Virulence of luminous vibrios to Artemia franciscana nauplii

From healthy and diseased penaeid shrimp from Asia and the Americas, 25 luminous and 2 non-luminous bacterial strains were isolated, and 14 were phenotypically identified as Vibrio harveyi; 9 isolates produced significant mortalities (45 to 80%) in Artemia franciscana nauplii at inoculation densities of 10(5) to 10(6) CFU ml(-1) compared to the controls (unchallenged nauplii). The maximum number of bacteria ingested (bioencapsulated) by the Artemia nauplii varied from less than 10 to 10(3) CFU nauplius(-1) and no significant relationship was observed between the density of bacteria inoculated, the amount of bacteria ingested, and naupliar mortality. Significant correlations were obtained between naupliar mortality and production of proteases, phospholipases or siderophores, but not between mortality and lipase production, gelatinase production, hydrophobicity or hemolytic activity. The results suggest that virulence of the strains tested was more related to the production of particular exoenzymes than to the measured colonization factors.     

Quantitative oral dosing of water soluble and lipophilic contaminants in the Japanese medaka (Oryzias latipes)

Quantitative oral dosing in fish can be challenging, particularly with water soluble contaminants, which can leach into the aquarium water prior to ingestion. We applied a method of bioencapsulation using newly hatched brine shrimp (Artemia franciscana) nauplii to study the toxicokinetics of five chlorinated and brominated halogenated acetic acids (HAAs), which are drinking water disinfection by-products. These results are compared to those obtained in a previous study using a polybrominated diphenyl ether (PBDE-47), a highly lipophilic chemical. The HAAs and PBDE-47 were bioencapsulated using freshly hatched A. franciscana nauplii after incubation in concentrated solutions of the study chemicals for 18 h. Aliquots of the brine shrimp were quantitatively removed for chemical analysis and fed to individual fish that were able to consume 400-500 nauplii in less than 5 min. At select times after feeding, fish were euthanized and the HAA or PBDE-47 content determined. The absorption of HAAs was quantitatively similar to previous studies in rodents: rapid absorption with peak body levels occurring within 1-2 h, then rapidly declining with elimination half-life of 0.3-3 h depending on HAA. PBDE-47 was more slowly absorbed with peak levels occurring by 18 h and very slowly eliminated with an elimination half-life of 281 h.     

Biological and biochemical quality of the Artemia (Anostraca: Artemiidae) population from Real de Salinas saltworks, Calkiní, Campeche, Mexico

Cysts of Artemia spp. collected from February 1997 to February 2000 in the Real de Salinas solar saltworks, Campeche, Mexico, were compared with Artemia franciscana (batch number 8,131 Microfeast Artemia Cysts, Texas, USA). The variables determined in these two populations were: number of cysts per gram, hatching percentage, hatching efficiency, hatching rate, hatching synchrony and hatching biomass, as well as diameter of the cysts and length of the nauplii (instar I). For Salinas, the average diameters of the encapsulated and decapsulated cysts were 230.5 +/- 4.14 and 221.8 +/- 3.39 microm, respectively. The thickness of the cyst shell was 4.35 +/- 0.68 microm and the length of the nauplii was 388.11 +/- 4.39 microm, this last value is among the smallest reported in the literature. For the commercial population of A. franciscana, the average diameters of the encapsulated and decapsulated cysts were 230.21 +/- 12.49 and 216.96 +/- 13.71, respectively. With respect to the corion thickness and length of the nauplii the values were 6.62 +/- 2.72 and 424.70 +/- 30.08, respectively. The protein value of the cysts (47.91 %) and nauplii (50.5 %) of Artemia population from Real de Salinas, are considered adequate to be used as food in aquaculture. The results indicate that the population from Real de Salinas presents positive features for its use in aquaculture in the region.     

Evaluation of the probiotics Bacillus subtilis and Lactobacillus plantarum bioencapsulated in Artemia nauplii against vibriosis in European sea bass larvae (Dicentrarchus labrax, L.)

