some folk may like to have a look at it.

I have only 2 copies left at present, but am happy to get a few more printed/photocopied (probably the latter if that's OK) if there is some interest, and I'd like to get it all done in one hit. I'll have to charge something for these copies to cover costs, as the original 1984-86 grant money has definitely run out :-). Probably in the order of Aust$10-15 depending on copying and posting costs (incl P&P).

If anyone else is interested, please let me know directly and I'll get moving on it.


INDIAN SHRIMP FARMING HIT BY DISEASE


An outbreak of the so-called white patch disease has taken a heavy toll on shrimp farming, resulting in a loss of about Rs180 million ($5.7m) in coastal India's Andhra Pradesh last year, according to the Press Trust of India.

The mysterious disease hit about 7,060 hectares of land in East and west Godavari and Krishna districts along the east coast of India, destroying 1,686 tonnes of shrimp, according to official estimates. There was also a recurrence of the outbreak earlier this summer. Though a detailed report was not available, examination of a few samples by pathologist professor Donald Lightner of Arizona University, suggested that the cause was 'China virus'. The unauthorised import of shrimp larvae from where the disease was known to exist is said to be a possible cause of the outbreak.

(excerpt from article in Seafood International, September 1995) Bioresource Technology, 51 (1995):265-267


LABORATORY STUDIES ON THE CULTURE OF THE BRINE SHRIMP ARTEMIA USING ORGANIC WASTES


Short Communication by J.A. Basil, V.K.S. Nain and A.J. Thatheyus School of Biological Sciences, Madurai Kamaraj University, Madurai 62501, India.

Abstract :

Three different Artemia strains (Belgian, USA and Tuticorin) were tested for their culture potential when offered five different diets comprising cabbage leaves, cow dung, poultry manure and pig dung in varying proportions at 45,60, 75 and 100 ppt salinities. One mixture of the ingredients gave the best results among the five diets tested, while the optimum salinity was found to be 75 ppt. Among the three strains the Tuticorin strain gave the best results.


COLLECTION OF WILD SHRIMP SEED IN INDIA


In India, the shrimp farming boom has prompted school children to collect wild shrimp seed from small saline embankments. They collect the seed and sell them to seed collectors (middlepersons) at 0.6-1.5 Rupees/piece for Penaeus monodon and 0.2-0.5 Rupees/piece for P. indicus (US$1=3D Rp31.75). Simple hand-made scoop nets of different sizes are used. A middleperson prepares lots of about 5,000-10,000 seed and supplies them to the growout ponds (improved extensive and extensive systems). The children collect 30-40 P. monodon and P. indicus seed per day, and earn about US$1 for about one hour's work at both dawn and dusk. The cost of P. monodon seed is three times more than the P. indicus seed. The size of the collected postlarvae varies and may reach 7 g.

(excerpt from article in NAGA, ICLARM QUARTERLY Vol 18, No 2)


TECHNICAL APPRAISAL MANUAL - SHRIMP AND FRESHWATER PRAWN HATCHERY- 1994


S.K. Gosh, K. Planisamy and S.C. Pathak (Eds), published by the National Bank for Agriculture and Rural Development (NABARD), India, 87 pp.

The manual has been designed to fill the gap in shrimp and freshwater prawn hatchery information. It gives information on project formulation, designing, appraisal and implementation of hatchery projects.

Contents :

Overseas price: US$ 10. For information, contact the General Manager, GAPD, NABARD, PB No. 6552, Worli, Bombay 400018, India.


LARVAE AND BACTERIA

By
Scott Feindel

To add to the thread on larval mortalities and bacteria in various systems... A grad student from Northeastern University analyzed our seawater, larval, and algal systems for Vibrio and total viable bacteria a couple of years ago. At the time we were having high losses in larval cultures of C. virginica. Our filtration system for larvae consisted of a rapid sand filter for primary filtration followed by charcoal, and a 20um nominal cartridge filter and finally- a diatomaceous earth filter and UV treatment before filling larval tanks.

The student found that bacteria counts (for both vibrio type and general) typically rose an order of magnitude higher from bulk water after passing through the filters. UV was effective in reducing vib and total counts 80 to 95%, and there was seasonal variation (positive correlation with temperature) of both vibrios and totals. She found no direct evidence that Vibrio type bacteria were suppressed by non-vibrio background levels. Nontheless -we were still experiencing losses even with the UV treatment.

After numerous experiments- bypassing certain filters, UV- non UV treatment etc.... we ripped out the entire filtration system and switched to a two stage filter bag system (25-50um primary and 1um point of use) and have had excellent survival ever since. Having a filtration system that allowed colonization of bacteria and reducing bacteria levels to too low a concentration apparently both played a role in poor larval survival. A good example of KISS I guess (Keep it simple...)

I also agree that too high a concentration of even background bacteria is a bad thing- but I'm wondering about Tom Lewis's number of app 2x10^6/ml. Our student found that epifluorescence counts were usually two orders of magnitude higher than plate (viable) counts. Is the 2E+06 based on plating?

Scott Feindel
Nantucket Shellfish Hatchery
Nantucket, MA


LARVAE METAMORPHOSIS 1


By
Tom Lewis

In reply to Will Borgeson's comments (italics)

Most commercial and research hatcheries feed bivalve larvae live algae cells of species that have been shown to have good nutritive characteristics (tropical *Isochrysis,* *Chaetoceros* spp, etc.).

I think the most significant nutrition variable for larvae may well be what is in the egg. A typical pattern for crashing larvae is that they survive well to straight-hinge, then crash, apparently unable to feed on the *ad libitum* ration of live algae that is provided. Other times the crash happens later; around 10 days post-fert is not uncommon.

