Regulation of virulence factors by quorum sensing in Vibrio harveyi.

Vibrio harveyi is an important aquatic pathogen that produces several virulence factors. In this study, the effect of quorum sensing, bacterial cell-to-cell communication, on the production of the virulence factors caseinase, gelatinase, lipase, hemolysin, and phospholipase, was investigated. The activity of virulence factors was studied through enzymatic plate assays using V. harveyi wild type and mutants with constitutively maximal or minimal quorum sensing activity. The results showed that quorum sensing negatively regulates phospholipase activity as higher activity was observed in mutants with minimal quorum sensing activity than in the mutant with maximal quorum sensing activity. Reverse transcriptase real-time PCR with specific primers revealed that the expression level of three phospholipase genes was 2-fold lower in the mutant with minimal quorum sensing activity than in the mutant with maximal quorum sensing activity. As far as we know, this is the first report of quorum sensing regulation of phospholipase. Finally, caseinase and gelatinase activity were positively regulated by quorum sensing, which is consistent with previous reports, and lipase and hemolysin activity were found to be independent of quorum sensing. Hence, the regulation is different for different virulence factors, with some being either positively or negatively regulated, and others being independent of quorum sensing. This might reflect the need to produce the different virulence factors at different stages during infection

Microbial community management in aquaculture

Microbial community management in aquaculture creates benefits at the nutritional as well as at health level for cultured species. In addition, in case of biofloc application, it allows to link species at different trophic levels, making bioflocs the potential link in integrated multispecies aquaculture

Prospects for management of whitefly using plant semiochemicals, compared with related pests

Whitefly (Hemiptera: Sternorrhyncha: Aleyrodidae) pests are economically important in agriculture, including the tobacco whitefly, Bemisia tabaci, and the greenhouse whitefly, Trialeurodes vaporariorum. Whiteflies are mainly controlled by synthetic insecticides but resistance to these insecticides is rapidly evolving. A semiochemical-based management strategy could provide an alternative to the use of insecticides, by exploiting natural volatile signalling processes to manipulate insect behaviour. Whitefly behaviour is affected by differences in plant odour blends. Selected compounds have been suggested as putative semiochemicals, but in only a few studies, potential volatiles were eventually characterised by electrophysiology or olfactometry. The application of antennal preparation methods from the closely related families, the aphids (Hemiptera: Aphididae) and psyllids (Hemiptera: Psyllidae), may help to facilitate whitefly electroantennography. Behavioural bioassays are essential to identify the repellent or attractant effect of each semiochemical. The relevance of the semiochemicals in whitefly management needs to be evaluated in the respective cultivation system. Although the value of semiochemicals has not been demonstrated in the field against whiteflies, there is an emerging range of possible field applications and some promising prospects. Overall, the olfactory system of whiteflies needs to be elucidated in more detail

Expression of virulence genes of vibrios belonging to the Harveyi clade in the brine shrimp Artemia

