Antibiofilm potential of a tropical marine Bacillus licheniformis isolate: role in disruption of aquaculture associated biofilms

Microbial biofilms are important in aquaculture industries as they resist antibiotic treatments. In this study, we have investigated the antibiofilm potential of a tropical marine culture Bacillus licheniformis D1 (containing an antimicrobial protein BLDZ1) against two aquaculture associated pathogens namely, Vibrio harveyi and Pseudomonas aeruginosa. Both the test cultures formed biofilms on polystyrene and glass surfaces. The cell free supernatant (CFS) of B. licheniformis inhibited V. harveyi and P. aeruginosa biofilms on polystyrene surfaces up to around 80% and 78% respectively. In addition, the CFS disrupted pre‐formed biofilms of test cultures by about 73%. Fluorescence and scanning electron microscope analysis confirmed the antibiofilm potential of the CFS. The cell free supernatant displayed antiadhesive activity that inhibited the initial attachment of the bacteria during the process of biofilm formation. In addition, the CFS exhibited antimicrobial activity and mediated cell death via cytoplasmic membrane disruption.     

Pathogenic threats and probiotic use in larviculture of the scallop,Pecten maximus

During a series of experiments, bacteriological elements in scallop larval rearing were investigated: larvae susceptibility to pathogens as a function of their age, and the use of probiotic bacteria during larviculture. Younger larvae (d5 PF) were highly more susceptible to pathogenic‐challenge than their older siblings, which were challenged at an older age (d15 PF). A challenge with 10⁴ CFU mL^?1 of V. pectenicida killed 100% of d5 PF larvae 7 days following challenge, yet killed only 9% of d15 PF larvae 9 days following challenge. Use of the probiotics Phaeobacter gallaeciensis, Alteromonas macleodii 0444 and Neptunomonas sp. 0536, provided for larger larvae, a high yield of competent larvae and, perhaps more importantly, protection against pathogen‐challenge similar to levels achieved from antibiotic use. When challenged with V. pectenicida, d29 survivals were 20.3%, 85.1% and 75.0% respectively for control (no probiotic), antibiotic treated, and ‘probiotic mix’ administered larvae. Use of potential probiotic Pseudoalteromonas sp. D41 appeared to hinder scallop larvae. Future use of probiotics in scallop larval rearing would benefit from combined use of P. gallaeciensis, A. macleodii 0444 and Neptunomonas sp. 0536.     

培养基对微生物被膜形成和厚壳贻贝附着的影响

为探讨微生物被膜生物学特性、培养基以及大型生物附着三者之间的相互关系,本实验通过微生物分子生态学和海洋贝类生态学等方法调查了不同培养基对海洋细菌所形成微生物被膜的影响及其对厚壳贻贝幼虫附着的影响。结果显示,厚壳贻贝稚贝率与培养基类型和细菌初始密度显著相关,进一步分析表明,Zo Bell 2216E和Seawater Luriabertani(SWLB)条件下分别有6株和9株细菌形成的微生物被膜密度与稚贝率显著相关。扫描电镜结果显示,Staphylococcus sp.3在Zo Bell 2216E培养基条件下形成微生物被膜的细菌分布较为紧密,Pseudoalteromonas sp.8则在SWLB培养基条件下微生物被膜细菌分布较为紧密,且形态变为短杆状。SDS-PAGE结果显示,相比Zo Bell 2216E培养基,Staphylococcus sp.3在SWLB培养基条件下9条条带蛋白显著下降,其中2个条带完全消失;Pseudoalteromonas sp.8则在SWLB培养基条件下明显增加5条蛋白条带。研究表明,微生物被膜的形成受到培养基的影响,培养基的不同导致微生物被膜的形态结构、分布和蛋白有所差异,最终导致微生物被膜诱导厚壳贻贝幼虫附着变态的活性差异,本研究为后续开展厚壳贻贝附着的分子机制奠定良好基础。     