This 10-day crash often involves just a percentage of the larvae. Other times the major mortality is delayed until metamorphosis.

A group of researchers in Tasmania did some work about ten years ago which showed a positive correlation between bacterial levels in algal cultures (tropical *Isochrysis,* *Chaetoceros* spp, etc.) fed to Pacific oyster larvae (in a commercial hatchery) and crashes. (Lewis et al. 1988 Aquaculture 69: 241-251).

We found that algal cultures "seemed" to be safe until culturable bacterial counts exceeded ~2 x 10^6/mL, then any amount of said cultures fed to larvae (at any time during the larval cycle) resulted in mortalities within days.

Similar circumstances of bacteriologically -unsafe algal cultures could also explain the crashes mentioned by Will.


LARVAE METAMORPHOSIS 2


By
Will Borgeson

I've had a chance to read the paper Tom referred to on this subject, and on which he was major author. A good piece of work!

A couple of comments:

Perhaps the reason I have had good luck with pasteurization is that I deliver the seawater to the (previously bleached and debleached) algae culture vats when the water is still at 75 degrees C. Submicron filtered air is bubbled into the vats overnight to help cool the water and to maintain positive pressure in the (covered) vats so that pathogens cannot enter. Typically an inoculum and nutrients are added the next morning. Within a few days the *Isochrysis* cultures are in the range of 1.5 million to 2 million cells/ml, and are ready to feed to larvae.

A final note - in Tom's study larvae started out at 16 d-stage per ml. In part due to advice from commercial hatchery personnel and in part due to our own observations, we feel that, at least in static cultures, this is a bit high. We start at 5/ml and usually end up with around 1/ml worth of spat. This varies greatly however depending on genetic group, degree of inbreeding, season, etc.


WATER TREATMENT IN ALGAE CULTURE 1


By
Will Borgeson

In reply to Daniel E. Gruenberg (italics) Will,

Could you please explain to me why you chose to pasteurize the water instead of using more typical techniques for creating sterile water? With milk the reason for pasteurization is to try to preserve certain nutritional factors and protein structures. Is there someting you are trying to protect in the water.

I apologize if this is an ignorant question, but larviculture is not my specialty.

Not at all Daniel. Some years ago I had been having periodic problems with contamination and resultant algal crashes; *Paramecium*-like protozoans were usually responsible. I had been using nominal 1 micron filtration and UV to prep the seawater for algal culture.

In setting up a hatchery for larviculture, I chose a plate/frame heat exchanger to bring the seawater up to 25 C for water changes. In the face of the recurrent algal crashes, I decided to see how hot the exchanger could get seawater. It brought it to 70-80 C with reasonable flow. This led me to try this water, once cooled, for algae culture. It worked extremely well, and I've been using it ever since.

I also tried .2 micron filtration. This also worked very well, but the filter cartridges I was using were very expensive, needed to be backflushed with water that also needed to be submicron filtered, and only let me know if they were blown out when contamination again occurred. The pasteurization technique is far more reliable and virtually free. I do however look forward to hearing more about Tom's filtration setup.

In answer to your question re: protection, I'm protecting the algae cultures, and perhaps selecting for certain bacteria. I'm aware that some bacteria are getting through, just as some bacteria survive milk pasteurization.

The heating no doubt does things to the seawater chemistry (as does UV, bleach/debleach). It just kills all the protozoans that eat algae. I'm now intrigued by Tom's work re: bacteria, and may find a way to more closely examining what pasteurization is doing to them. It's interesting to note that even the .2 micron filtration allows some bacteria to pass.


WATER TREATMENT IN ALGAE CULTURE 2


By
Robert Rheault

I would just like to echo Will's comments of UV/Chlor-dechlor and pasteurization for phytoplankton culture.

I also have had problems w/ UV and found that while it may kill some or most- you never get all, and you do leave a nice enrichment that is out of balance in terms of microbial ecology- in my experience often leading to a bloom of vibrios.

Chlorination leaves chlorinated organics if you have any organics (who doesn't) and if you have nitrate you can even generate toxic levels of nitrosamine. (algae don't seem to mind, but bivalve lavae do) I have tried all three and had strongest algal cultures w/ pasteurization. Pasteurization may even be preferable to autoclaving because certain metals will precipitate or form bizare complexes at higher temperatures.

Also- for large batch or semi-continuous algal cultures I have had good sucess w/ 0.2um FSW. Filter costs and success vary greatly w/ manufacturer and real porosity of cartridge.

One last observation- Healthy algal cultures are usually nearly bacteria free in my experience. I would not say axenic, because I know my stock cultures were contaminated w/ many different bacteria. However, once up in significant numbers in a large healthy culture, I could never plate anything out! If I did, it usually preceeded a crash in the algal culture.

I would love to see an equipment supplier come out with a plate heat exchanger to heat the water up and hook it up to a counter current heat exchanger to both take advantage of the waste heat in the post-pasteurized SW in heating up incomming SW and to speed up cooling off the pasteurized SW so that it could be used sooner.

Bob Rheault
Moonstone Oysters


WATER TREATMENT IN ALGAE CULTURE 3


By
Will Borgeson

I already have the two heat exchangers (ITT) and beaucoups cold seawater to do the cooldown. Up to 80C, down to 25C for culture. Now it's just a matter of plumbing (time & energy). Hope to vary the dwell time, do plating work on the water and see what gives.