Two potential probiotics Bacillus subtilis and Lactobacillus plantarum were evaluated for use in aquaculture as preventive measures against vibriosis. In vitro evaluation of the probiotics using co-culture assays with the pathogen Vibrio anguillarum and testing for the production of antibacterial substances showed the presence of antagonism and confirmed the production of antibacterial substances. Both potential probiotics were administered to the live fish feed Artemia franciscana nauplii, offering protection against a subsequent challenge of the nauplii with the fish pathogen V. anguillarum, with best survival rates of the nauplii and the most efficient protection offered by B. subtilis. Nauplii enriched with B. subtilis were further used to evaluate the protection of sea bass larvae against vibriosis. The untreated group of fish challenged with V. anguillarum presented low survival of 36.7 %, while the fish treated with nauplii enriched with the probiotic B. subtilis showed significantly increased survival rates of 86.7 % after challenge with the pathogen. The survival of healthy unchallenged fish treated with the probiotic was not significantly different from control unchallenged fish (90-94 %). Our results indicate that B. subtilis is a probiotic suitable to be used for the prevention of vibriosis in fish larvae and can be safely administered through their live feed Artemia nauplii.     

Inbred strains of brine shrimp derived from Artemia franciscana: lineage, RAPD analysis, life span, reproductive traits and mode, adaptation, and tolerance to salinity changes

Inbred strains of the brine shrimp were developed from dry dormant cysts of wild-type Artemia franciscana produced in the Great Salt Lake, U.S.A. The established strains were named GSL2, 4, and 7. They were raised in 2% natural sea salt solution at 28 degrees C under a long-day condition, and fed on food sold for Artemia. Ovoviviparous offspring (free-swimming nauplii) in each brood derived from full sib (sister x brother) matings were used for succeeding generations. The ordinal number of the filial generation increased at a rate of ten generations per year. The number was over 60, and the lineage was recorded. Random amplified polymorphic DNA (RAPD) analyses of the inbred strains revealed the uniqueness, homogeneity, and genetic similarity among them. Their life span, the time required to become sexually mature, brood size, mode of reproduction, and adaptation and tolerance to salinity changes were investigated. The inbred strains usually released free-swimming nauplii rather than spawning encysted gastrulae (dormant cysts). On the other hand, the opposite results were obtained from wild-type Artemia under the same conditions. Both adults and nauplii of the inbred strains appeared to be less adaptive and less tolerant to salinity changes compared to those of the wild type. The established inbred strains should provide a wider and deeper scope for Artemia biology in particular, and the life sciences in general.     

beta-Hydroxybutyrate in developing nauplii of brine shrimp (Artemia franciscana K.) under feeding and non-feeding conditions

Body content of beta-hydroxybutyrate, and individual dry mass, carbon content, and survival rate, were studied in developing nauplii of the brine shrimp Artemia franciscana K. from hatching to 96-97 h post hatching at 27 +/- 1 degrees C. The effect of two diets was studied in the experiment: Super Selco (SS) with a high lipid content; and Protein Selco (PS) with a high protein content. A starving group (S) was used as reference. The level of beta-hydroxybutyrate at hatching was 0.6 nmol.ind-1; it increased to 1.0-1.5 nmol.ind-1 in the SS- and S-groups, while in the PS-group it remained stable between 0.6-0.8 nmol.ind-1. At 60-80 h post hatch in the SS- and S-groups, the levels of beta-hydroxybutyrate were similar to the initial levels. The survival rate remained higher than 95% until 24 h post hatching in all groups. At the end of the experiment, the survival rate was 63% in the PS-group, 13% in the S-group and 3% in the SS-group. The Artemia nauplii individual dry mass and carbon content remained relatively stable in the SS-group; both parameters showed a significant increase in the PS-group and a significant decrease in the S-group. The results suggest that Artemia nauplii utilise ketone bodies as a fuel during development and growth, but that ketone catabolism may be overloaded by excessive lipid feeding resulting in increased mortality and possibly a ketotic acidosis.     

Intestinal uptake of lipovitellin from brine shrimp (Artemia franciscana) by larval inland silversides (Menidia beryllina) and striped bass (Morone saxatilis)

