Virulence of luminescent and non-luminescent isogenic vibrios towards gnotobiotic Artemia franciscana larvae and specific pathogen-free Litopenaeus vannamei shrimp

Aims:  This study was conducted to test the virulence of luminescent (L) and non-luminescent (NL) isogenic strains of Vibrio campbellii LMG21363, Vibrio harveyi BB120 (wild type) and quorum-sensing mutant strains derived from the wild type such as Vibrio harveyi BB152, BB170, MM30 and BB886.
Methods and Results:  The NL strains could be obtained by culturing rifampicin-resistant luminescent strains in the dark under static condition. The virulence of the L and NL strains was tested in gnotobiotic Artemia franciscana larvae challenged with 10⁴ CFU ml⁻¹ of bacteria. All luminescent isogenic tested strains showed higher virulence compared to the NL strains. The virulence of L and NL V. campbellii and V. harveyi BB120 was also tested in specific pathogen-free juvenile shrimp upon intramuscular injection with 10⁶ CFU of bacteria. In contrast with Artemia , there was no significant difference in mortality between the groups challenged with L and NL strains ( P  > 0·05). The non-luminescent strains were not able to revert back to the luminescent state and quorum sensing did not influence this phenotypic shift.
Conclusions:  Luminescent Vibrio strains can switch to a non-luminescent state by culturing them in static conditions. The NL strains become less virulent as verified in Artemia .
Significance and Impact of the Study:  The luminescent state of Vibrio cells in a culture needs to be verified in order to assure maintenance of virulence.     

Pathogenicity of vibrios to rainbow trout (Oncorhynchus mykiss, Walbaum) and Artemia nauplii

The taxonomy of marine vibrios has changed rapidly over the last two decades, and a wealth of new species have been identified. Many Vibrio species are pathogenic to fish and crustaceans; however, little is known about the virulence of many of the novel species. The purpose of this study was to determine the ability of various recent isolates of vibrios to cause disease in rainbow trout (Oncorhynchus mykiss, Walbaum) and crustacea, i.e. Artemia nauplii. Of 56 isolates, representing 26 species of Enterovibrio, Photobacterium and Vibrio, obtained from a diversity of healthy and diseased aquatic animal hosts and water samples from many geographical locations, Vibrio brasiliensis, V. coralliilyticus, V. ezurae, V. fortis, V. kanaloaei, V. neptunius, V. rotiferianus and V. tubiashii were pathogenic to rainbow trout and Artemia nauplii with mortalities of up to 100%. The extracellular products of these pathogenic isolates were harmful to the animal models. In contrast, cultures of Enterovibrio norvegicus, E. coralii, Photobacterium rosenbergii, Vibrio campbellii, V. chagasii, V. cyclitrophicus, V. gallicus, V. gigasii, V. hepatarius, V. hispanicus, V. lentus, V. nereis, V. pacini, V. pomeroyi, V. shilonii, V. superstes, V. tasmaniensis and V. xuii demonstrated either non- or low virulence in the animal models.     

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.     

Stereology and computer assisted three-dimensional reconstruction as tools to study probiotic effects of Aeromonas hydrophila on the digestive tract of germ-free Artemia franciscana nauplii

Aims: Validation of stereology and three‐dimensional reconstruction for monitoring the probiotic effect of Aeromonas hydrophila on the gut development of germ‐free Artemia franciscana nauplii. Methods and Results: Germ‐free Artemia nauplii were cultured using Baker’s yeast and dead Aer. hydrophila. Live Aer. hydrophila were added on the first day to the treatment group. The gut length and volume were monitored on days two and four using stereology and three‐dimensional reconstruction. Both methods showed comparable results. Stereology was least labour intensive to estimate volumes, while three‐dimensional reconstructions rendered architectural and topographical data of the gut. Moreover, a positive effect of probiotic bacterium, Aer. hydrophila is likely. Conclusion: Slight increment in the growth of the digestive tract of A. franciscana nauplii exerted by probiotic bacteria could be detected using stereology and three‐dimensional reconstruction. Significance and Impact of the Study: The gnotobiotic Artemia rearing system is unique to investigate the effects of micro‐organisms on the development of nauplii. However, in the base of this model system, only survival counts and length measurements exist as monitoring tools. Therefore, additional tools such as stereology and three‐dimensional reconstruction are prerequisite to obtain more powerful analysis.     

The heat shock response of adult Artemia franciscana

1. We studied responses of adult brine shrimp, Artemia franciscana, to high temperature, including LT₅₀ determination, induced thermotolerance (ITT), the Hsp-70 family of stress proteins and protein synthesis before and after heat shock.

2. Adults were grown in laboratory cultures from encysted embryos (cysts) obtained from San Francisco Bay (SF) and much warmer culture ponds in Vietnam (V).

3. Adults from V cysts were more tolerant of high temperatures than those from SF cysts, but this difference essentially disappeared in the second generation of adults.