Use of Ox‐Aquaculture© for disinfection of live prey and meagre larvae,Argyrosomus regius (Asso, 1801)

Live prey used for marine larval fish (rotifers and Artemia) as well as intensive larval rearing conditions are susceptible to the proliferation of bacteria that are the cause for reduced growth and larval mortality. Hydrogen peroxide has been recently proved a good disinfectant in aquaculture, either for eggs, larvae or live prey. In this study the effects of a hydrogen peroxide‐based product, Ox‐Aquaculture^©, on live prey (rotifers and Artemia) and meagre larvae bacterial load, composition and final status have been tested. A 34.6% reduction of total heterotrophic bacteria and 59.7% of Vibrionaceae were obtained when rotifers were exposed for 15 min to 40 mg L^?1 of the product. A 34.3% reduction of total heterotrophic bacteria and 37.7% of Vibrionaceae were obtained when Artemia were exposed for 5 min to 8000 mg L^?1 of the product. More than 95% reduction of total heterotrophic bacteria and 75% of Vibrionaceae were obtained when meagre larvae were exposed for 1 h to 20 mg L^?1 of the product. Furthermore, disinfection of enriched live prey with the product did not change the fatty acid composition and survival of the live prey and improved final larval survival.     

The use of diatom Skeletonema costatum on aquaculture‐produced purple sea urchin (Paracentrotus lividus) larvae and post‐larvae diet

The quality of the microalgae provided on Paracentrotus lividus larvae rearing is a primordial factor having a direct (nutritional properties) and indirect (water quality) impact on growth, competence and survival. Skeletonema costatum is a diatom commonly used in the bivalve cultivation. However, the use of this diatom in P. lividus larval cultivations is poorly known. The Rhodomonas spp. is a microalgae commonly used in sea urchin larvae culture. Three different diets were tested on P. lividus larvae and post‐larvae cultivation (D1—Rhodomonas marina, D2—S. costatum, D3—mixture of both algae). Larvae fed with the D2 diet (55.8%) and D3 (39.9%) had a survival at 15 DAH higher than D1 (5.5%). The low survival in D1 could be due to the higher microbiological load on microalgae (Vibrio alginolyticus and V. pectenicide). Larvae fed with S. costatum (D2) showed a lower development than other diets. The competency index was lower for larvae fed with the D2. These results show that microalgae diversified diets contribute to a better development of P. lividus larvae. During the settlement and post‐settlement phase, there was also a lower growth of the sea urchin fed with the D2 and a higher survival for D3.     

Effects of a mix of Bacillus sp. as a potential probiotic for Florida pompano,common snook and red drum larvae performances and digestive enzyme activities

This study examined the effect of a commercial mix of Bacillus sp. on survival, growth and digestive enzyme activities of Florida pompano, red drum and common snook. Larvae were fed either live feed enriched with Algamac 3050 (Control), Algamac 3050 and probiotics (PB), or the previous diet combined with a daily addition of probiotics to the tank water (PB+). Survival was not affected by the treatments for any of the species. At the end of the pompano and snook trial, standard lengths of larvae from the PB and PB+ treatments were significantly greater than for the control larvae. Microbiological analyses were performed at the end of the pompano trial, and numbers of presumptive Vibrio were not a concern in the system. For both pompano and snook, trypsin‐specific activity was higher in PB and PB+ larvae compared with the control larvae. Similarly, alkaline phosphatase activity was higher for the pompano larvae fed the PB and PB+ treatments and for the snook larvae fed the PB+ treatment compared with the control larvae. This experiment suggests that a mix of Bacillus sp. can promote growth through an early maturation of the digestive system during the early larval stages of pompano and snook.     

