222 the Israeli Journal of Aquaculture Bamidgeh 55(4), 2003, 222-242
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222 The Israeli Journal of Aquaculture – Bamidgeh 55(4), 2003, 222-242 THE 7TH ANNUAL DAN POPPER SYMPOSIUM OF THE NATIONAL CENTER FOR MARICULTURE ISRAEL OCEANOGRAPHIC & LIMNOLOGICAL RESEARCH LTD. May 8, 2003 Eilat, Israel Abstracts of Oral Presentations David Sklan. Tools for studying intestinal function ...... Ingrid Lupatsch and G. Wm. Kissil. Modeling of energy and nutrient budgets in intensive aquaculture systems...... Daniel Conijeski, Noam Mozes and Israel Haddas. The Mega-flow® recirculating aquaculture system for intensive fish farming: performance results from two pilot operations ...... Micha Eshchar, Noam Mozes, Michael Fediuk, Adi Peduel, Mirit Gada, Keren Bressler and Benny Ron. Growing fish in high ammonia concentrations ...... Amit Gross, Anna Nemirovsky, Dina Zilberg, Anna Khaimov, Asher Brenner, Eviatar Snir, Zeev Ronen and Ali Nejidat. Soil nitrifying enrichments as biofilter starters in intensive recirculating saline water aquaculture ...... Amir Neori, Vladimir Odintsev and Jaap van Rijn. Zero-effluent gilthead sea bream mariculture in Eilat: growth and nutrient budgets ...... Isashar Ben-Atia, Sigal Lutzky, William Koven, Oria Nixon, Michal Torten and Amos Tandler. New findings in larviculture of the white grouper (Epinephelus aeneus)...... R. Segev, S. Shoval, B. Ron, A. Peduel, A. Barki, S. Harpaz and I. Karplus. Introduction of the barramundi (Lates calcarifer) to aquaculture farms in the Negev and Arava regions in Israel. . . . Bina Zilberman-Peled, Benny Ron and Yoav Gothilf. The characterization of two melatonin generating systems in sea bream: the pineal gland and retinal AANATs ...... Alon Naor, Nitzan Segev, Keren Bressler, Adi Peduel, Eran Hadas and Benny Ron. The influence of the pineal organ and melatonin on the reproductive system and of light intensity and wavelength on melatonin in the gilthead sea bream (Sparus aurata) ...... Ariel Ronen, Ayana Perelberg, Julia Abramovitz, Marina Hutoran, Simon Tinman, Izhak Bejerano, Michael Steinitz and Moshe Kotler. An efficient vaccine against a virus that causes a lethal disease in cultured Cyprinus carpio ...... Asaf Lipshitz, Anton Post and Arik Diamant. Parasites of deep sea fishes in the Gulf of Eilat...... Eliahu D. Aflalo, Isam Khalaila, Simy Weil, Carmen Segall, Claytus Davis and Amir Sagi. The vitellogenin gene in crayfish: expression and encoded proteins mobilized to the ovary . . . . Shmuel Parnes, Carmen Segall, Shaul Raviv, Dudu Azulay, Tal Goshen, Claytus Davis and Amir Sagi. Gonad maturation and vitellogenesis in a marine shrimp (Litopenaeus vannamei) grown in geothermal brackish water ...... Yiftach Yashuv, Ulrike Klenke, Yonathan Zohar and Yoav Gothilf. Development of gonadotropin-releasing hormone systems in hybrid striped bass ...... Tatyana Zak, Ofer Sachs and Itai Tzchori. Sex-reversal of genetically female common carp (Cyprinus carpio L.) by dietary administration of an aromatase inhibitor...... Lena Plesser, Miri Lapidot, Yacob Weinstein and Shoshana (Malis) Arad. Isolation of a promoter with high expression activity from the red microalga Porphyridium sp...... Hadas Broshy, D. van-Moppes, M. Keidan and S. (Malis) Arad. Assimilation of sulfur into the cell wall of the red microalga Porphyridium sp...... Marina Keidan, Y. Weinstein and S. (Malis) Arad. Brefeldin A influences cell-wall production in the red microalga Porphyridium sp. through its effect on the Golgi apparatus ...... Daniel Golani and Amit Lerner. Was the ichthyofauna of the sandy shore of the northern Gulf of Eilat influenced by adjacent mariculture activity? ...... Avigad Vonshak. Are outdoor algae cultures light-limited, light-saturated or light-inhibited? . . .
Posters G. Wm. Kissil and I. Lupatsch. Replacement of fishmeal by plant protein sources in gilthead seabream (Sparus aurata) diets ...... J.-C. Avarre, M. Khayat, P. Babin, R. Michelis, H. Nagasawa, A. Tietz and E. Lubzens. Molecular characterization and gene expression of two proteins from penaeid shrimp (Penaeus semisulcatus) ovaries during oocyte development...... Rivka Manor, Ran Segev, Eliahu D. Aflalo, Dudu Azulay and Amir Sagi. Does the androgenic gland promote growth in the faster-growing male crayfish in an intensive separated cell system? ...... S. Gorshkov, I. Meiri, H. Rosenfeld, S. Ben-Atia, S. Lutzki, A. Peduel, B. Ron, G. Gorshkova, A. Skvortzov and A. Tandler. Parental influence on sex ratios in progeny of the European sea bass (Dicentrarchus labrax) ...... Keren Bressler and Benny Ron. The effect of anesthetics on stress and the innate immune system in the gilthead sea bream, Sparus aurata ......
