Vitellin and vitellogenin incorporation by isolated oöcytes of Locusta migratoria migratorioides (R.F.)

Vitellin and vitellogenin labelled in vitro with ¹²⁵I and in vivo with ³H were incorporated into yolk by locust oöcytes incubated in an in vitro system. This incorporation was specific and linear with the duration of incubation. Uptake of vitellin by oöcytes was 3–4 times higher than ¹²⁵I-bovine serum albumin in 2.1-mm oöcytes and 20 times higher than ¹²⁵I-bovine serum albumin in 4.0-mm long oöcytes. The uptake of the albumin was enhanced by the presence of vitellin in the incubation medium. ³H-labelled yolk protein was incorporated at higher rates than that labelled with ¹²⁵I. The addition of the juvenile hormone analogue ZR 515, caused the incorporation rates of vitellogenin to be increased. The amount of vitellin or vitellogenin taken up by the oöcytes increased with their length, and the rate of incorporation per unit surface area was highest in 3–4-mm long oöcytes. These results corroborate previously reported in vivo patterns of incorporation rates of developing oöcytes.     

Response of Penaeus indicus females at two different stages of ovarian development to a lethal infection with Vibrio penaeicida

An association between vitellogenesis and the immune system was suggested in crustaceans from studies on plasma lipoproteins. The present research studies the effect of an experimentally induced bacterial infection on vitellogenesis in females of the shrimp Penaeus indicus, as a model for penaeid species. Pre-vitellogenic and vitellogenic P. indicus females were experimentally infected with an extremely pathogenic bacterium, Vibrio penaeicida. The peak in mortality occurred earlier in pre-vitellogenic animals than in vitellogenic ones, although the final mortality level ( approximately 64-74%) 52h post-infection was nearly the same for the two groups. Twenty hours after infection, the total number of haemocytes was significantly reduced in vitellogenic females while there was no change in the pre-vitellogenic group. Protein synthesis in ovaries was not significantly affected by infection, at the two stages of ovarian development. No differences were found in mRNA levels of shrimp ovarian peritrophin protein (SOP), but preliminary results showed that mRNA expression of vitellin (VT) was reduced in a heavily infected vitellogenic female. The total amount of lipids in the haemolymph of vitellogenic females was almost twice higher than that of pre-vitellogenic ones. However, there was no change in the total content of lipids, lipid classes and fatty acid distribution in haemolymph or hepatopancreas following infection. Although vitellogenic and pre-vitellogenic females probably respond differently to a lethal bacterial infection, physiological differences may be concealed by the rapid onset of mortality.     

Protein synthesis in vitro in cultures of the subepidermal adipose tissue and the ovary of the shrimp Penaeus semisulcatus

In vitro culture systems were developed for subepidermal adipose tissue (SAT) and ovary of the shrimp Penaeus semisulcatus. Both tissues were cultured in sea water-based media buffered with HEPES to pH 7.4 in an oxygen enriched atmosphere. Various incubation conditions were tested in order to define those supporting optimal rates of protein synthesis. Best results for de novo protein synthesis were obtained when amino acids and other supplements were added according to Landureau's medium composition for the SAT and Eagle's MEM for the ovary. Streptomycin was found to inhibit protein synthesis in SAT cultures. These in vitro systems are appropriate for future studies of serum lipoprotein synthesis and its hormonal control.     

Induction of sexual reproduction and resting egg production in Brachionus Plicatilis reared in sea water

Brachionus plicatilis raised in our laboratory in sea water reproduces asexually even under high crowding conditions (at least 40 individuals per ml). Amictic females were induced to produce mictic females, males and resting eggs by reducing the concentration of the sea water culture medium. Mictic females and males appeared predominantly among the progeny produced by the amictic females during 4 days following their transfer into 25% sea water. Resting eggs appeared first 5–12 days after the onset of the experiment. Following the disappearance of males, the culture consisted of amictic females.Resting eggs produced by the method described above may be preserved for at least three months at –14°C or by desiccation at room temperature. Under the appropriate experimental conditions, resting eggs hatch into amictic females. Since B. plicatilis is one of the most commonly used food sources of fish larvae in aquaculture, the methods reported here may offer an easy and versatile way of preserving rotifer culture stock to be used on demand.     