Intestinal uptake of lipovitellin (LV) from brine shrimp (Artemia franciscana) in larval inland silversides (Menidia beryllina) and striped bass (Morone saxatilis) was described using immunocytochemistry. Polyclonal antisera were raised against two subunits of LV (LV68 and LV190). When tested by immunocytochemistry, anti-LV68 showed cross-reactivity with some of the pancreatic cells especially in inland silversides. Therefore anti-LV190 was used to localize immunoreactive LV. Inland silversides at 14 days after hatching were fed Artemia nauplii and then sampled 4, 8, 12 hr after feeding. Similar experiments were carried out by using striped bass at 5 days and 15 days of age. They were sampled at 2, 4, 8, and 12 hr after feeding. Anterior enterocytes showed no evidence of uptake; however, the brush border of the cells of inland silversides reacted with the antiserum. Posterior enterocytes took up the LV and/or, possibly, their immunoreactive breakdown products. The pattern of uptake included accumulation in supranuclear vacuoles and digestion in supranuclear vacuoles, as suggested by the decay of the immunoreactivity over time. Thus, the posterior intestine of these larval fishes is the site of uptake and digestion of LV, an important nutritive component in the food of many larval fishes; this supports earlier findings using non-nutritive marker proteins.     

Cytogenetic proof that the brine shrimp Artemia franciscana (Crustacea, Branchiopoda) is found in Argentina

Artemia brine shrimps are commercially important and they have been extensively studied. This branchiopod crustacean is extensively used in aquaculture and other commercial and applied practices. The genus also awakes an increasing interest worldwide as an experimental model in other basic areas of research such as evolution and cytogenetics. In the present work adult male meiosis and nauplii mitotic cells in two Artemia populations from Argentina (Mar Chiquita and Las Tunas, Córdoba Province) are analyzed and compared to the reference strains A. franciscana (Great Salt Lake, Utah, USA) and A. persimilis (Salinas Grandes de Hidalgo, La Pampa Province, Argentina). The Mar Chiquita population shows the diploid and haploid numbers characteristic of A. franciscana, and a regular male meiosis. In the Las Tunas population diverse diploid and haploid numbers are encountered, and an irregular meiosis is observed in some individuals. Fluorescent DAPI and CMA banding shows bright chromocenters of intermediate size and number in both populations. Cytogenetic and phenotypic data show that the population of Mar Chiquita could be assigned to A. franciscana; the variation found in chromocenter size and number reflects an heterochromatin polytypism already observed in this species. The cytogenetic and morphological traits in Las Tunas population suggest an occasional hybridization between A. franciscana and A. persimilis.     

4种生态因子对虎斑乌贼幼体生长与存活的影响

旨在研究温度、盐度、饵料种类、投饵量对虎斑乌贼（Sepia pharaonis）幼体生长与存活率的影响,以确定其生长发育的最佳生态条件,为人工育苗提供理论依据。在室内控制条件下,采用单因子试验研究了不同温度（18、21、24、27、30和33℃）、不同盐度（18、21、24、27、30和33）、不同饵料种类（虾糜、活糠虾、卤虫无节幼体、桡足类、死糠虾、虾糜＋强化卤虫后无节幼体、强化卤虫后无节幼体）、活糠虾不同投饵量（0、2、4、6、8和10 g/d）对虎斑乌贼幼体生长发育的影响。结果显示：不同温度对虎斑乌贼幼体生长影响显著（P〈0.05）,最适温度为24-27℃,在最适温度下,存活率为84.4%-91.1%,特定生长率为4.82%-6.13%,存活率（y）与温度（x）的函数关系为y=-0.15x3＋30.637x-447.002（r2=0.923）。幼体适宜盐度为24-33,最适盐度为27,在最适盐度条件下,其存活率为（90.0±5.29）%,特定生长率为（3.71±0.34）%。投喂7种开口饵料,以活糠虾效果最佳,存活率为（96.7±2.94）%,特定生长率为（3.77±0.23）%;强化卤虫后无节幼体效果次之,存活率为（95.6±2.31）%,特定生长率为（2.54±0.15）%,其余各组培养效果均不理想。投喂活糠虾,随着投饵量的增加,其存活率随之提高,摄食量（y）与个体重（x）的回归方程为y=0.227x-0.063（r2=0.921）。     

Effects of salinity on growth and survival in five Artemia franciscana (Anostraca: Artemiidae) populations from Mexico Pacific Coast