Abstracts of Oral Presentations TOOLS FOR STUDYING INTESTINAL FUNCTION David Sklan* Faculty of Agriculture, Hebrew University, Rehovot 76100, Israel The major purpose of fish cultivation is the production of high quality protein for human consumption. The predominant expense is feed, therefore improvements in uptake and efficiency of utilization will decrease production costs. It is important to know the proportion of the feed that is indigestible. This cannot be conveniently determined by simple input-output measurements but generally requires the use of markers to determine the availability of individual ingredients. Digestion commences with luminal hydrolysis, mainly by pancreatic enzymes in the gastrointestinal tract, and digestion of macromolecules at uptake sites, processes that are often rate-limiting steps in intestinal absorption. Synthesis, secretion and activation of pancreatic enzymes cannot be determined directly in the intestinal contents since the latter are not static. These factors have been addressed by determining mRNA expression for enzymes in the pancreas and by luminal marker studies. Further digestion and uptake occur on and within the brush border by membrane anchored enzymes and transporters which were examined by determining the expression of mRNA for the specific proteins and by measuring the digestion and uptake of labeled substrates. * E-mail: [email protected]
MODELING OF ENERGY AND NUTRIENT BUDGETS IN INTENSIVE AQUACULTURE SYSTEMS Ingrid Lupatsch* and G. Wm. Kissil Israel Oceanographic and Limnological Research Ltd., National Center for Mariculture, P.O. Box 1212, Eilat 88112, Israel The quantification of feed, fish growth and metabolism represent the primary processes for which recirculating aquaculture (RAS) fish culture systems are designed and managed. As fish feed is considered the only source of nutrients, total food input equals retention (fish growth) plus feces (solid waste) and excretion (dissolved waste). This paper describes a model that can be used to quantify the necessary energy and nutrient intake of gilthead sea bream for optimal growth and predict the retention efficiency and outputs of solid and dissolved nutrients. The total energy and nutrient requirements in growing fish are the sum of those needed for maintenance and growth. The requirement for maintenance is mainly a function of the size of the fish and water temperature, and is proportional to the metabolic body weight (kg)0.80. The requirement for growth, on the other hand, depends mainly on the weight gain and the composition of that gain. This model can be used to formulate practical feeds for gilthead sea bream and determine optimal feeding tables that supply the necessary daily amount of energy and protein. Once the digestibility of the feed is determined, solid waste production can be estimated. Total dissolved 224 The 7th Annual Dan Popper Symposium 225 ammonia nitrogen can be calculated as the difference between nitrogen intake and retention (as growth) plus fecal matter. By incorporating the oxygen equivalent per unit of energy required by the fish, O2 demand and subsequent CO2 excretion can be determined for increasing fish weights and different production levels. The equations can easily be incorporated into a computer program and used to predict fish production, feed demand, FCR, oxygen demand and waste production. The output of the model is the basis for designing culture system volumes, water flow rates, solid filters, biofilters, oxygenation and CO2 stripping devices in RAS. * Corresponding author. E-mail: [email protected]
THE MEGA-FLOW® RECIRCULATING AQUACULTURE SYSTEM FOR INTENSIVE FISH FARMING: PERFORMANCE RESULTS FROM TWO PILOT OPERATIONS Daniel Conijeski1*, Noam Mozes1 and Israel Haddas2 1 Israel Oceanographic and Limnological Research Ltd., National Center for Mariculture, P.O. Box 1212, Eilat 88112, Israel 2 Kora (1980) Architect Engineers Consultants Ltd., P.O. Box 38, Zofit 44925, Israel Expansion of the aquaculture industry in Israel has been constrained by increasing water shortages and environmental restrictions. Today, prospects for enhancing local fish production rely on the implementation of recirculating aquaculture systems (RAS). Although recirculating fish farms are on the rise in Europe, the USA and Israel, RAS projects remain challenged by economic performance. Recently in Israel a "low-head" RAS was developed. The system proved more compatible to local conditions and has already made vast improvements towards economical goals. The recent introduction of an innovative system, Mega-Flow®, will further extend the economic feasibility of intensive fish production in land-based RAS. Mega-Flow® is a patent-pending technology developed by Kora Ltd. in cooperation with the National Center for Mariculture (NCM). The low-head system, combined with the Mega-Flow® technique, enables an intensive RAS to be driven by only one air-blower. The Mega-Flow® system is based on state-of-the-art advanced design airlifts. The system includes two water circuits: (1) the main loop, where pond water is pumped through airlift pumps several times per hour creating a massive water flow (hence the name “Mega-Flow”). This loop fills three functions: aeration, CO2 stripping and the scouring of settled wastes and uneaten feed from the system. (2) The second loop is performed by smaller airlifts that circulate the water once or twice an hour through an up-flow non-pressurized filter for suspended solid removal and a moving bed biofilter for nitrification. In both filters, a “macaroni” type plastic substrate was used. The remarkable advantages of the low-head Mega-Flow® system are the simplicity and reliability of its water treatment components, the major reduction in the mechanical and electrical infrastructures and the lower risk of system failure. These advantages result in a considerable reduction of fish production costs by eliminating the use of expensive pure oxygen, increasing energy efficiency and lowering initial capital, operational and maintenance costs. The presentation will show performance results of intensive sea bream culture in two Mega-Flow® pilot systems, one at the NCM, Eilat, and the other at Gan Shmuel near Hadera. * Corresponding author. E-mail: [email protected] The 7th Annual Dan Popper Symposium
GROWING FISH IN HIGH AMMONIA CONCENTRATIONS Micha Eshchar, Noam Mozes, Michael Fediuk, Adi Peduel, Mirit Gada, Keren Bressler and Benny Ron* Israel Oceanographic and Limnological Research Ltd., National Center for Mariculture, P.O. Box 1212, Eilat 88112, Israel One of the major production costs in recirculating systems is the removal of ammonia to maintain a sub-toxic concentration. This research, financed by the Chief Scientist of the Ministry of Agriculture, suggests an innovative and generic approach. It may allow the total ammonia nitrogen (TAN) concentration to be greater by an order of magnitude than conventional concentrations for fish growth. The main principle is that the fraction of toxic unionized ammonia (UIA) of the TAN, inversely depends on the pH of the water. As CO2 excreted by fish lowers the water pH, the UIA fraction is reduced. Theoretically, this may allow the elevation of TAN to high concentrations. Prospected benefits of this approach are considerable savings in investment costs in biofilters in recirculating systems and a significant reduction of water consumption in flowthrough systems. Another advantage may be reduction of the environmental impact through higher nitrogen assimilation rates in algae ponds that treat fish tank effluents. Last year, experiments were conducted in the engineering and physiology departments at NCM. Gilthead sea bream (Sparus aurata) were reared in high ammonia concentrations. The fish tested in the physiology department were 1 g, whereas those tested in the engineering department weighed 40-80 g. In low pH (≤7.0-7.1), fish may be reared in TAN concentrations 5- 10 times higher than conventional levels without a significant reduction in fish growth performance. When the make-up sea water flow in a flow-through system was about 20% of the rate in conventional systems, fish grew without a biofilter. Equations were developed to show the relationship between inputs such as feed, the CO2-stripping and water exchange rates and water quality parameters such as pH, TAN, UIA, CO2 and alkalinity. * Corresponding author. E-mail: [email protected]