Experimental culture of larvae,post‐larvae and juveniles of the West Indian top shell,Cittarium pica (Linnaeus 1758)

The West Indian top shell, Cittarium pica, is an endangered vetigastropod of ecological and commercial value from the Caribbean. In order to assess the use of aquaculture as a tool for its sustainable production and conservation, embryos were produced in hatchery and experimentally cultured until juveniles under different conditions. Embryos were incubated under two temperatures (25 and 29°C) and six densities (0.3, 0.6, 1.0, 4.0, 9.0 and 37.0% of bottom‐coverage). Larvae were reared under different temperatures (25 and 27°C), densities (0.5, 1, 1.5 and 10 larvae/ml) and culture systems (static and down‐welling). Post‐larvae were obtained in three different settling conditions, and the early juveniles were cultured supplying fresh seaweed (Laurencia obtusa and Padina gymnospora), natural multi‐specific biofilm and Cylindroteca sp. biofilm. The growth and survival of embryos and larvae were not affected by temperature, but they were affected by density. Higher values were obtained at low densities (<1% of embryos bottom‐coverage and <1 larvae/ml), except for the growth of larvae, which was similar among treatments. Higher larval survival was recorded using the static culture system, and higher percent of post‐larvae recovered was associated with lower density of crawling veliger (0.2 and 0.1/ml), while its higher growth rate was related to the use of biofilms with conspecific mucus and low water‐flow (50%/h). The type of food tested did not affect the juvenile growth, but higher survival was obtained in those fed with seaweed than with multi‐specific biofilm.     

The release and uptake of metals from potential biofilm inhibition products during spiny lobster (Sagmariasus verreauxi,H. Milne Edwards 1851) culture

Zinc (Zn) and copper (Cu) are strong inhibitors of bacterial biofilms in aqueous solutions, but are known toxins of crustaceans. A new metal application method; cold‐sprayed metal embedment, known to modulate metal release, was tested for its applications in crustacean larval culture systems. Cold‐spray technology allows metal particles to bond to plastics, while modulating metal ion release and biocide activity to the substrate boundary. In this study, Eastern spiny lobster (Sagmariasus verreauxi) larvae (phyllosoma) were cultured in the presence of cold‐sprayed Zn and Cu metal surfaces. Metal loss was monitored gravimetrically on embedded surfaces, assessment of water ion concentrations and analysis of phyllosoma body content were undertaken. Phyllosoma moulting, deformity and mortality patterns were monitored. Cold‐sprayed Zn‐ and Cu‐embedded surfaces were depleted with losses of 0.69% and 31.2% noted respectively. Culture water concentrations of these metals were elevated and accumulation by phyllosoma occurred. Water Zn concentrations of 18.5 μg L^?1 were associated with chronic eyestalk moult deformities; the first report of Zn causing a non‐lethal moult deformity in crustacean larvae. The Cu surface lost a third of its metal mass with a water concentration of 40 μg L^?1 causing acute toxicity and localization of composite granules in the midgut gland. Cu associated mortality was noted by Day 2 of culture with a LD 50 experienced by Day 9. Future work on the use of bioactive metals in aquaculture systems will focus on a range of different metal alloys, and improved modulation of ion release mechanisms through increased particle embedment depth and separation.     

Effect of temperature on survival,growth and development of Mytilus galloprovincialis larvae

Mussel aquaculture is widely prevalent worldwide, but generally relies on natural seed collection, which does not always meet the needs of the producers. Thus, development of mussel hatcheries is of economic interest in some parts of the world, such as Europe; it provides opportunities not only on annual reliability of seed but also on genetic improvements. To broaden knowledge on mussel larval physiology, we carried out temperature treatments (17, 20 and 24 °C) on Mytilus galloprovincialis larvae under laboratory conditions. The trials ended when 30% of the larval population was in the post‐larval stage. The temperature coefficient Q₁₀ indicated a strong relationship between temperature and increase in growth from 17 to 20 °C, but not between 20 and 24 °C. Exposure of M. galloprovincialis larvae to 17 °C resulted in poor growth, low survival and a delayed development and was considered to be inadequate for M. galloprovincialis larval culture. Rearing the larvae at 20 or 24 °C produced better growth, higher survival rates and faster metamorphosis as compared with 17 °C. The temperature region within 20 and 24 °C was suggested as adequate for the mussel M. galloprovincialis larval culture, and implications of these results on the development of commercial hatcheries were discussed.     