Salinity is an important factor influencing growth and survival of aquatic organisms such as Artemia, a valuable aquaculture species. This study evaluated the effects of salinity on A. franciscana populations from different water bodies in Mexico's Pacific Coast. With this purpose, five autochthonous bisexual Artemia populations were tested to assess their survival and growth values against salinities of 40, 60, 80, 100 and 120 g/l, under laboratory conditions (25 +/- 2 degrees C; pH 8-10; constant light and aeration). The organisms were fed with 100 mL of rice bran and 2L of Tetraselmis suecica (500 000 cel/ml). The culture experiments were made in 200L plastic tanks, and survival and growth final values were obtained after 21 culture days. Survival and growth curves were determined by a regression analysis (R2). The significant differences between salinities were determined by ANOVA test (p < 0.05). The best survival and growth rates were found at salinities of 100-120 g/l. When the Mexican Artemia populations were cultivated at 40 g/l of salinity, 100% mortality was observed in the juvenile stage. This study determined that survival and growth values of A. franciscana populations increased with salinity. The five A. franciscana populations presented significant differences in their survival rate under various salinity regimes. The studied populations experienced high mortality at salinities under 60 g/l and over 200 g/l, and especially during the metanauplius stage. The present study confirms that growth rates in Mexican A. franciscana populations from Pacific Coast habitats are not inversely proportional to salinity. These A. franciscana populations should be cultured at 100-120 g/l of salinity to obtain better survival and growth rates. This data is useful to improve culture systems in aquaculture biomass production systems.     

Isolation of a cDNA encoding a putative SPARC from the brine shrimp, Artemia franciscana

SPARC (Secreted protein, acidic, rich in cysteine) is an extracellular matrix-associated and anti-adhesive glycoprotein extensively studied in vertebrates. Its presence among invertebrates has been reported in nematodes and flies. We cloned a cDNA containing a complete open reading frame for SPARC from the brine shrimp, Artemia franciscana. The amino acid sequence identity between the Artemia and the fly SPARCs was 55%, whereas that of the Artemia and the nematode proteins was 45%. Artemia and vertebrates exhibited a sequence identity of 30% in the predicted aa sequences. The SPARC consisted of four domains commonly found among reported SPARCs. The protein comprised 291 amino acids, having a signal peptide, a follistatin-like domain, one N-glycosylation site and one calcium-binding EF-hand motif. Fourteen cysteine residues conserved among all the secreted forms of SPARCs were present in the Artemia SPARC, and four extra cysteine residues were also found in it. The extra residues were conserved among SPARCs of the arthropods and the nematode. Phylogenetic analyses showed that the sequences of SPARCs were grouped into those of vertebrates and invertebrates. Though the structural organization of SPARC was conserved among all the species studied, SPARC within a group was highly conserved within that group, but divergent between the two. Northern blots revealed the presence of a 1.1 kb mRNA, which was faintly expressed in embryos and considerably detected in prenauplii and nauplii. The isolation of a SPARC cDNA from Artemia franciscana provides intriguing features of the divergent protein, SPARC.     

Non-lethal heat shock protects gnotobiotic Artemia franciscana larvae against virulent Vibrios

Brine shrimp Artemia were exposed under gnotobiotic conditions to a non-lethal heat shock (NLHS) from 28 to 32, 37 and 40 degrees C. Different recovery periods (2, 6, 12 and 24h) and different heat-exposure times (15, 30, 45 and 60 min) were tested. After these NLHS, Artemia was subsequently challenged with Vibrio. Challenge tests were performed in stressed and unstressed nauplii at concentrations of 10(7) cells ml(-1) of pathogenic bacteria, Vibrio campbellii and Vibrio proteolyticus. A NLHS with an optimal treatment of 37 degrees C for 30 min and a subsequent 6h recovery period resulted in a cross-protection against pathogenic Vibrio. A 100% increase in the larval survival (P < 0.05) was observed. We have also demonstrated by Western blot that a NLHS increases the expression of HSP-70 in heat-shocked (HS) treated animals. This report is the first to reveal a cross protection of a NLHS against deleterious bacterial challenges in living crustaceans. The putative role of heat shock proteins (HSPs) in this process is discussed.     

Review of the biogeography of the genus Artemia (Crustacea, Anostraca)

In this study, we report on the known Artemia habitats worldwide. Recent literature information is incorporated about the taxonomic status of the various populations studied. The genus is composed of di-, tri-, tetra- and pentaploid parthenogenetic populations and of the following bisexual species: A. franciscana franciscana , A. franciscana monica , A. franciscana sp., A. persimilis , A. salina , A. urmiana , A. sinica and A. sp. from Kazakhstan. The problems of characterizing new brine shrimp populations are discussed. In view of the great importance of Artemia as part of the live food chain for the culture of fish and shellfish larvae and the present cyst shortage from the market, the need for commercial exploitation and development of new Artemia sources is now, more than ever, necessary.     