SOIL NITRIFYING ENRICHMENTS AS BIOFILTER STARTERS IN INTENSIVE RECIRCULATING SALINE WATER AQUACULTURE Amit Gross1*, Anna Nemirovsky1, Dina Zilberg2, Anna Khaimov1, Asher Brenner3, Eviatar Snir4, Zeev Ronen1 and Ali Nejidat1 1 Department of Environmental Hydrology and Microbiology, Jacob Blaustein Institute for Desert Research, Ben Gurion University of the Negev, 84990 Israel 2 Department of Dry Land Biotechnologies, Jacob Blaustein Institute for Desert Research, Ben Gurion University of the Negev, 84990 Israel 3 Department of Environmental Engineering, Ben Gurion University of the Negev, 84990 Israel 4 Negev Shrimps Ltd. (Matan), D.N. Haluza, Ramat Negev, 85515 Israel Intensive recirculating aquaculture relies on biofilters to maintain satisfactory water quality in the ponds. Without a start-up, establishment of new biofilters in aquaculture ponds requires a long time, causing significant losses and environmental harm from the discharge of nitrogen-rich effluents. A laboratory scale setup (7 l aquaria stocked with shrimp and fish) demonstrated that an 226 The 7th Annual Dan Popper Symposium The 7th Annual Dan Popper Symposium 227 external start-up nitrifying enrichment culture performed similarly to the natural bacterial population of an established pond biofilter, and better than similar biofilters without the start-up culture (control). The ammonia concentration in the control treatment increased daily and reached 18 mg/l during a 14-day experiment whereas in the treated aquaria it averaged less than 2 mg/l. Fish growth and survival were similar in the established pond and the start-up treatments (average growth 0.45 g/14 days; 95% survival) and significantly higher than in the control (average growth 0.0 g/14 days; 80% survival). The source of the enrichment cultures was soil samples collected from the region where the farm is located. This approach may lead to the development of bacterial amendments (probiotic products) that can be used as start-up cultures for new operations or damaged filters, and potentially enhance nitrification in established filters. Since the cultures are collected from soil, it is unlikely that they will be contaminated with agents that cause diseases in fish. This will improve water quality and, consequently, aquatic animal production. * Corresponding author. E-mail: [email protected]
ZERO-EFFLUENT GILTHEAD SEA BREAM MARICULTURE IN EILAT: GROWTH AND NUTRIENT BUDGETS Amir Neori1*, Vladimir Odintsev1 and Jaap van Rijn2 1Israel Oceanographic and Limnological Research Ltd., The National Center for Mariculture, P.O. Box 1212, Eilat 88112, Israel 2Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel A 12 m3 pilot mariculture system for sea bream has been in operation for three years in Eilat. Aerobic and anoxic treatment loops, foam fractionation, oxygenation and ozonation allowed 100% recirculation. From May 2000, the system was gradually stocked (total stocking 696 kg), reaching 60 kg/m3. By August 2001, 490 kg fish (35 kg/m3/year; FCR = 2.3) were produced, while at the same time 789 kg fish died, mostly of power cuts and diseases. In total, 397 kg of the fish were exported live and sold. Ninety cubic meters of tap water and 21 m3 of sea water were used (227 l/kg production). The nitrification-denitrification processes gasified about 85% of the unassimilated organic C and N. The remaining solids contained only about 15% of the unassimilated organic C and N, but all of the unassimilated P and ash. In a second trial, 6000 sea bream fingerlings (average weight 1 g) were stocked in mid- December 2001. In mid-August (when the trial was cut short by a mechanical incident), the average fish weighed 91 g and the biomass produced was 485 kg (extrapolated to 61 kg/m3/year; FCR = 1.6). Of this 107 kg were exported live and 67 kg died of various causes. Water use (194 l/kg production) consisted of 47 m3 tap water and 47 m3 seawater. The data demonstrate the biotechnological ability to commercially culture sea bream in completely recycled sea water. * Corresponding author. E-mail: [email protected] 228 The 7th Annual Dan Popper Symposium
NEW FINDINGS IN LARVICULTURE OF THE WHITE GROUPER (EPINEPHELUS AENEUS) Isaschar Ben-Atia, Sigal Lutzky, William Koven, Oria Nixon, Michal Torten and Amos Tandler* Israel Oceanographic and Limnological Research Ltd., National Center for Mariculture, P.O. Box 1212, Eilat 88112, Israel Larvae that ingest insufficient nutrients from hunting inappropriately-sized prey may quickly pass the “point of no return” where the digestive tract irrevocably regresses, leading to rapid mortality. In larvae of the white grouper (Epinephelus aeneus), the period between the onset of exogenous feeding and the point of no return is thought to be extremely short, stressing the importance of successful first feeding. In light of this, the importance and duration of feeding small, homogeneously sized (<100 μm) rotifers (Brachionus rotundiformis) vis a vis survival, population structure and ingestion rate in first feeding white grouper were tested. Two-day-old yolk sac larvae were stocked a 50 larvae/l in six 1500 l conical V-tanks with a 100% daily water exchange rate of filtered (10 μm) sea water (25‰) at 27±1ºC. After eye pigmentation and the opening of the mouth (three days after hatching), three rotifer feeding protocols were tested in duplicate tanks. The treatments differed in the duration of time that small homogeneously sized and enriched rotifers were fed to the larvae (until 5, 7 and 9 days after hatching). After these feeding periods, the larvae were fed heterogeneously sized unfiltered rotifers (ca 120 μm) until 30 days after hatching. The survival and ingestion rate significantly (p<0.05) increased with the increasing duration of feeding filtered rotifers. This was due mainly to the markedly (p<0.05) improved survival of the smaller larvae (55 mg wet wt), which represented the most substantial segment of the population in the nine-day treatment, compared to their larger cohorts (96 and 200 mg wet wt). The improved performance of the 55 mg larvae was likely a consequence of the higher capture rate of the small rotifers shortly after the onset of exogenous feeding. These findings emphasize the critical importance of ingesting nutrients immediately following the onset of exogenous feeding in white grouper larvae. * Corresponding author. E-mail: [email protected]
INTRODUCTION OF THE BARRAMUNDI (LATES CALCARIFER) TO AQUACULTURE FARMS IN THE NEGEV AND ARAVA REGIONS IN ISRAEL R. Segev1*, S. Shoval1, B. Ron2, A. Peduel2, A. Barki3, S. Harpaz3 and I. Karplus3 1 Desert Aquaculture, Arava Research and Development, Sapir, Israel 2 Israel Oceanographic and Limnological Research Ltd., National Center for Mariculture, P.O. Box 1212, Eilat 88112, Israel 3 Department of Aquaculture, Agricultural Research Organization, Beit Dagan, Israel The barramundi, Lates calcarifer, is a fast growing, high quality, predatory fish, recently introduced in several aquaculture farms in the Negev and Arava regions of Israel. Total production is currently about 30 tons per year. The introduction of the barramundi was accompanied by R&D The 7th Annual Dan Popper Symposium 229 activities which aimed at (a) developing practical interfaces to lessen cannibalism among fry and promote uniform and rapid growth; (b) finding alternative sources of supply of fingerlings for commercial farms; and (c) tracking activities at the commercial farms. The research focused on the effects of various factors on survival and growth of small fish as well as the breeding performance of a brood stock. The tested factors were crowding, the complexity of the breeding environment, the social environment, feeding management, salinity and photoperiod. This presentation summarizes the results of the experiments as well as production from the local farms. * Corresponding author. E-mail: [email protected]