Aquaculture, 134 (1995):257-268


LARVAE AND BACTERIA


M. Yufera, C. Fernandez-Diaz, E. Pascual
Instituto de Ciencias Marinas de Andalucia (C.S.I.C.), Apartado
Oficial, 11510 Puerto Real, Cadiz, Spain
Abstract:

Food consumption rates of gilthead seabream larvae from first feeding to 20 days of age were studied by feeding larvae on two types of microcapsule (hard- and soft-walled) having diameters ranging from 25 to 400 micrometer. Experiments were carried out in 15-litre beakers using larvae cultured with standard techniques and fed on rotifers and Artemia sp. nauplii. Ingestion rate ranged from 3 to 35 particles/larva/h but, except for some increase during the first days of feeding, no consistent trend was observed in relation to larval weight. However, when values were converted to volume or dry weight units, food consumption rate increased progressively with increasing larval weight. On a volume basis, the rate of this increase was different for the two microcapsule types, being generally higher with soft microcapsules, probably due to its better accessibility and to their easier progress through the digestive tract. However, on a dry weight basis the rates of food consumption were similar with both types of microcapsule because of the higher density of hard-walled microcapsules. Ingestion rates increased from 0.5-3 microgram/larva/h at first feeding up to 18-25 microgram/larva microgram/h in older larvae. The daily specific ration increased from O.5 to 2.5 microgram food/microgram larva/d at first feeding to a maximum of 3-5 microgram food/microgram larva/d at a larval dry weight of 70-110 microgram. Above this larval weight, the specific ration tended to remain constant. The results show that gilthead seabream larvae can be transferred directly from live to microencapsulated food without appreciable variation in the incidence of feeding or in the rate of food consumption from first feeding onwards.

Aquaculture, 134 (1995):269-278


CAPACITY OF GILTHEAD SEABREAM, SPARUS AURATA L., LARVAE TO BREAK DOWN DIETARY

MICROCAPSULES
C. Fernandez-Diaz, M. Yufera
Instituto de Ciencias Marinas de Andalucia (C.S.I.C.), Apartado
Oficial, 11510 Puerto Real, Cadiz, Spain
Abstract:

The capacity of hatchery-reared gilthead seabream larvae to disintegrate mixed-walled protein/carbohydrate microcapsules in their midgut was studied. The response of feeding larvae to eight types of microcapsules was examined. The effects of the following factors, which could potentially influence capsule breakdown, were evaluated: (a) larval age; (b) addition of enzymes to the diet; (c) concentration of cross-linking agent; and (d) procedure for isolating microcapsule after formation.

The results revealed that larvae belonging to the same population exhibited substantial individual variability in their capacity to disrupt the microcapsule wall. Despite this, there were clear differences in the ability of the larvae to break down different types of microcapsules, depending principally on the technique followed during the isolation phase of microcapsule elaboration. Thus, capsules isolated in gelatin (type G) were more easily broken down than those isolated in alcohol (type A),irrespective of larval age ( P<0.001 ) . Addition of enzymes to capsules did not result in any significant changes (P>0.05) in the degree of capsule breakdown. Likewise, the concentration of cross-linking agents used to form the capsule walls had no effect on capsule disruption by the larvae.

The present results suggest that seabream larvae are able to digest inert food from the onset of exogenous feeding. Their capacity to do so is, however, influenced by the thickness of the capsule coating and by their age. The results of this study also contribute to our knowledge of the behaviour of a cultured larval population in the presence of microencapsulated food.

Aquaculture, 134 (1995):307-311


FLUOROMETRIC DETERMINATION OF SELECTIVITY BETWEEN LIVE AND INERT FOOD BY PENAEUS JAPONICUS LARVAE


V. Marin-Magan, J.P. Canavate
CICEM 'El Toruno' Junta de Andalucia, P.O. Box 16, 11500, Puerto
de Santa Maria, Cadiz, Spain
Abstract:

Preferences between microencapsulated particles and phytoplankton cell food particles by P. japonicus larvae were studied. The gut pigment content method was used when both types of particle, in the same proportions, were offered suspended in the medium. A lack of feeding selectivity between both types of particles was found for zoea stages, whereas mysid feeding was based on a preferential ingestion of microencapsulated particles. The feeding mechanism and the effects of particle morphology are discussed.

Aquaculture, 134 (1995):325-337


VARIATION OF ASCORBIC ACID CONTENT IN DIFFERENT LIVE FOOD ORGANISMS


G. Merchie, P. Lavens, Ph. Dhert, M. Dehasque, H. Nelis, A. De Leenheer, P. Sorgeloos
Laboratory of Aquaculture & Artemia Reference Center, Rozier 44, B-9000 Gent, Belgium
Abstract:

Ascorbic acid (AA) is an essential nutrient both in particle and live aquafeeds. In order to better assess the needs for this nutrient during larviculture the AA content of algae, rotifers and Artemia was studied with respect to their suitability at startfeeding. In general, the microalgae evaluated were rich in AA (1000-4000 microgram AA/g DW), but showed a considerable variability among the different species: e.g. the concentration in Isochrysis and Chlorella reached values 3-to 4-fold the percentage of Tetraselmis (0.11% of DW). Brachionus routinely cultured on Chlorella contained 2300 microgram AA/g DW. Cysts of various batches and strains of Artemia differed considerably in ascorbic acid-2-sulphate (AAS) concentration (296-517 microgram AA/g DW).The amount of AA available in the freshly-hatched nauplii reflected exactly the AAS reserve present in the cysts, what evidences the complete conversion of AAS to free AA during completion of embryonic development into nauplii.

Boosting techniques both for Brachionus and Artemia using ascorbyl palmitate (AP) as the vitamin C source were established. The addition of 20% AP in the diet of Brachionus enhanced their AA content 10-fold over 3 days of culture. Supplementation of the enrichment emulsion for Artemia with 20% AP increased the AA content up to 2000 microgram/g DW after 24 h enrichment. This lipophilic derivative of AA appeared to be a stable form of vitamin C for enhancing AA levels in the live diets during culture and/or enrichment. This bioencapsulation method provides a tool for hatcheries to build up high AA concentrations in the live prey administered to first feeding larvae of aquaculture organisms in case of specific requirements (e.g. with respect to handling stress, deformities).

