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

Probing the phenomenon of trained immunity in invertebrates during a transgenerational study, using brine shrimp <i>Artemia</i> as a model system

The invertebrate’s innate immune system was reported to show some form of adaptive features, termed trained immunity. However, the memory characteristics of innate immune system and the mechanisms behind such phenomena remain unclear. Using the invertebrate model Artemia, we verified the possibility or impossibility of trained immunity, examining the presence or absence of enduring memory against homologous and heterologous antigens (Vibrio spp.) during a transgenerational study. We also determined the mechanisms behind such phenomenon. Our results showed the occurrence of memory and partial discrimination in Artemia’s immune system, as manifested by increased resistance, for three successive generations, of the progenies of Vibrio-exposed ancestors towards a homologous bacterial strain, rather than to a heterologous strain. This increased resistance phenotype was associated with elevated levels of hsp70 and hmgb1 signaling molecules and alteration in the expression of key innate immunity-related genes. Our results also showed stochastic pattern in the acetylation and methylation levels of H4 and H3K4me3 histones, respectively, in the progenies whose ancestors were challenged. Overall results suggest that innate immune responses in invertebrates have the capacity to be trained, and epigenetic reprogramming of (selected) innate immune effectors is likely to have central place in the mechanisms leading to trained immunity

Influence of different yeast cell-wall mutants on performance and protection against pathogenic bacteria (<i>Vibrio campbellii</i>) in gnotobiotically-grown <i>Artemia</i>

A selection of isogenic yeast strains (with deletion for genes involved in cell-wall synthesis) was used to evaluate their nutritional and immunostimulatory characteristics for gnotobiotically-grown Artemia. In the first set of experiments the nutritional value of isogenic yeast strains (effected in mannoproteins, glucan, chitin and cell-wall bound protein synthesis) for gnotobiotically-grown Artemia was studied. Yeast cell-wall mutants were always better feed for Artemia than the isogenic wild type mainly because they supported a higher survival but not a stronger individual growth. The difference in Artemia performance between WT and mutants feeding was reduced when stationary-phase grown cells were used. These results suggest that any mutation affecting the yeast cell-wall make-up is sufficient to improve the digestibility in Artemia. The second set of experiments, investigates the use of a small amount of yeast cells in gnotobiotic Artemia to overcome pathogenicity of Vibrio campbellii (VC). Among all yeast cell strains used in this study, only mnn9 yeast (less cell-wall bound mannoproteins and more glucan and chitin) seems to completely protect Artemia against the pathogen. Incomplete protection against the pathogen was obtained by the gas1 and chs3 mutants, which are lacking the gene for a particular cell-wall protein and chitin synthesis, respectively, resulting in more glucan. The result with the chs3 mutant is of particular interest, as its nutritional value for Artemia is comparable to the wild type. Hence, only with the chs3 strain, in contrast to the gas1 or mnn9 strains, the temporary protection to VC is not concomitant with a better growth performance under non-challenged conditions, suggesting non-interference of general nutritional effects

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

Norepinephrine and dopamine increase motility, biofilm formation, and virulence of <i>Vibrio harveyi</i>

Vibrio harveyi is one of the major pathogens of aquatic organisms, affecting both vertebrates and invertebrates, and causes important losses in the aquaculture industry. In order to develop novel methods to control disease caused by this pathogen, we need to obtain a better understanding of pathogenicity mechanisms. Sensing of catecholamines increases both growth and production of virulence-related factors in pathogens of terrestrial animals and humans. However, at this moment, knowledge on the impact of catecholamines on the virulence of pathogens of aquatic organisms is lacking. In the present study, we report that in V. harveyi, norepinephrine (NE) and dopamine (Dopa) increased growth in serum-supplemented medium, siderophore production, swimming motility, and expression of genes involved in flagellar motility, biofilm formation, and exopolysaccharide production. Consistent with this, pretreatment of V. harveyi with catecholamines prior to inoculation into the rearing water resulted in significantly decreased survival of gnotobiotic brine shrimp larvae, when compared to larvae challenged with untreated V. harveyi. Further, NE-induced effects could be neutralized by α-adrenergic antagonists or by the bacterial catecholamine receptor antagonist LED209, but not by β-adrenergic or dopaminergic antagonists. Dopa-induced effects could be neutralized by dopaminergic antagonists or LED209, but not by adrenergic antagonists. Together, our results indicate that catecholamine sensing increases the success of transmission of V. harveyi and that interfering with catecholamine sensing might be an interesting strategy to control vibriosis in aquaculture. We hypothesize that upon tissue and/or hemocyte damage during infection, pathogens come into contact with elevated catecholamine levels, and that this stimulates the expression of virulence factors that are required to colonize a new host

Non-lethal heat shock protects gnotobiotic <i>Artemia franciscana</i> larvae against virulent <i>Vibrios</i>

Brine shrimp Artemia were exposed under gnotobiotic conditions to a non-lethal heat shock (NLHS) from 28 to 32, 37 and 40°C. Different recovery periods (2, 6, 12 and 24 h) 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 107 cells ml-1 of pathogenic bacteria, Vibrio campbellii and Vibrio proteolyticus. A NLHS with an optimal treatment of 37°C for 30 min and a subsequent 6 h 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