CHARACTERIZATION OF TWO MELATONIN GENERATING SYSTEMS IN SEA BREAM: THE PINEAL GLAND AND RETINAL AANATS Bina Zilberman-Peled1, Benny Ron2 and Yoav Gothilf1* 1Department of Zoology, George S. Wise Faculty of Sciences, Tel Aviv University, Tel Aviv 2 Israel Oceanographic and Limnological Research Ltd., National Center for Mariculture, P.O. Box 1212, Eilat 88112, Israel Melatonin is an important component of the vertebrate circadian system. It is produced during the night at two main sites: the pineal gland and the retina. Pineal-derived melatonin is the source for circulating melatonin which provides a hormonal signal that regulates circadian and seasonal physiological rhythms, especially those involving photoperiodicity. Retinal melatonin, on the other hand, is thought to have a local paracrine function related to photic adaptation. The rate of melatonin production in the retina and the pineal gland is controlled by the enzymatic activity of serotonin-N-acetyltransferase (AANAT). The existence of two AANAT genes, AANAT1 and AANAT2, has been demonstrated in teleost fish. However, the biological significance of this duality is unknown. Toward answering this question, we compared and characterized the two AANATs in the gilthead sea bream (Sparus aurata). cDNA encoding the two sea bream AANATs – sbAANAT1 and sbAANAT2 – has been cloned using PCR amplification. Comparison of their deduced amino acid sequences demonstrate that they share 66% homology. Northern blot analysis demonstrated that AANAT1 is expressed only in the retina, while AANAT2 is expressed only in the pineal gland. To characterize the enzyme kinetics of the two AANATs, we prepared two recombinant proteins by isolating the full length of the AANAT1 and AANAT2 cDNAs and cloned their open reading frames into a prokaryotic expression vector. The two recombinant proteins were purified and their activity were characterized using a colorimetric assay. The enzymes demonstrated different activity characteristics, including optimal pH, molarity and temperature. Substrate specificity was determined by incubating the sbAANATs with a variety of substrates. We found that while sbAANAT2 preferentially acetylates indoleethylamines (especially serotonin, the precursor of melatonin), AANAT1 acetylates a variety of arylalaylamines. Given the differences in the temporal expression pattern and the substrate preferences of the two AANATs, we hypothesize that a gene duplication event results in the formation of two somewhat different systems: AANAT2, expressed in the pineal gland, is exclusively involved in melatonin production whereas AANAT1, expressed in the retina, may be involved in other biological processes. * Corresponding author. E-mail: [email protected] 230 The 7th Annual Dan Popper Symposium
THE INFLUENCE OF THE PINEAL ORGAN AND MELATONIN ON THE REPRODUCTIVE SYSTEM AND OF LIGHT INTENSITY AND WAVELENGTH ON MELATONIN IN THE GILTHEAD SEA BREAM (SPARUS AURATA) Alon Naor1, Nitzan Segev1, Keren Bressler1, Adi Peduel1, Eran Hadas2 and Benny Ron1* 1 Israel Oceanographic and Limnological Research Ltd., National Center for Mariculture, P.O. Box 1212, Eilat 88112, Israel 2 Suf Fish Farm Ltd., North Shore, Eilat 88000, Israel Effects of full-spectrum white (at normal room intensities) and monochromatic light (at various wavelengths) on pineal (in vitro) and plasma (in vivo) melatonin were investigated in the gilthead sea bream (Sparus aurata) during the naturally dark hours of the day (18:00 to 06:00). The evaluated zenith wavelengths (lambda max) of the intensity treatments tested in the in vivo experiment were 434 nm (blue) with a light intensity of 5 lux, 548 nm (green) with a light intensity of 20 lux, 614 nm (red) with a light intensity of 50 lux and white light with an intensity of 550 lux. The treatments for the in vitro experiment were 434 nm (blue) with a light intensity of 60 lux, 548 nm (green) with a light intensity of 100 lux, 614 nm (red) with a light intensity of 80 lux and white light with a light intensity of 800 lux. The potency of the tested lights to suppress melatonin levels in the plasma were ranked in the following order: white > green > blue > red. On the other hand, light suppression of melatonin levels in the pineal organ ranked: white > blue > green > red. The results demonstrate that the suppression of melatonin by light depends on the wavelength of the light and the circadian phase. We tested the consequences of applying monochromatic light in gilthead sea bream aquaculture and found that the growth rate improved with white and blue light before and during the reproductive season. There were significant differences between the gonad weights of the treated groups and those of the control groups. Another experiment tested the effect of pinealectomy on gonad maturation. The results showed that male maturation in pinealectomized fish was delayed almost three weeks compared with that of sham-operated and control animals. In addition, we investigated the effects of melatonin given in the food on gonad maturation. Our findings showed that the melatonin treatment affected the gonadosomatic index and that the pineal organ has an important role in the alteration of the reproductive system in fish via pineal melatonin. * Corresponding author. E-mail: [email protected] The 7th Annual Dan Popper Symposium 231
AN EFFICIENT VACCINE AGAINST A VIRUS THAT CAUSES A LETHAL DISEASE IN CULTURED CYPRINUS CARPIO Ariel Ronen1, Ayana Perelberg1,3, Julia Abramovitz1, Marina Hutoran1, Simon Tinman2, Izhak Bejerano2, Michael Steinitz1 and Moshe Kotler1* 1 Department of Pathology, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel 2 Central Fish Health Laboratory, Nir David 19150, Israel 3 Aquaculture Research Station Dor, Ministry of Agriculture and Rural Development, 30820, Israel Massive mortality of koi and common carp of the Cyprinus carpio species has been observed in many farms throughout Israel, resulting in severe financial losses. This lethal disease is highly contagious and extremely virulent, but morbidity and mortality are restricted to koi and common carp. The virus propagates and induces severe cytopathic effects five days after infection in fresh carp fin cell cultures, but not in epithelioma papillosum cyprini cells. The virus harvested from the fin cell cultures was inoculated into naive koi and common carp and induced the disease with a mortality rate of 75-85%. Using PCR with authentic primers provided final proof that the isolated virus is indeed the etiological agent causing mortality in both food and ornamental carp. Electron microscopy revealed viral cores with icosahedron morphology of 100-110 nm resembling those of the herpes virus. However, the genome of the isolated virus is a doublestranded DNA molecule of 250-300 Kbp, larger than that of other known Herpesviridae members. The viral DNA seems highly divergent and bears only small fragments (16-45 bp) homologous to the genomes of several DNA viruses. We suggest, therefore, that the etiological agent of this disease may represent an as yet unclassified virus species endemic to carpinoids. Carps exposed to the virus at 23°C for 3-5 days and transferred to the non-permissive temperature of 30°C became resistant to the disease and their sera contained a high level of virus specific antibodies. We isolated the attenuated non-pathogenic virus that rendered virus-vaccinated carp resistant to the disease. The vaccinated fish developed high levels of antibodies against the virus. We suggest, therefore, that this attenuated virus could be used as a live vaccine to eradicate this lethal disease that afflicts common and ornamental carp fisheries in many countries. * Corresponding author. E-mail: [email protected]