A survey of commercial hatcheries indicated that a wide range of products is used for the cultivation and boosting of rotifers, which consequently affect their AA levels. In general, the AA content in the algae and, consequently, the algal enrichment of Brachionus tended to score lower in the hatcheries than under lab conditions.

Aquaculture, 134 (1995):351-359


THE EFFECTIVENESS OF TRICAINE, QUINALDINE SULFATE AND METOMIDATE AS ANESTHETICS FOR LARVAL FISH


K.C. Massee, M.B. Rust, R.W. Hardy, R.R. Stickney
School of Fisheries, WH-IO, University of Washington Seattle WA 98195,USA
Abstract:

Tricaine, quinaldine sulfate, and metomidate were compared as anesthetics for larvae of two species of fish, red drum (Sciaenops ocellatus) and goldfish (Carassius auratus). Larvae were exposed to various concentrations of each anesthetic and the percentages of fish reaching stage 4 of anesthesia, post-exposure recovery, and survival were recorded. Effective concentrations were defined as those which induced stage 4 of anesthesia within 3 min after exposure with a recovery time of 10 min or less. Post-exposure survival of 100% was an additional criteria used to define effective anesthetic concentrations. The lowest effective concentration of tricaine for red drum was 55 mg/l (26=F8C), while 50 mg/l (24=F8C) was the lowest effective concentration for goldfish. The lowest effective concentration of quinaldine sulfate for red drum was 35 mg/l (26=F8C), while 60 mg/l (24=F8C) was the lowest effective concentration for goldfish. Metomidate was found to be an ineffective anesthetic for both red drum and goldfish larvae based upon survival and recovery times. Mortality occurred in red drum larvae at all tested concentrations of metomidate. Larvae of both species that survived anesthesia with metomidate had longer induction and recovery times compared to larvae exposed to tricaine and quinaldine sulfate.


ARTEMIA CULTURE IN THE MEKONG DELTA OF VIETNAM


(correspondence from Cao Thang Binh to Shrimp News International, Sept./Oct. '95)

(the author is lecturer at the Faculty of Fishery of Can Tho University in Vietnam, and has been working in shrimp farming for over seven years, at hatcheries, nurseries and farms. He has also worked with Artemia at small-scale salt farms in the Mekong Delta. His research covers shrimp farming systems, the technical and socio-economic aspects of shrimp farming, and the environmental impact of shrimp farming on mangroves in the Mekong Delta For more information on this subject, contact Cao Thang Binh, Aquaculture Program, AIT. G.P.O. 2754, Bangkok 10501, Thailand (phone 66-2-524-5485,fax 66-2-524-6200, email 949658@ recvax.ait.ac.th).

Artemia culture in Vietnam was initiated in 1983 when researchers at Can Tho University imported cysts and began experiments in Vinh Chau District, where farmers produced salt during the dry season and shrimp during the wet season. Artemia production was integrated into this cycle and became profitable in 1988.

Artemia are cultured in salt evaporation ponds during the dry season. Every day, high salinity water is removed from the Artemia ponds and channeled to nearby salt extraction ponds. It is replaced with water from a low-salinity reservoir. During the rainy season, when salinity levels are too low for Artemia production, the ponds are used for shrimp culture.

Either dried or wet Artemia cysts can be used to inoculate the ponds. Wet cysts are dehydrated by immersion in salt-saturated water for a few hours before incubation. Incubation is carried out in conical jars or buckets supplied with strong aeration. Incubating density ranges from 3 to 5 grams of dried cysts per liter and inoculating densities ranged from 200 to 300 grams of dried cysts per 0.5-hectare pond (approximately 1 kg of dried cyst equals 2.5 kg of wet cysts). The inoculation of nauplii was usually carried out in the early morning or late evening when the water temperature was low. Nauplii were gently released along all four dikes.

In nearby reservoirs, we culture algae to feed the Artemia. Chicken manure and rice bran are also used to feed adult Artemia. Chicken manure is placed in bags or bamboo baskets and immersed in water for 2 days. Then it's spread over the pond. Rice bran is mixed with water in a bucket and fed directly to the Artemia. Feed amount is based on the Artemia population, pond water color, and the amount of algae pumped per day. On average. about 80-100 kilograms of chicken manure and 10-20 kilos of rice bran were fed per hectare per day. Trials with pig and cattle manure resulted in lower cyst production.

Annually, with this integrated system, farmers averaged 84 kilograms of Artemia cysts (wet weight), 371 tons of salt, and 164 kilograms of shrimp per hectare. More intensive systems produced up to 150-200 kilograms of wet cysts per hectare. From 1990 to 1994, approximately 150 hectares in Vinh Chau District (Soc Trang Province) and Vinh Hau District (Minh Hai Province) produced 8-10 tons of wet cysts annually. If Artemia biomass is used for nursing shrimp fry or for semi-intensive culture, more profits and higher shrimp production can be expected. The results indicate a great potential for developing this system, although, at the present time, it's confined to a small area because Artemia culture requires know-how and investment.

The integration of Artemia production with traditional methods of salt/shrimp production offers many advantages. Artemia biomass is used to feed the shrimp. The high densities of phytoplankton in the waste water of the shrimp ponds is pumped to the Artemia ponds, where the Artemia consume the algae and, in the process, clean the water. Then, the high saline "algae-free" water from Artemia ponds is channeled to salt extraction ponds.

