Standardization of protocol for <i>Vibrio</i> challenge in specific pathogen-free (SPF) shrimp (<i>Litopenaeus vannamei</i>)

This study was conducted to standardize the protocol for Vibrio challenge in specific pathogen-free (SPF) shrimp (Litopenaeus vannamei). Shrimp, from postlarvae15 to juvenile, were challenged with Vibrio campbellii, V. harveyi 642, V. harveyi E022, V. harveyi E2, and V. penaeicida by immersion and injection in normal as well as stress conditions. For immersion challenge, shrimp were immersed in seawater containing 103, 105, and 107 CFU mL-1 of bacterial cells. They were fed with Artemia nauplii, Artemia charged with V. campbellii, a piece of shrimp meat which was injected with the Vibrio campbellii or artificial feed during five days experiment. Bacterial density in the water and shrimp samples was determined at the end of experiment. There was no significant difference in mortality between the control and Vibrio challenged groups in normal conditions (p>0.05). Therefore, different stress factors were tested such as starvation, salinity and ammonium. 12h exposure to 40 and 50 mgL-1 NH4 + (pH = 7.9- 8.1) were found as sublethal doses for postlarvae and juvenile shrimps respectively. Starvation, ammonium stress (before and during challenge periods), and salinity stress at 5, 10, 20 gL-1 did not enhance the susceptibility of shrimp to Vibrio challenge by immersion. In absence of stress, SPF shrimp is not susceptible to Vibrio either by immersion or injection. Only in ammonium stress, SPF shrimp is susceptible to Vibrios to a large extent by injection with 106 CFU shrimp-1. Vibrio campbellii was found as the most virulent strain towards SPF shrimp among five tested strains

Expression and quorum sensing regulation of type III secretion system genes of <i>Vibrio harveyi</i> during infection of gnotobiotic brine shrimp

Type III secretion systems enable pathogens to inject their virulence factors directly into the cytoplasm of the host cells. The type III secretion system of Vibrio harveyi, a major pathogen of aquatic organisms and a model species in quorum sensing studies, is repressed by the quorum sensing master regulator LuxR. In this study, we found that during infection of gnotobiotic brine shrimp larvae, the expression levels of three type III secretion operons in V. harveyi increased within the first 12h after challenge and decreased again thereafter. The in vivo expression levels were highest in a mutant with a quorum sensing system that is locked in low cell density configuration (minimal LuxR levels) and lowest in a mutant with a quorum sensing system that is locked in the high cell density configuration (maximal LuxR levels), which is consistent with repression of type III secretion by LuxR. Remarkably, in vivo expression levels of the type III secretion system genes were much (> 1000 fold) higher than the in vitro expression levels, indicating that (currently unknown) host factors significantly induce the type III secretion system. Given the fact that type III secretion is energy-consuming, repression by the quorum sensing master regulators might be a mechanism to save energy under conditions where it does not provide an advantage to the cells

Genetic characterization of <i>Artemia tibetiana</i> (Crustacea: Anostraca)

The brine shrimp Artemia consists of a number of bisexual species and a large number of parthenogenetic forms, which collectively, inhabit a wide range of hypersaline habitats. A recently described species (A. tibetiana) from a carbonate lake (Lagkor Co) in Tibet at an altitude of 4490 m has been tested with New World (A. franciscana USA, and A. franciscana feral population Vietnam) and Old World species (A. salina, A. urmiana, A. sinica) for cross fertility. These tests show complete infertility between A. tibetiana and A. franciscana. Between A. tibetiana and A. urmiana, A. sinica partial fertility through to F-2 and F-3 generations is evident. Allozyme and RAPD comparison of A. tibetiana with A. franciscana (USA), A. franciscana (Vietnam), A. sinica (Mongolia) and A. urmiana (Iran) show that A. tibetiana is similar to other bisexual species in mean heterozygosity (0.074) but has a somewhat higher proportion of polymorphic loci (40%, similar to that of A. urmiana). The genetic distance between A. tibetiana and A. franciscana is 0.730, between A. tibetiana and A. urmiana is 0.475 and that between A. tibetiana and A. sinica is 0.114. F-is estimates for A. tibetiana differ significantly from zero for six loci, mainly because of lack of fit to Hardy-Weinberg expectations. This may suggest that even within the limited area of Lagkor Co there are genetically distinct populations

The bacterial storage compound PHB protects <i>Artemia franciscana</i> from pathogenic <i>Vibrio campbellii</i>

Infections caused by luminescent vibrios can cause dramatic losses in aquaculture. These infections are often hard to treat with antibiotics because of the spread of resistant strains and therefore, alternative control strategies are urgently needed. We previously found that the short-chain fatty acid 3-hydroxybutyrate protects Artemia from pathogenic Vibrio campbellii. In this study, we investigated whether the homopolymer of the fatty acid, the well-known bacterial storage compound poly-3-hydroxybutyrate (PHB), could be used to protect the nauplii from the pathogen. A starvation experiment learned us that the addition of 1000mg.l-1 PHB particles (average diameter 30μm) to the culture water of starved Artemia nauplii significantly enhanced their survival. This indicated that the nauplii could obtain energy from the PHB. In order to provide the nauplii from energy, the PHB must have been at least partially degraded into water-soluble products (i.e. 3-hydroxybutyrate monomers and oligomers). Subsequently, an in vivo challenge test was performed with the PHB particles and Artemia nauplii challenged to Vibrio campbellii LMG21363. The addition of the PHB particles (at 100mg.l-1 and 1000mg l-1) to the Artemia culture water together with the pathogen significantly enhanced the survival of the infected nauplii. A complete protection (no significant difference in survival with uninfected nauplii) was observed at the highest concentration. If the PHB particles were added 1 day after the addition of the pathogen, a similar but less pronounced effect was noticed. In a second in vivo challenge test, we investigated the effect of the addition of a PHBaccumulating Brachymonas denitrificans strain on the survival of infected nauplii. The strain, added either untreated or pasteurised at 107CFU ml-1, completely protected the nauplii from the pathogen if it had a high PHB content (32% of the VSS). No protection was observed if the strain had a low PHB content