Bioencapsulation of Two Different Vibrio Species in Nauplii of the Brine Shrimp (Artemia franciscana)

Two groups of nauplii from the brine shrimp (Artemia franciscana) were enriched with different bacteria, and the dynamics of bacterial uptake by the nauplii were observed. This study showed that the efficiency of Artemia nauplii in bioencapsulating bacteria strongly depends on the type of bacteria used, time of exposure, and status (live or dead) of the bacteria.     

Effects of experience on feeding success by larval white seabass, Atractoscion nobilis

Laboratory experiments on larval white seabass, Atractoscion nobilis , showed that first feeding success on mixed zooplankton was zero, and less than 5% on Artemia nauplii , for larvae of different sizes. Experience increased success from 20% after 2 days to 69% after 14 days when larvae fed on Artemia nauplii , and to 42% after 4 days of feeding on zooplankton. This increase was attributed to learning in larvae less than 5·0 mm s.l. , and in part to size-related factors in larvae greater than 5·0 mm s.l.     

Assessment of fluorescent-labeled bacteria for evaluation of in vivo uptake of bacteria (Vibrio spp.) by crustacean larvae

Available methods to study crustacean digestive tract colonization by bacteria are laborious, time-consuming, and do not permit in vivo assays and observation. This paper reports on a rapid and consistent technique to apply a fluorescent label to bacteria, which can then be presented to filter-feeding crustacea such as Artemia and penaeid larvae for later in situ bacterial distribution observation. Three luminescent Vibrio spp. were stained and observed inside Artemia nauplii, shrimp zoea and mysis stages, Vibrio harveyi type strain ATCC 14126, M(1) (pathogenic) and Ea (non-pathogenic). Factors such as dye (DTAF) concentration, exposure time/temperature and sonication time were evaluated. Viability of the dye and stained bacteria were tested at 4, -20 and -70 degrees C storage temperatures for up to 81 days. Results show that 4 and -20 degrees C storage temperatures are not recommended. At -70 degrees C, both bacteria and dye are optimally preserved. Monodispersed fluorescent-labeled bacterial cells can be observed inside the digestive tract of crustacean larvae at a density of inoculation as high as 5.2 x 10(6) CFU ml(-1). After 2 to 4 h, some leaching occurs, increasing difficulty in observation, although after 24 h, it is still possible to observe monodispersed FLB inside the digestive tract of crustacean larvae. Autofluorescence may complicate observation when filter-feeding crustacean larvae are co-fed with microalgae.     

Regiospecific distribution of fatty acids in triacylglycerols of Artemia franciscana nauplii enriched with fatty acid ethyl esters

This paper reports the positional distribution of fatty acids in triacylglycerols (TAG) of Artemia franciscana nauplii enriched with each of palmitic (16:0), oleic (18:1n-9), linoleic (18:2n-6), linolenic (18:3n-3), eicosapentaenoic (20:5n-3), and docosahexaenoic (22:6n-3) acid ethyl esters. TAG extracted from the enriched and unenriched nauplii were subjected to regiospecific analysis to determine the fatty acid compositions of the sn-1(3) and sn-2 positions of TAG. In the unenriched nauplii, 16:0, 18:1n-9, and 18:2n-6 were preferentially located in the sn-1(3) position followed by the sn-2 position [i.e. sn-1(3)>sn-2], whereas 18:3n-3 was concentrated in the sn-2 position [i.e. sn-2>sn-1(3)]. Contents of 20:5n-3 and 22:6n-3 were low. After the nauplii were enriched with each of the ethyl esters for 18 h, fatty acid fed to the nauplii showed higher content in the sn-1(3) position than in the sn-2 position [i.e. sn-1(3)>sn-2]. Distribution pattern of 18:3n-3 changed from sn-2>sn-1(3) to sn-1(3)>sn-2 during the enrichment with 18:3n-3 ethyl ester. Increases in all of the fatty acids in TAG were attributed to that in the sn-1(3) position much more than that in the sn-2 position. Artemia nauplii appear to be characterized by preferential incorporation of exogenous fatty acids into the sn-1(3) position of TAG, even though endogenous fatty acids are esterified in the opposite position.