Although Artemia cysts could be exported at high prices, the farm-gate price is still quite low. In 1989, it was only 0.5 U.S. dollar for a kilogram of wet cysts. After that it increased to 3-4 U.S. dollar in 1990, 5 U.S. dollar in 1991, and 10 U.S. dollar in early 1995. Obviously, the expansion of Artemia culture in the Mekong Delta heavily depends on the market for Artemia cysts, farmers'investment ability, and the extension of Artemia culture techniques.


TAURA DISEASE


In mid-June 1995, survivals of wild-caught seed were ranging from 50% to 60%; survivals of hatchery-produced seed, 20% to 30%. Some evidence suggest that good old vannamei has already become more resistant to the virus, in the same way that stylirostris appears to have become more resistant to the IHHN virus in Mexico's Gulf of California.

Wild Postlarvae: In pond systems affected by TS, the survival of captured wild postlarval Penaeus vannamei has been apparently higher than that found for postlarvae which have come out of hatcheries. Thus, if available, wild postlarvae may be the most appropiate to stock on farms with a history of losses from TS.

Alternate Species: Transmission (infectivity) trials reported in this paper which compared the relative sensitivity of juvenile, SPF P.vannamei and P. stylirostris indicated that P. stylirostris was far less sensitive to TSV than P. vannamei. This provides a clue that P.stylirostris may serve as an effective, alternate shrimp species which can be farmed instead of P.vannamei in areas affected by TS. However, P.stylirostris will probably not be effective in those shrimp farming areas where IHHNV is endemic, unless a specific pathogen resistant (SPR) strain of P.stylirostris is available.

TSV Resistant P.vannamei: Taura Syndrome results in a marked mortality of exposed populations of farmed P.vannamei. We have observed in raceway populations in Hawaii that within a few weeks of a TS epidemic the survivors display no outward indications of TS. These shrimp go on to grow and perform normally. The mechanism for this apparent 'resistance' is unknown. However, perhaps, these shrimp have a tendency that is inheritable. If this is the case, then, potentially, these animals have been rigorously selected for and could serve as a founder stock for a program to selectively breed for resistance to TSV. Another possibility is that the survivors received a sublethal exposure to TSV and were able to develop an induced (immune mediated) resistance to the agent.

Hatchery managers used to be skeptical about stylirostris, but now (September 1994) they realize it is easier to work with than vannamei. Hatcheries are now producing equal amounts of vannamei and stylirostris. Some hatcheries produce 100% stylirostris.

Ecuador: Jerome Thompson owner of Intensive Culture Systems,a consulting firm which specializes in closed system shrimp farming, reports:

When I arrived [1994] in Guayaquil, Ecuador, I noticed that most of the mortality from Taura seemed to occur during the first week that the animals were exposed to pond/river water (PL-12 to PL-20). By the end of the second week most of the mortality seemed to have subsided. I put together a small closed system consisting of 2 holding tanks and an interconnected biofilter tank. The idea was to protect the PLs during what I believed to be the period of maximum susceptibility. The first experiment consisted of stocking the holding tanks and dividing the population into three groups (each with three replicates). The first group, the control, was held for three days in the system (typical acclimation time at this farm) and then stocked in growout ponds. The second group was held in the system for 15 days and then stocked, while the third group was held in the system for 30 days and then stocked. Each group was grown for 120 days. At harvest, the control group was 123-days-old, the second group was 135-days-old, and the last group was 150-days-old. Survival in the control group was about 25%, in the second group about 48%, and in the third group about 58%. Average survival at this time for animals acclimated without closed system was between 12% and 15%. Not only did the control group have the poorest survival, but it also had the smallest shrimp. The animals in the control group weighed about 11 grams at harvest, while the 30 day animals were between 15 and 16 grams.

(excerpts from article "Taura, Taura, Taura" in Shrimp News International, Sept./Oct. 1995)


HATCHERY DEVELOPMENTS IN HONDURAS


At the Symposium, Jim Norris, manager of GMSB's big hatchery in Florida, talked about hatchery developments in Honduras. He said, in April 1995, there were approximately 11,050 hectares of shrimp ponds in Honduras, requiring approximately 3.5 billion postlarvae a year. Here are some excerpts from his presentation:

Since 1991, fourteen hatcheries have received permits to operate on the Gulf of Fonseca. Nine are currently in operation. They could supply 32% of the country's seed requirement. In addition, each year, GMSB, Inc,. a Honduran owned hatchery in Florida, supplies an additional 34% of the country's requirement, or 1.2 billion Penaeus vannamei postlarvae, to its parent company in Choluteca, Honduras. The remaining 36% of the seedstock is supplied by fishermen and other imports.

Of the nine hatcheries in operation, four produce less than 10 million postlarvae per month; the remaining five produce between 20 and 25 million per month. Currently, there are no combination hatchery/maturation systems in Honduras.

Close communications between Grupo Granjas Marinas, S.A., in Honduras, and GMSB, Inc., the hatchery in Florida, allow us to coordinate stocking schedules and hatchery production. These communications address questions about the age and size of postlarvae and help relieve the tension that surrounds postlarvae transactions. The farms want the largest postlarvae possible and the hatchery wants to ship the smallest animal possible. Several tools allow us to attach a value to the quality of a group of postlarvae, thus eliminating as much as possible, any subjective grading by hatchery technicians and farm personnel.

One of the first criteria used in the hatchery is gill development. Gills must demonstrate secondary branching before a group of animals is considered ready to acclimation prior to shipping. After acclimation a group of postlarvae to salinity conditions on the farm, a sample of these animals is challenged with a stress test at the hatchery. The stress test consists of a mixture of salinity, temperature and pH adjustments and closely matches the stress test given on the farm prior to introducing the animals into the ponds. The results from these two tests should compare favorably and depending on the percent of animals that survive a grade for the group of postlarvae is calculated. Depending upon the grade, the postlarvae can either be stocked directly into a production pond or put through the nursery system if quality appears marginal.