PARASITES OF DEEP SEA FISHES IN THE GULF OF EILAT Asaf Lipshitz1, Anton Post1 and Arik Diamant2* 1Interuniversity Institute for Marine Science, Hebrew University of Jerusalem 2 Israel Oceanographic and Limnological Research Ltd., National Center for Mariculture, P.O. Box 1212, Eilat 88112, Israel Parasites of coral reef fishes and commercially grown finfish have been studied for three decades in Eilat. However, fish that inhabit the deeper waters of the Gulf of Eilat (the dysphotic zone at depths >150 m, where illumination is too slight and brief for effective photosynthesis) have generally been overlooked. Practically no information is available on fish parasites at this depth. Sixty-nine species of fish have been recorded in this habitat. Most are atypical of deepsea species and, therefore, are considered to have evolved from shallow water species. The absence of true deep-sea species in the gulf is attributed to shallow sills at Bab el Mandeb and Tiran, which effectively exclude invasion by deep-sea species from the Indian Ocean. The present study is the first to characterize the parasites of endemic Red Sea fish fauna that inhabit this isolated and unique deep habitat. We encountered a parasitic fauna that differs significantly from those of the typical ocean abyss and of Red Sea shallow-water fish. During 14 months, we collected parasites from 283 fish specimens belonging to 13 species. The overall prevalence of digestive tract helminths was 70.4-100%, with the infraclass Cestodaria being the most abundant group among the demersal and benthic host species. Four orders of cestodes were represented: Tetraphyllidea, Trypanorhyncha, Diphyllidea and Pseudophyllidea. Another highly prevalent group, particularly in demersal species (e.g., Sparidae), was the phylum Nematoda. In contrast, other groups of gut helminths (e.g., subclass Digenea and phylum Acanthocephala) and ectoparasites (infraclass Monogenea and subclass Copepoda) were extremely rare. In this ongoing study, we are attempting to trace the origin of the parasites using both morphological characteristics and molecular phylogeny tools. We are examining the population biology and ecology of deep-sea organisms which are generally inaccessible for direct observation and, consequently, poorly known. The life cycles of selected parasites are being studied in vivo and molecular sequence analyses are being employed to overcome the constraints of experimental infections and artifacts such as near-identical morphology of unrelated species. This approach will likely form the basis for future studies of deep-sea fish and population-monitoring in the dysphotic and aphotic zones, as there is a regional trend to develop the deep-sea fishery. * Corresponding author. E-mail: [email protected]
THE VITELLOGENIN GENE IN CRAYFISH: EXPRESSION AND ENCODED PROTEINS MOBILIZED TO THE OVARY Eliahu D. Aflalo1, Isam Khalaila2, Simy Weil1, Carmen Segall1, Claytus Davis3 and Amir Sagi1* 1Department of Life Sciences and the Institute for Applied Biosciences, Ben Gurion University of the Negev, P.O. Box 653, Beer Sheva, Israel 2Institute for Medical Physics und Biophysics, University of Muenster, Robert-Koch-Str. 31, D-48149 Muenster, Germany 3Department of Molecular Genetics of Development, Ben Gurion University of the Negev A complete cDNA sequence of the crayfish Cherax quadricarinatus vitellogenin (CqVg) was cloned and sequenced in our laboratory (Genbank accession number AF306784). The CqVg gene was expressed in vivo in the hepatopancreas of secondary-vitellogenic females with the onset of vitellogenesis and in vitro in hepatopancreas primary cell culture up to seven days. Male hepatopancreatic cells did not express this gene in vivo or in vitro (negative control). Sequence similarities between the complete deduced amino acid sequence of the CqVg cDNA and its presumably high density lipoprotein (HDL) products, i.e., vitellogenin in the 232 The 7th Annual Dan Popper Symposium 233 hemolymph and vitellin in the ovary, were examined. Predominant proteins from the hemolymph (208, 196, 177 and 80 kDa) and different components of yolk protein vitellin in the ovary (predominantly 177, 155, 106, 95, 86, 75) were separated, analyzed by mass spectrometry for peptide sequences and compared. The observed similarity suggests that HDL proteins from both locations are derived from the same CqVg gene expressed in the hepatopancreas, the protein products of which are transported through the hemolymph and accumulate in the developing oocytes. * Corresponding authors. E-mail: [email protected]
GONAD MATURATION AND VITELLOGENESIS IN A MARINE SHRIMP (LITOPENAEUS VANNAMEI) GROWN IN GEOTHERMAL BRACKISH WATER Shmuel Parnes, Carmen Segall, Shaul Raviv, Dudu Azulay, Tal Goshen, Claytus Davis and Amir Sagi* Department of Life Sciences and the Institute for Applied Biosciences, Ben Gurion University of the Negev, P.O. Box 653, Beer Sheva 84105, Israel A unique case of the full development of female gonads was discovered during a routine check of Litopenaeus vannamei shrimp broodstock grown in geothermal brackish water at a commercial shrimp farm in the Negev. The gonads of the female were vitellogenic, based on morphological and physiological examinations. Using PCR and specific vitellogenin primers, we found that the vitellogenin gene, which was partially sequenced in our laboratory, was expressed in the female. We therefore decided to probe the issues of gonad maturation and the timing of vitellogenin gene expression in this marine shrimp when it is grown in geothermal brackish water from the Negev in southern Israel. Female shrimp were sampled from the farm at various times. Vg gene expression was not detected in young previtellogenic females using the same set of primers, however, it was detected after the females reached a certain size and age threshold, without clear signs of secondary vitellogenesis. Older female shrimp that grew in brackish water (2-3 ppt) and were fed a commercial pelleted diet could be manipulated to start vitellogenic processes by transferring them to a marine-like environment (30 ppt, enriched diet). Most of these females developed a full vitellogenic ovary with clear expression of the vitellogenin gene in both the hepatopancreas and the ovary. * Corresponding author. E-mail: [email protected] The 7th Annual Dan Popper Symposium