Pater Dr.h.c. Josef Donner (28.2.1909–8.1.1989) in memoriam

Pater Dr.h.c. Josef Donner (28.2.1909–8.1.1989) in memoriam     

Mariculture in Israel – past achievements and future directions in raising rotifers as food for marine fish larvae

Marine fish production is now being carried out afteralmost two decades of research. The production ofseabream (Sparus aurata), which reached over 750tons in 1995, is expected to reach an annualproduction ranging between 4000 - 12 700 metric tonsby year 2010. The anticipated introduction of newspecies and its expansion to the Mediterranean shoreline will help in leading the increased maricultureproduction. Two marine fish hatcheries that operatetoday in Israel produce 7 million fingerlings a year.Traditionally, aquaculture in Israel raises fish ininland freshwater ponds and irrigation reservoirs. Inaddition, Lake Kinneret, the only freshwater lake inIsrael, is stocked yearly with juvenile fish raised inlocal hatcheries (tilapia) or imported fromMediterranean countries (mugil). While culture offreshwater teleost species (carp) was introduced morethan fifty years ago, mariculture started on acommercial scale less than 5 years ago. The limitedsupply of freshwater will accelerate the futureculture of marine species.The bottleneck of almost all marine finfish productionlies in obtaining adequate numbers of fingerlings, dueto their high mortality at early life stages. Theproduction is hindered by inadequate supply of food toearly larval stages which require live food.Development of technologies in Israel for masscultivation of food chain organisms including algae,rotifers and brine shrimp followed their developmentin other parts of the world, most notably thoseachieved in Japan. The local commercial scaleproduction of rotifers relies on several batch orsemi-continuous cultures in conical or flatbottomrectangular containers that supply daily 0.6-4billion rotifers in each hatchery. Originally arelatively large local Brachionus plicatilisstrain was used, but later smaller B.rotundiformis strains were introduced, resulting ina mixture of undefined strains. The incorporation ofalgae (Nannochloropsis sp.) generated in highyield raceways contributes to the reliability ofrotifer cultures. Algae are supplied directly from theraceways or centrifuged and stored as a frozen pasteuntil required in the hatchery. The current dependablesupply of live cultures reduces the need for preservedstocks of rotifers, either as resting eggs or keptalive at low temperatures. To the fish grower,rotifers are live food capsules that deliver essentialnutrients (e.g. long chain unsaturated fatty acids)for growth and survival of fish larvae. Research aimedat replacing live food with chemically definedmicrodiets could reveal physiological principles inprey recognition and digestion of food by marine fishlarvae.     

Insight into molecular pathways of retinal metabolism, associated with vitellogenesis in zebrafish