For the stress test, postlarvae are maintained in a tank with the following environmental characteristics for four hours. Water 10 degrees lower than ambient, salinity 15 parts per thousand lower than ambient and pH 0.5 lower than ambient. The percentage of live and active animals after four hours is the grade for the group. Healthy animals larger than an eight day old postlarvae survive the challenge quite well. If there were no unanticipated stresses during shipment, the hatchery test and the farm test will give similar results. If there are significant differences between hatchery and farm results, it is relatively easy to trace them.

Our hatchery ships to five different farms, each having specific salinity requirements that vary through the year. The task of acclimating postlarvae several days prior to shipping is a real challenge. Proper acclimation, however, is very beneficial because it means stocking a stronger animal. Hatcheries, for the most part, have more controls and equipment for safely acclimating postlarvae than farms, where transfers may have to be done on a pond bank, at night, and in the rain.

(from article by Bob Rosenberry in Shrimp News International, July 24, 1995)


MOLECULAR BIOLOGY AND GENETIC ENGINEERING OF FISH


by Benzion Cavari and Bruria Funkenstein

a) Growth hormone and acceleration of fish growth rate A major contribution to the growing worldwide demand for seafood will undoubtedly come from aquaculture of finfish and shellfish. One possible method to increase the efficiency of fish production is the application of growth stimulating hormones. Although several hormones have the disadvantages of acting on the human consumer, and like steroids, are slowly biodegraded, growth hormones are not. Cross-activity between different species is relatively rare. In the gilthead seabream Sparus aurata it was shown that growth hormones from vertebrate sources have low or no effect on growth rate. In addition, growth hormone (GH) is a protein which undergoes quick denaturation by heat and the physiological conditions in the mammalian digestive tract. Therefore, it can be expected that the use of this protein in edible fish will have no effect on the consumer.

GH can be produced in large quantities only by recombinant DNA technology. To circumvent all problems which arise from introducing a foreign gene into edible fish, it is proposed to produce the GH in bacteria and then purify it by standard biochemical techniques. The pure protein will then be introduced into the fish.

Several experiments were carried out on the growth rate of Sparus aurata, using recombinant growth hormones from human, bovine, porcine and chicken. The GH was injected intramuscularly or intraperitoneally twice each month. Only a slight increase of about 15% in fish growth was obtained when human or bovine GHs were administered.

The phylogenetic tree of GH consists of several trunks, and the four GHs examined are on one trunk which is different from the teleost trunk. Additional experiments ware therefore performed with recombinant fish GHs.

In one experiment, implants of salmon GH were prepared, and were introduced subcutaneously. The implants were programmed to release the hormone in about 100 days. No acceleration of growth resulted from such treatment.

The salmon GH is on the teleost trunk of the phylogenetic tree; it is, however, not on the same branch as the S. aurata. Another experiment was performed with tuna GH, which is very closely related to the S. aurata . Weekly injection of this hormone to S. aurata fish accelerated the growth rate of the fish by about 50%.

It is now planned to produce the S. aurata GH, to inject it into fish, and also to prepare implants with this hormone to be introduced subcutaneously. It is believed that the growth acceleration will be at least as good as that obtained with the tuna GH. The S. aurata GH cDNA was cloned by us, and an expression construct was made. It was shown that this construct produces the GH of S. aurata in large quantities.

In order to be able to market fish which were treated with the GH, it is required to show that when the fish are marketed, the GH level in their blood is similar to that in nontreated fish. For this reason, a native GH was isolated and HPLC purified from pituitaries of fast growing S. aurata. Antibodies were prepared to this GH, and served in Western blots to detect S. aurata GH. It is planned to increase the sensitivity of the detection by preparing a radioimmunoassay for S. aurata GH.

b) The role of growth factors in early embryonal development Experimental evidence suggests that the action of GH in mammals, and probably in fish as well, is not direct, but is mediated by a group of growth factors: somatomedins or insulin-like growth factors (IGF), especially IGF-I. Very little is known regarding control of growth and differentiation in fish during early stages of development. In order to study the expression of this gene during early development, a cDNA coding for S. aurata IGF-I was cloned.

Poly (A ')RNA, which was prepared from livers of adult S. aurata fish during their fast growing period, was used for cDNA synthesis. Sparus IGF-I was then cloned using PCR techniques. Sequences for the 5' untranslated region, the signal peptide and part of the beta domain sequence were determined from the genomic clone. Based on these data, the remaining coding sequence was obtained by anchored PCR and 3' RACE. The composite sequence of Sparus IGF-I cDNA contains a 546 nucleotides open reading frame and encodes 182 amino acids of the pre-proIGF-I.

Based on the sequence of this cDNA, two primers were designed in order to use the highly sensitive reverse transcription-polymerase chain reaction (RT-PCR). It was found that a fragment of 237 nt was amplified from total RNA prepared from unfertilized eggs, and also in embryos at 4, 8, and 12 hours after fertilization as well as in larvae on the day of hatching. It was verified that the amplified fragment was IGF-I. These results suggest that maternal IGF-I transcript is present in fish eggs.