DEVELOPMENT OF GONADOTROPIN-RELEASING HORMONE SYSTEMS IN HYBRID STRIPED BASS Yiftach Yashuvi1,3, Ulrike Klenke2, Yonathan Zohar2 and Yoav Gothilf1* 1Department of Zoology, George S. Wise Faculty of Sciences, Tel Aviv University, Tel Aviv, Israel 2Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland, USA 3Madan, Ma'agan Michael Fish Breeding Center, Israel Gonadotropin releasing hormone (GnRH) is a key hormone in the control of reproduction. Three kinds of GnRH neurons are distributed in the adult brain. GnRH-1 neurons are located in the preoptic area of the hypothalamus where they project axons into the pituitary gland and stimulate production and release of gonadotropin. GnRH-2 neurons, located in the midbrain, are believed to play a roll in reproductive behavior. GnRH-3 neurons are located in the terminal nerve where they presumably transduce olfactory signals into reproductive information. During embryonic development, GnRH-1 and -3 neurons originate in the nasal region and migrate along olfactory axons to the brain. In this study we determined the onset of expression and the migration pattern of the GnRH neurons in hybrid striped bass larvae. Using whole mount in situ hybridization, GnRH-3 neurons were first localized in the nasal region two days after fertilization. Throughout the first 14 days of development, GnRH-3 mRNA signals expanded along the ventral telencephalon, reflecting the developmental migration of the GnRH-3. The GnRH-2 signal was detected in the midbrain two days after fertilization but no migration pattern was detected. GnRH-1 mRNA expression began 4-5 days after fertilization, as determined by PCR amplification analysis (GnRH-1 was undetectable using in situ hybridization.) Efforts are now being made to characterize its migration pattern using a recently cloned specific GnRH-1 cDNA. Understanding the mechanisms that control the early migration and establishment of the GnRH neuronal systems may lead to the development of new approaches for inducing sterility in striped bass and other aquacultured fish, resulting in overall enhanced performance and profitability. * Corresponding author. E-mail: [email protected] 234 The 7th Annual Dan Popper Symposium The 7th Annual Dan Popper Symposium 235 SEX-REVERSAL OF GENETICALLY FEMALE COMMON CARP (CYPRINUS CARPIO L.) BY DIETARY ADMINISTRATION OF AN AROMATASE INHIBITOR Tatyana Zak1, Ofer Sachs1* and Itai Tzchori2 1 Aquaculture Research Station Dor, Ministry of Agriculture and Rural Development, 30820, Israel 2Department of Zoology, George S. Wise Faculty of Sciences, Tel Aviv University, Tel Aviv, Israel The influence of a nonsteroidal aromatase inhibitor (Fadrozole™) on a genetically female population of Cyprinus carpio was studied. Fry were treated by dietary administration of an aromatase inhibitor during sexual differentiation (30 and 23 days after beginning exogenous feeding) and in an average temperature of 25±1.5°C (Exp I) or 28°C with fluctuations up to 35°C (Exp II). The duration of the treatments was 40 days in Exp I and 36-50 days in Exp II. At 25°C, the aromatase inhibitor (200 mg/kg feed) resulted in greater masculinization of females than methyltestosterone (58% and 3-6%, respectively). In Exp II, masculinization of females was even better: 63-100% males at a dose of 100-400 mg Fadrozole per kg feed. Masculinization was not observed to be dose-dependent. Within each treatment there was a positive correlation between the final weight of the fish and the proportion of masculinized fish. The greater masculinization of genetic females at the higher temperature probably indicates the presence of a temperature-dependent mechanism in sex determination. * Corresponding author. E-mail: [email protected]
ISOLATION OF A PROMOTER WITH HIGH EXPRESSION ACTIVITY FROM THE RED MICROALGA PORPHYRIDIUM SP. Lena Plesser1,2, Miri Lapidot1, Yacob Weinstein3 and Shoshana (Malis) Arad1* 1 The Institute for Applied Biosciences, Ben Gurion University of the Negev, Beer Sheva, Israel 2 Department of Biotechnology Engineering, Faculty of Engineering Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel 3 Department of Microbiology and Immunology, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel Red microalgae constitute a valuable resource that will find application in a range of biotechnological products such as pigments, unsaturated fatty acids and sulfated polysaccharides. However, the lack of basic molecular tools, such as mutants, genomic libraries and transformation systems, is currently limiting the biotechnological exploitation of these microorganisms. Our group recently developed a novel chloroplast transformation system with the AHAS gene as a selectable marker for sulfometuron methyl (SMM) resistance. We also established an expressed sequence tag (EST) project that contains cDNA libraries obtained from red microalgae grown under different physiological conditions. As a tool for biotechnological exploitation, we sought an endogenous promoter that would give a high level of foreign protein expression in the algal cells. To this end, we cloned a homologous promoter of the actin depolymerization factor (ADF) gene, which had been found to be highly expressed in various cDNA libraries of the red microalga Porphyridium sp. With the inverse PCR method, we isolated a 1.1-kb fragment containing the promoter of the ADF gene. The promoter contains the TATA box and GC box and can be used for the construction of red microalgae expression vector. Characterization of additional promoters is underway. * Corresponding author. E-mail: [email protected]
ASSIMILATION OF SULFUR INTO THE CELL WALL OF THE RED MICROALGA PORPHYRIDIUM SP. Hadas Broshy1,2, D. van-Moppes2, M. Keidan1,2 and S. (Malis) Arad2* 1Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel 2Institute for Applied Biosciences, Ben Gurion University of the Negev, Beer Sheva, Israel The main difference between the cell walls of land plants and those of algae lies in the presence of sulfated polysaccharides, which are common in algae but absent in higher plants. The cells of marine red unicellular alga Porphyridium sp. are encapsulated within such a sulfated polysaccharide. This complex heteropolymer (3-5 kDa) is composed of 10 different sugars and contains a non-covalently bound cell-wall glycoprotein. The polysaccharide is anionic due to the presence of glucuronic acid and sulfate ester groups. Very little is known about sulfation pathways of polysaccharides. Current knowledge of these pathways is based mainly on studies of higher plants and mammals where sulfur is assimilated mainly through the N-sulfation of proteins. In some mammalian cells, the Golgi apparatus is involved in the sulfation of proteins and proteoglycans. In experiments designed to investigate the sulfation pathway(s) in Porphyridium sp., the uptake of organic (cysteine) and inorganic (sodium sulfate) sources of sulfur by cells starved of sulfate was monitored. The following results were obtained from uptake and pulse-chase experiments on sulfur-starved cells. 35S from organic and inorganic sources was incorporated into the polysaccharide of Porphyridium sp. cells. Although the uptake of 35S into the cells was 26.5 times higher when Na235SO4 was used as the label source, 35S accumulation into the soluble polysaccharide was twice as high when [35S]cysteine was used. SDS-PAGE analysis showed that the 35S label resides in the sugars of the soluble polysaccharide and not in the glycoprotein. 35S incorporation into Porphyridium sp. polysaccharide was not affected by sodium chlorate, a chemical known to inhibit sulfation reactions in mammalian cells. 35S incorporation to Porphyridium sp. polysaccharide was reduced by Brefeldin A, a fungal metabolite that disassembles the Golgi apparatus into a tubular network and causes the dissociation of coatomer proteins from Golgi membranes. The increase in 35S labeling in the polysaccharide correlated with the decrease in 35S labeling of the low molecular weight fraction. An unidentified precursor was detected in the low molecular weight fraction. These findings suggest that both sources of sulfur can be used for the sulfation of the polysaccharide in Porphyridium sp. and, hence, more than one sulfation pathway for the polysaccharide exists. * Corresponding author. E-mail: [email protected] 236 The 7th Annual Dan Popper Symposium 237