Retinal is the main retinoid stored in oviparous eggs of fish, amphibians, and reptiles, reaching the oocytes in association with vitellogenins, the yolk precursor proteins. During early presegmentation stages of zebrafish embryos, retinal is metabolized to retinoic acid (RA), which regulates genes involved in cell proliferation, differentiation, and tissue function and is therefore essential for normal embryonic development. While synthesis of vitellogenin and its regulation by 17β-estradiol (E(2)) were extensively investigated, pathways for retinal synthesis remain obscure. We determined the expression pattern of 46 candidate genes, aiming at identifying enzymes associated with retinal synthesis, ascertaining whether they were regulated by E(2), and finding pathways that could fulfill the demand for retinoids during vitellogenesis. Genes associated with retinal synthesis were upregulated in liver (rdh10, rdh13, sdr) and surprisingly also in intestine (rdh13) and ovary (rdh1, sdr), concomitantly with higher gene expression and synthesis of vitellogenins in liver but also in extrahepatic tissues, shown here for the first time. Vitellogenin synthesis in the ovary was regulated by E(2). Gene expression studies suggest that elevated retinal synthesis in liver, intestine, and ovary also depends on cleavage of carotenoids (by Bcdo2 or Bmco1), but in the ovary it may also be contingent on higher uptake of retinol from the circulatory system (via Stra6) and retinol synthesis from retinyl esters (by Lpl). Decrease in oxidation (by Raldh2 or Raldh3) of retinal to RA and/or degradation of RA (by Cyp26a1) may also facilitate higher hepatic retinal levels. Together, these processes enable meeting the putative demands of retinal for binding to vitellogenins. Bioinformatic tools reveal multiple hormone response elements in the studied genes, suggesting complex and intricate regulation of these processes.     

A model evaluating the contribution of environmental factors to the production of resting eggs in the rotifer Brachionus plicatilis

The production of resting eggs by the rotifer Brachionus plicatilis was tested at four salinities (9, 18, 27 and 36\%) and six concentrations of the alga Chlorella stigmatophora (0.25, 0.5, 1.0, 2.0, 4.0 and 6.0 × 10⁶ cells ml^–1). The results indicated that resting eggs were produced only at two salinities (9\% and 18\%) and that their number was affected by the amount of food provided. A model consisting of two generalized linear sub-models was built to evaluate the contribution of each of the tested food concentrations at the two salinities. The sub-models were used to distinguish between two different components of resting egg production: one related to the presence or absence of resting egg production, and the other to the number of resting eggs produced, given that production had occurred. Besides indicating the best combination of salinity and food concentration for obtaining large numbers of resting eggs, they revealed the contribution of internal population factors that were not controlled in the course of the experiment. The model identified the positive contribution of the relative number of females to males, and the negative association between high rotifer densities and the production of resting eggs. The results of the present study help in defining the optimal conditions for mass production of resting eggs, which are of potential importance in aquaculture.Deceased, September 1991.     

Molecular characterization and high expression during oocyte development of a shrimp ovarian cortical rod protein homologous to insect intestinal peritrophins

Penaeoid shrimp oocytes nearing the completion of oogenesis are enveloped in an acellular vitelline envelope and possess extracellular cortical rods (CRs) that extended into the cortical cytoplasm. These cortical specializations are precursors of the jelly layer (JL) of the egg. In searching for highly expressed mRNAs during oogenesis in the marine shrimp (Penaeus semisulcatus), two related cDNAs have been isolated that encode a mature protein of 250 amino acid residues. The deduced amino acid sequences revealed the presence of repeated cysteine-rich domains that are related to the chitin-binding domains of insect intestinal peritrophins. Similar cysteine-rich domains were reported in insect intestinal mucin, crustacean tachycitin, and invertebrate chitinases. The shrimp ovarian peritrophin (SOP) is glycosylated and can bind chitin when extracted from CRs. Its apparent molecular mass in SDS-PAGE is 29-35 kDa and 33-36 kDa, under nonreducing or reducing conditions, respectively. SOP is a major protein of CRs and the JL, and was immunodetected in ovaries; purified CRs; fertilized eggs that were surrounded by a JL matrix; and in the cloudy, whitish flocculent material appearing in sea water immediately after spawning. Immunolocalization in tissue sections determined that SOP was present in oocyte cytoplasm and in extraoocytic CRs. Shrimp expressed SOP mRNA in ovaries at all oocyte developmental stages, whereas expression in the hepatopancreas was restricted to vitellogenic stages. SOP mRNA was abundant in the shrimp ovary and was detected before the presence of the corresponding protein. This is the first demonstration that a protein with similar features to insect intestinal peritrophins is a component of CRs and is therefore a main precursor of the JL of spawned shrimp eggs.