Vibrios belonging to the Harveyi clade are pathogenic marine bacteria affecting both vertebrates and invertebrates. These bacteria are severe threat to the aquaculture industry with multibillion dollar losses. Many virulence factors have been identified in Harveyi clade vibrios, including phospholipases, hemolysins, proteases, (cysteine protease, metalloprotease, serine protease), lipopolysaccharide, presence of bacteriophages, bacteriocin-like substance (BLIS) and chitinase. The virulence of vibrios is controlled by different regulatory systems, including quorum sensing and the transmembrane transcriptional regulator ToxR. However, detailed knowledge on the expression and regulation of virulence genes, especially under in vivo conditions (i.e. during infection of a host) is still lacking. For the development of specific biocontrol measures to overcome this major aquaculture disease, it is of utmost importance to understand the molecular basics of virulence expression. This study aimed at investigating virulence factors (virulence genes) and their expression in vitro and in vivo within isolates belonging to the Harveyi clade. In the first step of this study, an overview is given of the current knowledge on pathogenesis of bacteria belonging to the Harveyi clade, their impact on the aquaculture sector, the virulence factors that are involved in pathogenicity and their regulation. The first part of the research aimed at checking for the presence of several virulence genes that has been described before in V. harveyi in different isolates belonging to the Harveyi clade. The presence of virulence genes of other vibrios (i.e. atypical virulence genes) in the genomes of the isolates was also studied. It was found that all the virulence genes that are typical for the Harveyi clade vibrios (including the quorum sensing master regulator luxR, the virulence regulator toxRVh, vhp metalloprotease, chiA chitinase, srp serine protease and vhh hemolysin) were present in all the isolates tested and some atypical virulence genes were also found in some of the isolates tested. These results suggest that vibrios belonging to the Harveyi clade might have acquired virulence genes from other vibrios in the aquatic environment through horizontal gene transfer. As all tested Harveyi clade isolates have been positive for all typical virulence genes, their different specific virulence cannot be linked to the presence of (one of) these genes. In a further study, the expression of virulence genes in these bacteria was quantified both in vitro and in vivo and the relation between virulence gene expression and survival of challenged brine shrimp (Artemia franciscana) was determined. A standardized a method was developed for quantifying in vivo gene expression using Artemia as an invertebrate gnotobiotic host model, which facilitates the study of virulence gene expression and regulation under natural conditions in order to gain more relevant data. The results showed that there is variation in the expression level of genes coding for virulence regulators and virulence factors in Vibrio isolates belonging to the Harveyi clade. In vitro expression of the regulators luxR and toxR and the vhh hemolysin gene were correlated with the survival of challenged brine shrimp larvae, indicating that variation in the expression of these genes might be the cause of the variation in the virulence of the isolates. The in vivo virulence gene expression in three selected isolates (one avirulent, one moderately virulent and one strongly virulent) during infection of brine shrimp larvae revealed a peak in expression after 24h in the virulent isolates and a constantly low expression in the avirulent isolate. Therefore, it can be concluded that the expression levels of the virulence genes studied here are strongly linked to virulence of Harveyi clade vibrios towards brine shrimp. Quorum sensing is one of the regulatory mechanisms in V. harveyi and other vibrios. Quorum sensing regulation of virulence genes was investigated by determining the expression levels of virulence genes in wild type V. harveyi and different quorum sensing mutants, both in vitro and in vivo during infection of brine shrimp (Artemia franciscana) larvae. This study revealed that the vhp metalloprotease gene is positively regulated by quorum sensing. The vhh hemolysin gene did not show a regulation by the three-channel QS mechanism (no difference in expression in the mutants which have either maximum or minimum activity of the three-channel system). But there was a lower expression in the AI-2 synthase and receptor mutants when compared to the other strains. This suggests that vhh hemolysin might be specifically regulated by AI-2 quorum sensing via the AI-2 receptor LuxPQ but not via the three-channel signal transduction system. There was no difference between the strains in expression levels of the virulence regulator gene toxR and the srp serine protease gene, which indicated that these genes are independent of quorum sensing. Under in vivo conditions, the luxR and vhp metalloprotease genes also showed to be under quorum sensing regulation and the vhh hemolysin gene followed the same pattern of expression as in in vitro, having lower expression in an AI-2 deficient mutant. toxR and the srp serine protease gene did not show quorum sensing regulation under in vivo conditions. Further, the expression levels of the virulence genes in luminescent and non-luminescent isogenic strains were investigated. The study revealed that the non-luminescent variants produced lower levels of the quorum sensing master regulator luxR and the vhp metalloprotease gene. Challenge tests with gnotobiotic brine shrimp larvae showed that the non-luminescent strains are less virulent than their luminescent counterparts. In a last study, the quorum sensing regulation of the chitin-degrading enzyme chitinase, which might be involved in colonization and infection of host organisms, especially crustaceans (which contain chitin in their shells) was investigated. This study indicated that chitinase A is negatively regulated by quorum sensing

Volatile-Mediated Attraction of Greenhouse Whitefly Trialeurodes vaporariorum to Tomato and Eggplant

The behavior of the greenhouse whitefly, Trialeurodes vaporariorum Westwood (Hemiptera: Aleyrodidae), is known to be affected by plant volatile cues, but its attraction or repellent to specific volatile cues has not been deeply studied yet. Therefore, the aim of our study was to identify the most attractive plant among cultivars of tomato (Solanum lycopersicum) and eggplant (Solanum melongena) to evaluate the volatiles of plants to identify the chemical compound(s) that attract T. vaporariorum. We speculated that whitefly–host plant interaction primarily depends on plant volatile emissions and that once the plant is damaged, it might attract more whiteflies. Three intact (uninfested) tomato, four intact eggplant cultivars and whitefly infested plants of the most whitefly attractive tomato and eggplant cultivars were examined by behavioral assay experiments for attractiveness to T. vaporariorum and headspace volatile were determined by solid-phase microextraction (SPME) and gas chromatography-mass spectrometry. Whiteflies had the highest preference for the intact eggplant Kuai Yuan Qie (KYQ) among the eggplant and the tomato plant cultivars in bioassay experiments. Although both male and female whiteflies were significantly more attracted to infested KYQ plants than to intact plants, whitefly females did not select conspecific-infested YG plants. The volatile emissions among different plant cultivars in individual species and infested versus intact plants were significantly different. Among these volatiles, identified major green leaf volatiles [(Z)-3-hexen-1-ol] and terpenoids [α-pinene, (E)-β-caryophyllene, α-humulene, azulene] showed a constitutive relationship with the most whitefly preference plants. Our findings provide new insights into the chemical compounds that attract or repel whiteflies