Probiotic activity of bacteria associated with egg capsules of Concholepas concholepas (common name ‘Loco’)

Egg capsules of Concholepas concholepas were analysed microbiologically, showing the presence of bacteria inside the capsules. The bacteria were isolated and identified, and their probiotic potential on early larvae of C. concholepas was evaluated. From 32 capsules that were examined, 45 bacterial strains were isolated, of which 67% belonged to the genus Bacillus. Survival bioassays were carried out with all the isolated bacterial strains on C. concholepas larvae. It was found that 71% of the bacteria had positive effects on larval survival. This research determined that bacteria of the genera Bacillus and Vibrio present in the capsules have probiotic potential. The bacteria that led to the greatest larval survival were Bacillus pumilus, which may be used in the cultivation of C. concholepas, because one of the limiting factors for the cultivation of this species is larval survival; thus, we propose that the probiotic strains identified in this study will be used during the cultivation of C. concholepas to enhance the chances of larval survival.     

Bacteria in the culture of the scallop Argopecten purpuratus (Lamarck, 1819)

The scallop Argopecten purpuratus is one of themajor resources of commercial importance in Chilean aquaculture. One of themajor limiting factors for the culture of this species has been mass mortalityoccurring during production of juvenile seed organisms where massmortality of veliger larvae has been attributed to the presence of pathogenicbacteria in scallops and hatchery systems. Bacteriological studies havedemonstrated that in addition to bacterial pathogens, beneficial bacterialspecies capable of improving scallop larval survival may also be isolated fromscallops and hatchery systems. Research has been carried out on thedetermination of the feasibility of implementing beneficial bacteria in thecontrol of culture pathogens, thus reducing the need for chemotherapeuticmethods. The present review analyzes bacteriological research data on thedifferent roles of bacteria associated with scallop culture and discussesrecentdata on the implementation of beneficial bacteria in biological control ofpathogens in larval mass cultures of Argopectenpurpuratus.     

Bacterial communities and predicted nitrogen metabolism of heterotrophic‐ and probiotic‐based biofilms used for super‐intensive indoor shrimp culture

Biofilm‐based aquaculture systems constitute a promising alternative for intensive shrimp rearing. Microorganisms forming biofilms can recycle the nitrogen compounds the production units improving the water quality while using zero or limited water exchange. This study aimed to compare the taxonomic profiles and the predicted functions related to the transformation of nitrogenous compounds between a heterotrophic‐ (HtB) and a probiotic biofilm (PrB), and the effect of these on the water quality and the productive response of cultured shrimp. Libraries of the 16S‐rRNA gene (V3‐V4 region) were prepared and sequenced to be used as a taxonomic biomarker. Analysis of metagenomic datasets revelated that genera Halomonas, Planctomycetes and Rhodopirellula were the most abundant genera in HtB; meanwhile, Bacillus, Halobacillus and Flavobacterium dominated in PrB. Regarding nitrogen metabolism, the proportion of genes encoding enzymes catalyzing the six pathways shaping the nitrogen metabolism showed differences between biofilms, which could also explain the difference in water quality between treatments. Concerning the productive response of shrimp, no significant differences were detected except for survival, which was higher in PrB. Finally, the results suggest that biofilms harbour functions for nitrogen metabolism, including dissimilatory nitrate reduction, assimilatory nitrate reduction, denitrification, nitrification, nitrogen fixation and anammox; however, the balance of these functional capabilities seems to be relevant to maintain water quality.     