4. Levels of constitutive Hsc-70 were very low in adults of both groups, but were strongly upregulated by a sublethal heat shock (37°C, 30 min), with V adults showing the greater degree of upregulation. Heat shock also induced Hsp-67, to a greater extent in V compared to SF adults

5. Incorporation of ¹⁴C-leucine into protein did not result in the “classic” heat shock response, possibly due to increased permeability of heat-shocked animals to the tracer.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded^1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes³. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation⁴. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.     

Food size selectivity of Artemia franciscana at three developmental stages

Food size selectivity was examined in Artemia franciscana metanaupliiat three different developmental stages. Clearance rates weredetermined in short-term experiments either by measuring thedecrease in concentration of live particles and plastic beads,or by measuring the radioactivity accumulated in animals thatgrazed ¹⁴C-labelled live particles. The maximum clearance rateofA.franciscana metanauplii increased during development andwas measured at 50–63 µl ind.^–1 h^–1,254 µl ind.^–1 h^–1 and 1.48–2.10 ml ind.^–1h^–1 in 2-, 4- and 7-day-old metanauplii, respectively.A preference for particles with a diameter of 4–8 µmwas observed at all three developmental stages. The abilityof A.franciscana metanauplii to graze bacterial particles wasalso demonstrated, although the efficiency in grazing such smallparticles was low compared to microalgae (28, 20 and 9% of themaximum clearance rate in 2-, 4- and 7-day-old metanauplii,respectively). Electron microscopy showed that the inter-setulardistance in antennae and thoracopods was 0.20 ± 0.07,0.16 ± 0.05 and 0.18 ± 0.04 µm in 2-, 4-and 7-day-old metanauplii, respectively, and accordingly independentof stage.     

The complete mitochondrial DNA sequence of the crustacean Artemia franciscana

The complete mitochondrial DNA (mtDNA) sequence of the brine shrimp Artemia franciscana has been determined. It extends the present knowledge of mitochondrial genomes to the crustacean class and supplies molecular markers for future comparative studies in this large branch of the arthropod phylum. Artemia mtDNA is 15,822 nucleotides long, and when compared with its Drosophila counterpart, it shows very few gene rearrangements, merely affecting two tRNAs placed 3 downstream of the ND 2 gene. In this position a stem-loop secondary structure with characteristics similar to the vertebrate mtDNA L-strand origin of replication is found. This suggests that, associated with tRNA changes, the diversification of the mitochondrial genome from an ancestor common to crustacea and insects could be explained by errors in the mtDNA replication process. Although the gene content is the same as in most animal mtDNAs, the sizes of the protein coding genes are in some cases considerably smaller. Artemia mtDNA uses the same genetic code as found in insects, ATN and GTG are used as initiation codons, and several genes end in incomplete T or TA codons.Correspondence to: R. Garesse     

Functional morphology of the neck organ in Artemia salina nauplii

Fine structure of the ion transporting epithelium of the neck organ in the brine shrimp (Artemia salina) nauplius is described. The neck organ is a dome-like gland situated atop the cephalothorax of the larva and is composed of 50 to 60 cuboidal epithelial cells. These cells possess many of the characteristics of salt-secretory cells from other tissues. They contain many mitochondria and exhibit a high degree of plasma membrane elaboration. This membrane amplification takes two forms; the apical plasmalemma is infolded into irregular loops, while the basal and lateral membranes penetrate the cytoplasm in the form of branching sinusoids. The labyrinth of tubular reticulum thus formed fills most of the cell volume. Mitochondria in the labyrinth are often in intimate contact with these tubular membranes and regular arrays of parallel mitochondria with constricted intervening sinusoids are often observed. Other organelles including Golgi complexes, multivesicular bodies, and rough endoplasmic reticulum are also numerous, particularly in the narrow rim of cytoplasm which lies between the apical infolds and the labyrinth. Yolk platelets and glycogen fields are conspicuous in the basal perinuclear regions of the cells.     

The total and mitochondrial lipidome of Artemia franciscana encysted embryos

Encysted embryos (cysts) of the crustacean Artemia franciscana exhibit enormous tolerance to adverse conditions encompassing high doses of radiation, years of anoxia, desiccation and extreme salinity. So far, several mechanisms have been proposed to contribute to this extremophilia, however, none were sought in the lipid profile of the cysts. Here in, we used high resolution shotgun lipidomics suited for detailed quantitation and analysis of lipids in uncharacterized biological membranes and samples and assembled the total, mitochondrial and mitoplastic lipidome of Artemia franciscana cysts. Overall, we identified and quantitated 1098 lipid species dispersed among 22 different classes and subclasses. Regarding the mitochondrial lipidome, most lipid classes exhibited little differences from those reported in other animals, however, Artemia mitochondria harboured much less phosphatidylethanolamine, plasmenylethanolamines and ceramides than mitochondria of other species, some of which by two orders of magnitude. Alternatively, Artemia mitochondria exhibited much higher levels of phosphatidylglycerols and phosphatidylserines. The identification and quantitation of the total and mitochondrial lipidome of the cysts may help in the elucidation of actionable extremophilia-affording proteins, such as the ‘late embryogenesis abundant’ proteins, which are known to interact with lipid membranes.     