Nutritional Composition and Apparent Metabolizable Energy (AME) Value of Black Soldier Fly Larvae (Hermetia illucens L.) Full-Fat Meal for Broiler Chickens

Black soldier fly larvae (BSFL; Hermetia illucens L.) is a proven high-cost protein source replacer and could be grown in a range of bio-degradable waste materials where hardly incorporated into broiler diets locally. The present study was aimed to assess the nutritional composition of BSFL, and apparent metabolizable energy (AME) value of BSFL meal provided to broiler chickens. BSFL full-fat meal produced from kitchen waste as a substrate were examined for their proximate composition, minerals and fatty acid profile. Eighty, 21-d old unsexed Cobb-500 broiler chickens (BW±SD: 665.8 ±14.3 g) were assigned randomly into 16 battery cages (04 replicates, five birds/replicate). A maize-soybean meal-based diet was used as the basal diet which was partially substituted by pre-analyzed BSFL meal at the rates of 5%, 10%, and 15% to produce three test diets. Birds were fed in a completely randomized design for 7-d with a 4-d adaptation period. Excreta were collected for three days from day 25 to 28. The results envisaged that the crude protein (CP) and ether extract (EE) contents of the kitchen waste were 12.3%, and 10.5%, respectively. BSFL meal when analyzed had 34.4% CP and 47.3%, EE. The fatty acid (FA) profile of the kitchen waste was more or less similar to that of BSFL‘s meal. The estimated AME of the BSFL full-fat meal fed for broilers was estimated to be 15.7 MJ/kg. The BSFL full-fat meal can be utilized sustainably in feed formulation and has a high potential to replace costlier feed ingredients

Expression and Quorum Sensing Regulation of Type III Secretion System Genes of Vibrio harveyi 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

Quorum sensing negatively regulates chitinase in Vibrio harveyi

Quorum sensing, bacterial cell-to-cell communication, regulates the virulence of Vibrio harveyi towards different hosts. Chitinase can be considered as a virulence factor because it helps pathogenic bacteria to attach to the host and to penetrate its tissues (e.g. in case of shrimp). Here, we show that quorum sensing negatively regulates chitinase in V. harveyi. Chitinolytic activity towards natural chitin from crab shells, the synthetic chitin derivative chitin azure, and fluorogenic chitin oligomers was significantly higher in a mutant in which the quorum-sensing system is completely inactivated when compared with a mutant in which the system is maximally active. Furthermore, the addition of signal molecule containing cell-free culture fluids decreased chitinase activity in a Harveyi Autoinducer 1 and Autoinducer 2-deficient double mutant. Finally, chitinase A mRNA levels were fivefold lower in the mutant in which the quorum-sensing system is maximally active when compared with the mutant in which the system is completely inactivated. [Correction added on 25 September 2009, after first online publication: the preceding sentence was corrected from 'Finally, chitinase A mRNA levels were fivefold lower in the mutant in which the quorum-sensing system is completely inactivated when compared with the mutant in which the system is maximally active.'] We argue that this regulation might help the vibrios to switch between host-associated and free-living life styles

Quorum sensing regulation of virulence gene expression in Vibrio harveyi during its interaction with marine diatom Skeletonema marinoi

Communication between species from different kingdoms may be as important as intra-kingdom communication. It has recently been confirmed that co-existing bacteria and phytoplankton in aquatic ecosystems do cross-talk. This study examined the signs of possible cross signalling between V. harveyi, one of the predominant bacterial species of the marine ecosystem and a dominant diatom species, S.marinoi, to understand communication over species borders. It is known that V.harveyi employ quorum sensing for cell-to-cell communication, bioluminescence (luxR), and the regulation of the virulence gene (vhp, chiA). Former studies have also shown, this kind of interactions being disrupted by compounds secreted by a few algal species existing in the aquatic ecosystem. We investigated the QS communication by quantifying the expression levels of virulence regulator luxR and virulence factors metalloprotease (vhp) and chitinase (chiA) in four different V. harveyi strains grown in the presence of S. marinoi strain. Results obtained in this study indicate that quorum sensing was activated in strains of V. harveyi analysed but did not regulate the expressions of vhp and chiA virulence factors. This observation suggests that the existence of S. marinoi did not interfere with the QS behavior of V. harveyi and its interaction with marine diatom; it may be due to the commensalism relationship