BREFELDIN A INFLUENCES CELL-WALL PRODUCTION IN THE RED MICROALGA PORPHYRIDIUM SP. THROUGH ITS EFFECT ON THE GOLGI APPARATUS Marina Keidan1,2, Y.Weinstein3 and S. (Malis) Arad1* 1Institute for Applied Biosciences, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel 2Department of Life Sciences, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel 3Department of Microbiology and Immunology, Faculty of Health Sciences Ben Gurion University of the Negev, Beer Sheva, Israel The cells of red microalga Porphyridium sp. are encapsulated within a complex sulfated polysaccharide, the external part of which dissolves continuously in the medium. This cell-wall polysaccharide is composed of 10 different sugars and contains non-covalently bound cell-wall proteins. The Golgi apparatus of all eukaryotic cells is the site of glycosylation of proteins and biosynthesis of complex polysaccharides. In some mammalian cells the Golgi apparatus is involved in sulfation of proteoglycans. In this study, we investigated the effect of Brefeldin A on cell-wall synthesis in Porphyridium sp. Brefeldin A is a fungal metabolite that disassembles the Golgi apparatus into a tubular network and causes dissociation of coatomer proteins from the Golgi membranes. Brefeldin A acts on the ADP-ribosylation factor (ARF), the small G protein that plays a key role in vesicular transport processes. Treatment with Brefeldin A had the following effects on the Porphyridium sp. Cell growth was inhibited. Polysaccharide production was reduced. Golgi structure was disrupted (as shown by electron microscopy). Incorporation of 14C and 35S into the polysaccharide was reduced (pulse-chase experiments). ARF mRNA transcription ceased (Northern blot analysis). ARF level was reduced (Western blot analysis). These findings suggest that Brefeldin A affects polysaccharide biosynthesis and secretion in Porphyridium sp. cells through its action on the structure of the Golgi apparatus and through the ARF protein. * Corresponding author. E-mail: [email protected] The 7th Annual Dan Popper Symposium
WAS THE ICHTHYOFAUNA OF THE SANDY SHORE OF THE NORTHERN GULF OF EILAT INFLUENCED BY ADJACENT MARICULTURE ACTIVITY? Daniel Golani* and Amit Lerner Department of Evolution, Systematics and Ecology, The Hebrew University of Jerusalem The ichthyofauna of the sandy shore of the northern Gulf of Eilat was the subject of a long-term study whose objective was to assess the possible impact of mariculture activity adjacent to the study site. We used data from 1984-1986 that was collected prior to the commencement of local fish farm activity. A second set of data was collected during 1989-1994, when the nearby fish farms produced a low yield. The final studied period was 2000-2001 when the annual adjacent fish farm production was close to 2000 tons. Fish assemblages in the three periods were compared by number of individuals, number of species and biomass per sample and by accumulative number of species. Data were analyzed by time of day (morning, noon, evening, night) and season. Cluster analysis (UPGMA) was performed on the data. The number of species collected in all three periods totaled 93. Three of these species (e.g., Sparus aurata, Dicentrarchus labrax and Oreochromis mossambicus) were exotic to the region and their presence resulted from human activity. The results showed a high similarity in the structure of the fish assemblage during all three periods of the study. No significant difference was detected in any of the studied parameters. The same fish species remained significant and retained their level of relative importance throughout all three periods. * Corresponding author. E-mail: [email protected]
ARE OUTDOOR ALGAE CULTURES LIGHT-LIMITED, LIGHTSATURATED OR LIGHT-INHIBITED? Avigad Vonshak* Jacob Blaustein Institute for Desert Research, Microalgal Biotechnology, Ben Gurion University of the Negev, Sde Boker Campus 84990, Israel Outdoor algal cultures are exposed to two different rhythms of light and dark. The first is relatively fast. It is induced by the mixing and turbulent flow in the culture that dictates the frequency of the cycle. Algal cells shift between full solar radiation when they are located at the upper culture surface and complete darkness when they reach the bottom of the culture, usually at a depth of 12-15 cm. The second regime is relatively slower. It is the daily change in solar radiation from sunrise to sunset. Both light cycles impose unique physiological conditions in terms of adaptation or acclimation of the outdoor grown algal cells. At optimal concentrations for biomass production, light penetrates to a depth of only 3-5 cm, leaving the rest of the culture in complete darkness. In a regular pond with a depth of about 15 cm, more than 50% of the culture is in complete darkness. This situation, together with many other observations reported in the last 15 years, led to the conclusion that algal cultures grown outdoors are light-limited. More recent findings demonstrate that the maximum photosynthetic activity in outdoor cultures is not reached at the highest light intensity. We have indications that, by shading algal cultures, photosynthetic activity and productivity in outdoor cultures can be increased. Following 238 The 7th Annual Dan Popper Symposium 239 daily changes in variable fluorescence (Fv/Fm) in algae growing systems that differ in the amount of light to which the algal cells are exposed indicated that PS 2 activity decreases as a function of exposure to high solar radiance. This finding supports the idea that outdoor algae cultures are photo-inhibited. Can algal cultures be light-limited and photo-inhibited at the same time, or is something wrong in our interpretation? * E-mail: [email protected]
Posters
REPLACEMENT OF FISHMEAL BY PLANT PROTEIN SOURCES IN GILTHEAD SEABREAM (SPARUS AURATA) DIETS G. Wm. Kissil* and I. Lupatsch Israel Oceanographic and Limnological Research Ltd., National Center for Mariculture, P.O. Box 1212, Eilat 88112, Israel As part of a continuing study aimed at finding effective plant protein alternatives to fishmeal, soy protein concentrate, wheat gluten and corn gluten meal were evaluated in combination and as sole sources of dietary protein in diets for gilthead seabream. Both growth trials and digestibility determinations were employed to evaluate the effectiveness of these plant proteins. Digestibility trials indicated superior protein digestibility for soy protein concentrate (92%), wheat gluten (96%) and corn gluten meal (90%) in comparison to fishmeal (86%), while digestibility of energy varied with only wheat gluten (91%, as opposed to 75% for soy protein concentrate and 72% for corn gluten meal) being more available than fishmeal (84%). Diets used in growth trials were as isonitrogenous and isoenergetic as possible. The diets were supplemented with phosphorus and amino acids at levels determined in separate trials as being sufficient yet not growth-limiting. Growth of seabream (40-130 g) on diets containing soy protein concentrate or corn gluten meal as the sole protein source were inferior to the fishmeal only diet while the wheat gluten diet proved superior. Diets containing a mixture of equal portions of the three plant proteins replacing 25-100% of the fishmeal out-performed the all fishmeal diet by 9-16% greater weight gain. The feed conversion ratio was significantly superior to fishmeal in the all-wheat gluten diet and in the 50% and 75% mixture replacements. In these treatments, fish used 9-10% less feed for their weight gain. The use of soy protein concentrate or corn gluten meal as the sole protein source in diets for seabream is not recommended while their use in combination with wheat gluten can provide a partial or complete alternative to fishmeal. In a mixture, the wheat gluten is likely supplementing nutrients that may be limiting in the two other protein sources. The economic feasibility of using this mixture of plant proteins to replace fishmeal in seabream diets was evaluated, based on the costs of the feed ingredients in Israel. * Corresponding author. E-mail: [email protected] The 7th Annual Dan Popper Symposium