The presence of IGF-I gene in fish embryos may indicate that growth factors are important during early development. Presence or function of other growth factors has not been studied in fish. In addition to the importance of studying the function of growth factors in embryonic development of fish, fish may serve as a model system for studying the molecular biology of vertebrate development. A small aquarium fish, the Japanese medaka, was chosen for this study. The advantages of this fish system are as follows: they can be easily maintained in the laboratory, and large numbers can be bred on a routine basis; eggs and embryos can be made available all year round; fertilization and development are external; the eggs and embryos are translucent, and embryogenesis can be observed easily under a stereomicroscope; the medaka has a short life cycle of 3 to 4 months.

To examine whether sequences that are homologous to the growth factor cDNAs from different organisms are present in the genome of medaka, a probe specific for each growth factor was used. A number of hybridization bands were detected with each of the growth factor probes tested. These data strongly suggested that in medaka these growth factors are present.

A cDNA library was prepared from the whole body of medaka, and the cDNAs of basic fibroblast growth factor (bFGF), transforming growth factor-alfa (TGF-alfa) and transforming growth factor-beta (TGF-beta) are now cloned.

In addition, the effect of these growth factors on developing medaka embryos was studied. For assessing the rate of embryo development, the incorporation of radioactive labelled thymidine into the DNA fraction of the embryo was determined. It was found that addition of bFGF to the rearing medium caused an acceleration of 3H-thymidine incorporation into the embryos. It is known that embryos that grow faster are of better quality than slow growing embryos.

c) Somatolactin, a newly discovered pituitary hormone Somatolactin (SL) is a glycoprotein newly isolated from pituitaries of teleosts. It is related to the growth hormone/prolactin family, produced by the pars intermedia cells, and secreted to the blood. The physiological role of the somatolactin is currently unknown. However, the conserved nature of SL indicates that it may have a very important function.

It is planned to elucidate the physiological role of this hormone in S. aurata SL was isolated from the pituitary by alkaline extraction, gel filtration on a Sephadex G-100 column, and reversed-phase high-performance liquid chromatography (rpHPLC) on a TSK gel ODS-120T column. The purified protein was confirmed to be somatolactin by immunoblotting using chum salmon somatolact in antisera. It was found that S. aurata SL consists of at least two forms. One form (28 kDa) is probably a glycosylated form, while the other (25 kDa) is a simple protein form, as was also found in Atlantic cod.

For determination of the primary structure, the SL was digested with lysyl endopeptidase and with endoproteinase Asp-N, and cleaved with cyanogen bromide. The resulting fragments were separated by rpHPLC and subjected to sequence analysis on an automated gas-phase sequencer employing an Edman method. It was found that the S. aurata SL consists of 207 amino acids. Alignment of its sequence with the SL sequences of flounder, halibut, lumpfish, chum salmon and Atlantic cod, showed remarkable conservation of the SL sequence of 86%, 83.5%, 78%, 76% and 71.5%, respectively.

In order to clone the cDNA of SL, the cDNA library of S. aurata was screened with cod and flounder SL cDNA probes. A fragment of about 2.5kb was isolated from several recombinant phages, and it is now being analyzed for the nucleotide sequence. With the amino acid and nucleotide sequences in hand, it will be possible to study the function of SL in this fish.

(article in Israel Oceanographic & Limnological Research, Biennial Report 1992 -1993)


CRUSTACEAN REPRODUCTION STRUCTURE AND BIOSYNTHESIS OF LIPOPROTEINS AND ACCUMULATION OF LIPIDS IN THE OVARY


by Bruria Funkenstein and Esther Lubzens

The increased world wide demand for edible marine shrimp resulted in the development of large scale aquaculture of penaeid shrimp in South America (mainly Penaeus vannamei) and Southeast Asia (Penaeus monodon and P. japonicus). The species P. semisulcatus was found to be the most appropriate one for culture in the southern part of Israel.

Following the success in producing large quantities of post larvae with high survival rates, there remain two main problems which must be overcome in order to reach the stage of commercia1 production of the shrimp. The first one is the development of an adequate, cheap pellet food diet that will promote fast growth of post larvae to commercia1 size. Secondly, full control over the reproductive cycle is required to permit year-round production of good quality eggs. This necessitates research into the processes associated with oocyte development (vitellogenesis) and their hormonal control.

Lipids constitute up to 30% of the wet weight of ovaries. Phospholipids and triacylglycerols containing saturated and unsaturated fatty acids were found to accumulate in the ovaries of P. semisulcatus during vitellogenesis.

These lipids originate in the food and accumulate in hepatopancreas on route to the ovary. Triacylglycerols and phospholipids were found in the high density lipoprotein (HDL) fraction that was prepared by density gradient ultracentrifugation from the hemolymph of vitellogenic females. The triacylglycerols were not detected, however, in either the HDL of males or that of non-vitellogenic males. Two lipoproteins were identified in the HDL of vitellogenic females: vitellogenin (the female specific protein) and LP1. The LP1, which was also found in male HDL showed one ~ 100kDa polypeptide in SDS gel electrophoresis, under reducing conditions. The mode transport of lipids by the lipoproteins from the food consumed in gut and/or the lipids stored in the hepatopancreas is the main objective live of a current research project. The results are relevant for formulation of diets required to promote growth and reproduction.

Evidence accumulating in our laboratory points to intra-ovarian synthesis of vitellin, one of the most prominent proteins in developing oocytes. Immunoisolation from cell-free translation of poly(A+)RNA using antiserum against purified vitellin produced a 35 kDa polypeptide. It is assumed that the difference between this polypeptide and the larger 2-4 subunits (MW ranging from 80-158 kDa) found in the native Vt (MW =3D 283 kDa) is due to the non-glycosylated nature and lack of bound phosphate in cell-free translation.

Indeed, the removal of oligosaccharides from purified Vt by enzymatic digestion with N-glycosidase F resulted in a smaller polypeptide of 36 kDa, which is very close to the molecular weight of the translation product.