Poly-beta-hydroxybutyrate-accumulating bacteria protect gnotobiotic Artemia franciscana from pathogenic Vibrio campbellii

A poly-beta-hydroxybutyrate (PHB)-accumulating enrichment culture was obtained using activated sludge from a polyphosphate-accumulating reactor as inoculum. PHB accumulated by the enrichment culture significantly enhanced the survival of Artemia nauplii, infected with the virulent pathogen Vibrio campbellii LMG 21363. A strain was isolated from the enrichment culture, based on its ability to accumulate PHB, and 16S rRNA gene sequencing of the isolate revealed 99% sequence similarity to Brachymonas denitrificans AS-P1. The isolate, named PHB2, showed good PHB-accumulating activity (up to 32% of the cell dry weight). PHB accumulated by isolate PHB2 was able to protect Artemia completely from the V. campbellii strain. Our data indicate that PHB-accumulating bacteria, such as B. denitrificans PHB2, could be used as an an effective and economically interesting alternative strategy to control infections in aquaculture.     

Biochemical studies on sphingolipids of Artemia franciscana: complex neutral glycosphingolipids

Brine shrimp are primitive crustacean arthropodal model organisms, second to daphnia, which can survive in high-salinity environments. Their oviposited cysts, cuticle-covered diapausing eggs, are highly resistant to dryness. To elucidate specialties of brine shrimp, this study characterized glycosphingolipids, which are signal transduction-associated material. A group of novel and complex fucosyl glycosphingolipids were separated and identified from cysts of the brine shrimp Artemia franciscana by repeated lipid extraction, alkaline methanolysis, acid treatment, successive column chromatography, and post-source decay measurements by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Structures of the glycosphingolipids were elucidated by conventional structural characterization and mass spectrometry, and the compounds were identified as GlcNAcβ1-3GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer, GalNAcβ1-4(Fucα1-3)GlcNAcβ1-3GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer, and GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer. These compounds also contained a branching, non-arthro-series disaccharide with an α-GlcNAc terminus, similar to that found in a previously reported ceramide hexasaccharide (III³(GlcNAcα2Fucα)-At₄Cer). The glycans within these complex GSLs are longer than reported glycans of the animal kingdom containing α-GlcNAc terminus. These complex GSLs as well as the longest GSL with ten sugar residues, ceramide decasaccharide (CDeS), contain the fucosylated LacdiNAc sequence reported to associate with parasitism/immunosuppression and the α-GlcNAc terminus reported to show a certain antibacterial effect in other reports. CDeS, the longest GSL of this species, was found in the highest amount, which indicates that CDeS may be functionally important.     

Characterization of the microtubule proteome during post-diapause development of Artemia franciscana

The microtubule proteome encompasses tubulin and a diverse group of proteins which associate with tubulin upon microtubule formation. These proteins either determine microtubule organization and function or their activity is influenced by microtubule association. To characterize the microtubule proteome in Artemia franciscana, tubulin assembly was induced with taxol in vitro after 0 and 12 h of post-diapause development. Proteins obtained by extraction of microtubules with 0.5 M NaCl were electrophoresed in two-dimensional gels and analyzed by mass spectrometry. Fifty-five proteins were identified with 10 of these occurring at both developmental stages, and multiple isoforms were observed for some proteins of the Artemia proteome. Their functions include roles in membrane transport, metabolism, chaperoning and protein synthesis, thus reflecting physiological properties of encysted Artemia such as stress resistance and the ability to rapidly initiate post-diapause development. For example, chaperones may protect tubulin during encystment and facilitate folding in metabolically active embryos. Additionally, the interaction of metabolic enzymes with microtubules funnels reaction intermediates, potentially enhancing efficiency within biochemical processes. This study represents the first systematic characterization of a crustacean microtubule proteome. Although it is difficult to be certain that all protein associations documented herein occur in vivo, the results suggest how protein-protein interactions contribute to cytoplasmic organization while implying how Artemia embryos resist stress and remain capable of development once diapause terminates.     

Potential utilization of Artemia franciscana eggs as food for Coleomegilla maculata

A major hindrance to mass rearing predators for augmentative biological control is the limited availability of inexpensive, alternative foods in lieu of natural prey or target prey. We tested the hypothesis that brine shrimp eggs (Artemia franciscana) are a suitable alternative food that support the development of the predatory ladybird beetle, Coleomegilla maculata. Laboratory bioassays determined the effects of A. franciscana on C. maculata growth, development and reproduction. In comparison to Mediterranean flour moth eggs (Ephestia kuehniella), A. franciscana eggs were suitable for growth and development, but not for reproduction. C. maculata females oviposited less often when fed A. franciscana rather than E. kuehniella. One reason for the low oviposition rate could be less soluble protein in A. franciscana than in E. kuehniella, as determined by biochemical analysis. Further research is needed to identify all deficiencies in A. franciscana and explore the possibility of using supplemental nutrients to counteract them.