MOLECULAR CHARACTERIZATION AND GENE EXPRESSION OF TWO PROTEINS FROM PENAEID SHRIMP (PENAEUS SEMISULCATUS) OVARIES DURING OOCYTE DEVELOPMENT J.-C. Avarre1**, M. Khayat1***, P. Babin2, R. Michelis1***, H. Nagasawa3, A. Tietz4 and E. Lubzens1* 1 Israel Oceanographic and Limnological Research, P.O. Box 8030, Haifa 31080, Israel 2 Genomique et Physiologie des Poissons USC INRA, Universite Bordeaux 1, 33405 Talence Cedex, France 3 Department of Applied Biological Chemistry, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan 4 Department of Neurobiochemistry, Tel Aviv University, Tel Aviv 66978, Israel Two proteins contained in the ovaries of penaeid shrimps have been identified so far: vitellogenin/ vitellin (VT/VTG), the main protein component of egg yolk, and shrimp ovarian peritrophin protein (SOP), a component of the cortical rods and jelly layer formed around the eggs immediately after spawning. The cDNAs encoding of VT/VTG and SOP were cloned and sequenced in Penaeus semisulcatus. VT/VTG transcripts (~8 kb) were detected in the ovary and hepatopancreas and their level of expression was related to the stage of ovarian development and molt cycle. Although they are of the same length (2569 amino acids), VT from the ovary and VTG from the plasma were composed of three and two subunits, respectively. Cleavage occurred at a consensus motif for a subtilisin- like convertase in VT and VTG, and an additional cleavage occurred only in VT, by an unidentified endoprotease. Phylogenetic analysis indicated that crustacean VTGs are more closely related to insect apolipophorins and vertebrate apolipoprotein B-100, suggesting a possible role in the endogenous lipid transport pathway. SOP mRNA (~1 kb) was expressed in the ovaries at all oocyte developmental stages, whereas expression in the hepatopancreas was restricted to vitellogenic stages. SOP mRNA was abundant in the ovary and detected in previtellogenic stages before the occurrence of the corresponding protein. SOP showed the presence of repeated cysteine-rich domains, was glycosylated and could bind chitin when extracted from cortical rods. It shares these properties with insect intestinal peritrophins and mucins, crustacean tachycitin and invertebrate chitinases. * Corresponding author. E-mail: [email protected] ** Present address: DRIM, IFREMER/CNRS, 2 Place E. Bataillon, CP 80, 34095 Montpellier Cedex 5, France *** Present address: Eliachar Research Laboratory, Western Galilee Hospital, Nahariya 22100, Israel 240 The 7th Annual Dan Popper Symposium The 7th Annual Dan Popper Symposium 241
DOES THE ANDROGENIC GLAND PROMOTE GROWTH IN THE FASTER-GROWING MALE CRAYFISH IN AN INTENSIVE SEPARATED CELL SYSTEM? Rivka Manor1, Ran Segev2, Eliahu D. Aflalo1, Dudu Azulay1 and Amir Sagi1* 1 Department of Life Sciences and the Institute for Applied Biosciences, Ben Gurion University of the Negev, P.O. Box 653, Beer Sheva 84105, Israel 2 Aquaculture Unit Negev-Arava Research and Development, Yair, D.N. Arava, 86825, Israel In several crustacean species, such as the Australian red claw crayfish Cherax quadricarinatus, males grow faster than females. In the process of exploring ways to intensify crayfish culture, a growout system of individual cages was designed. The growth rate of the males in these systems was significantly higher than that of the females. These results suggest that C. quadricarinatus males grow larger than females, even when there is no social interaction. An intrinsic factor is thus suggested in the form of the androgenic gland hormone. It has been suggested that the androgenic gland is the exclusive source of the hormone responsible for sex differentiation in crustaceans. The androgenic gland in crustaceans promotes male sex differentiation and inhibits female characteristics. The androgenic gland of C. quadricarinatus was identified and described in our laboratory. To investigate the role of the androgenic gland on growth and development of secondary and primary sexual characters in C. quadricarinatus, androgenic glands were implanted into young females. The growth rate of the implanted females was significantly higher than that of the control females, and the implanted females developed secondary male characteristics such as the red patch on the propodas as early as the second molt after implantation. These results support the hypothesis that growth in male crayfish is promoted by the androgenic gland. * Corresponding author. E-mail: [email protected]
PARENTAL INFLUENCE ON SEX RATIOS IN PROGENY OF THE EUROPEAN SEA BASS (DICENTRARCHUS LABRAX) S. Gorshkov*, I. Meiri, H. Rosenfeld, S. Ben-Atia, S. Lutzki, A. Peduel, B. Ron, G. Gorshkova, A. Skvortzov and A. Tandler Israel Oceanographic and Limnological Research, National Center for Mariculture, PO Box 1212, Eilat, 88112, Israel Females of the European sea bass (Dicentrarchus labrax) grow 20-50% faster than males. Therefore, they are more in demand for commercial farming. This fact generated much interest in developing female monosex populations. The majority of recent studies focused on the influence of temperature on sex determination in sea bass. However, information on parental influences on the sex ratio is still limited and has been only partially documented. The present experiments aimed at studying the parental effects on sex ratios of the progeny in sea bass. The study was principally based on analyzing progeny of four families produced in a diallel crossing experiment (2 x 2 type or complete bi-factorial mating design), reflecting both the maternal and paternal genetic relatedness among progeny. The proportion of females varied significantly (p<0.05) among families (20.7-68.2%). There were significant maternal and paternal effects on the proportion of females within the progeny. The effect of parental interactions on the sex ratio of the progeny was also significant. Parents had a significant effect (p<0.05) on total length and body weight in the progeny. In addition to temperature manipulations proposed in our earlier studies, an improved female to male ratio of sea bass will probably be attained by directional selection of parents that produce the highest proportion of female progeny. Future use of DNA markers for both parents and progeny produced by mass spawning may substantially facilitate this selection. * Corresponding author. E-mail: [email protected]
THE EFFECT OF ANESTHETICS ON STRESS AND THE INNATE IMMUNE SYSTEM IN THE GILTHEAD SEA BREAM, SPARUS AURATA Keren Bressler and Benny Ron* Israel Oceanographic and Limnological Research, National Center for Mariculture, PO Box 1212, Eilat, 88112, Israel Anesthetics are frequently used in fisheries research and aquaculture to minimize stress response and prevent its negative impact on performance. Nevertheless, severe anesthesia may itself induce a stress response in fish that can lead to immunodepression and increase susceptibility to disease. In the present work, two anesthetics that are commonly used in aquaculture, clove oil (0.445 mM) and benzocaine (0.225 mM), were tested to observe their effects on the stress responses and innate immune system activity of the gilthead sea bream (Sparus aurata). Both anesthetics increased blood glucose and serum cortisol levels. In addition, benzocaine depressed lysozyme activity, production of reactive oxygen species and pinocytosis, while these results were not observed in fish anesthetized with clove oil. Benzocaine had a negative effect on the humoral and cellular responses. Clove oil, however, did not exert any immunodepressive effect on the innate immune system, suggesting that clove oil is the preferred anesthetic for sea bream aquaculture and research. * Corresponding author. E-mail: [email protected] 242 The 7th Annual Dan Popper Symposium