Synthesis of cDNA to poly(A+) RNA that was extracted from vitellogenic ovaries resulted in a major band which was detected by ethidium bromide staining.

This band, as well as a cDNA insert purified from the library, was estimated to have 1.1 kb. Both hybridize to mRNA prepared from ovaries or hepatopancreas of vitellogenic females, but did not hybridize to mRNA prepared from the hepatopancreas of males and non-vitellogenic females, testes or subepidermal adipose tissues. These results suggest that vitellin, which is produced in the ovary, and vitellogenin, which is synthesized in the hepatopancreas, are the gene products of one gene. We plan to study the regulatory mechanisms which determine the level of expression of vitellin and vitellogenin during the process of vitellogenesis.

(article in Israel Oceanographic & Limnological Research, Biennial Report 1992-1993)


THE SAMUEL LUNENFELD ROTIFER CENTER


by Esther Lubzens

A rotifer reference center was established in 1991 to offer scientists and fish growers help and advice in culturing the rotifer Brachionus plicatilis as food for marine fish larvae. Available information also includes an updated list of publications on B.plicatilis and a list of the locations in which it is cultured.

Several strains with inherent traits important for mariculture are maintained as a "live library" in our laboratory. These are sent to various locations on request. The strain selection includes large and small size rotifers which are suitable for culture at various temperatures and salinities. These strains were obtained from various zoogeographical regions.

The optimal conditions (temperature, salinity and type of food) for obtaining the highest reproductive performance was determined for each of the cultured strains or clones. Using this information, it is possible to recommend the most appropriate strain for culture in a specific hatchery.

Various strains have been sent to hatcheries in Greece, Spain, Belgium, Norway, Iceland, Portugal and Japan.

In order to help us in the identification of the various strains, a simple method for extraction of mt DNA was developed. The extracted mt DNA of each strain was subjected to digestion with restriction endonucleases, giving a specific pattern for each strain. These patterns were determined for over 20 strains now held in the live library.

Keeping live strain or clone cultures of rotifers is problematic. Due to the high reproductive rate of rotifers, a change in strain may result from random mutation. The periodic transfer of cultures may lead to accidental contamination. Also, death of cultures results in loss of characterized important strains. We developed methods for cryopreservation at -196=F8C of amictic eggs (which are actually embryos) that will ensure a long term supply of unchanged strains or clones.

(article in Israel Oceanographic & Limnological Research, Biennial Report 1992-1993)


A GAMETE AND EMBRYO BANK FOR CULTIVATED FISH


by Esther Lubzens

Future advances in aquaculture will depend on genetic improvement programs for currently cultured commercial finfish. These programs include the genetic engineering of fish for accelerated growth, whole chromosome set manipulations for the production of fast growing triploids and all female stocks, and selection for commercially important traits such as growth and disease resistance. However, the practical implementation of these genetic improvement programs requires the cryopreservation of gametes which is necessary for: (a) preservation of genetically improved stocks, especially valuable transgenic fish stocks; (b) transfer of genetic material between locations, with reduced chance of parasite or disease infections; (c) production of diploid individuals by androgenesis, which permits obtaining fish with exclusive inheritance of the paternal chromosomes; this is achieved by fertilization of eggs whose maternal genetic content is inactivated by irradiation; (d) self fertilization of fish changing their sex (e.g. Sparus aurata), thus promoting faster selection of genetic traits; (e) cross breeding between related species with non-overlapping reproductive seasons; and (f) obtaining fertilized eggs without the need to synchronize the reproductive phases of males and females.

The use of low temperature for live preservation of a variety of organisms, as well as cells and tissues of plant and animal origin, has gained broad acceptance in science and technology during the past 30-40 years. Cryopreservation will allow the maintenance of genetic material at a fraction of the cost involved in maintaining live fish. The advantages of using cryopreservation techniques have been widely recognized and successfully applied to cattle and other domesticated animals. Although the importance of applying this technique to marine and freshwater cultured fish has been recognized for many years, this is yet unavailable for widespread use. Furthermore, many nearshore areas and estuaries, where marine organisms (including invertebrates and fish) breed, are endangered today by increasing land-based pollution. This results in lower production of larvae, which may lead in extreme cases to extinction of species of ecological and/or economic importance. In the development of successful techniques for the cryopreservation of fish gametes, the following considerations must be taken into account:

Methods for cryopreservation of fish spermatozoa are numerous and well established for several freshwater and marine species. However, these methods must be developed and tested for each species, in order to have a fully operational technique on hand. Some of the required practical information includes the number of cryopreserved spermatozoa needed for fertilization of a known number of eggs, the variation among individual fish, and the effect during the breeding season on success of cryopreservation.

Commercially operating fish spermatozoa banks have not yet been established. In the past few years we have directed our efforts towards establishing the first such spermatozoa bank for the freshwater ornamental carp (Japanese koi), which is cultured and exported from Israel.

We plan to develop methods for cryopreservation of spermatozoa that could be used immediately for genetic improvement programs (as mentioned above) and as commercial products on their own. We are interested in forming a spermatozoa bank for species cultured in Israel (Epinephelus aeneus, Sparus aurata, Mugil cephalus, Dicentrarchus labrax and hybrid striped bass). The possibility of maintaining fish eggs or embryos at temperatures near the freezing point of water and/or in a cryopreserved state is now being investigated. The techniques developed will be useful also for preservation of yolk-containing eggs of other aquatic or marine organisms.

This project is carried out in collaboration with Dr. W. Knibb, National Center for Mariculture (IOLR) and Mr. Yoav Magnus (Kibbutz Maagan Michael).

(article in Israel Oceanographic & Limnological Research, Biennial Report 1992-1993)