Colorimetric assay for the in vitro evaluation of Saprolegnia biofilm inhibitors

A quantitative and reproducible 96‐well microtiter method that is easily adaptable for the screening of Saprolegnia biofilm inhibitors is described. As opposed to other methods previously developed for the screening of Saprolegnia inhibitors on spore germination or mycelial growth, this technique is of particular significance as it investigates potential inhibitors against surface‐attached mycelial mats of Saprolegnia spp. (biofilm). In this study, we have investigated the effects of propionic acid (PPA) on reducing the viability of induced Saprolegnia biofilms using colorimetric MTS assay based on the reduction of tetrazolium salts. Viability of Saprolegnia hyphae in treated biofilms was reduced significantly following treatment with different PPA concentrations. The effect was enhanced after combining each of the tested PPA concentrations with 500 mg/L of boric acid (BA). However, the percentage of non‐viable hyphae was still higher in 200 mg L^–1 bronopol‐treated biofilms (positive control) following 6‐ and 12‐hr exposure. Similar results were observed using other recently described fluorescence‐based assays for viability.     

Survival and growth of geoduck clam larvae (Panopea generosa) in flow‐through culture tanks under laboratory conditions

The geoduck clam, Panopea generosa, is a species from the west coast of Baja California, Mexico, and the optimization of seed production systems is still a limiting factor for its aquaculture. In this study, a flow‐through culture system was designed and tested in P. generosa larvae. Survival and growth was compared in triplicate 45‐L fiberglass tanks using three larval densities (5, 10 and 15 larvae/ml). A head tank kept constant the water inflow, with a daily renewal rate of 1.8× tank volume. The food (Isochrysis spp.) was dosed according to the ingestion rate of larvae and the dilution rate. Survival decreased linearly during the first 10 days and reached asymptotic values of ca. 20% (15 larvae/ml) and 50% (densities of 5 and 10 larvae/ml) afterwards. Mean shell length at the end of the experiment (243 ± 1.8 to 270 ± 0.7 μm) was not statistically different among treatments, even though a trend towards higher gross growth rate was observed in the treatment with the lowest density (9.5 μm/day) relative to the rest of the treatments (8.5 μm/day). It is concluded that P. generosa larvae can be successfully grown in flow‐through systems at maximum densities of 10 larvae/ml without significantly affecting their survival and growth rates. The system design was reliable, kept a constant water flow with reduced maintenance, and may represent an important option in the laboratory for increasing the stocking density of Panopea species during the larval phase.     

Microbial communities associated with early stages of intensively reared orange‐spotted grouper (Epinephelus coioides)

The gut microbiota plays key roles in the health and general welfare of fish larvae, the present study characterized the bacterial communities associated with grouper Epinephelus coioides larvae during a period of 22 days post hatch (DPH) in an intensive hatchery using both cultivation‐based and cultivation‐independent approaches. Both approaches confirmed that bacteria were present in the gut of larvae before and after the onset of exogenous feeding, and the number of cultiviable bacteria increased gradually from 2 DPH to 22 DPH. A more complex bacterial profile was present in larvae fed fertilizer oyster eggs for 4 days (8 DPH), probably as a result of the onset of exogenous feeding. Interestingly, similar internal microbiota were observed in larvae fed fertilized oyster eggs for 4 days (8 DPH) and rotifers for 2 weeks (22 DPH), although different microbial communities were present in the two feeds. This might suggest that the gut environment of E. coioides larvae selects for a common microbiota, which is more closely related with the rearing water than the two feeds. Therefore, bacterial community of the rearing water may play a critical role in the establishment of gut microbiota of fish larvae and more attention should be paid to its practical modulation by using probiotics. In addition, some potentially beneficial bacteria, such as Lactococcus spp., were the major components of the microbiota associated with fertilized oyster eggs, while these bacteria were not detected in larvae samples.     

Effect of live algae used as green water on survival,growth, behaviour,ontogeny and bacterial profile of lobster larvae (Homarus americanus Milne Edwards)

Green water is a technique commonly used in aquaculture, that consists of adding live algae in the water culture and its benefits have been shown for several species. Several hypotheses exist to explain the benefits of green water: increase in nutritional value; action as a probiotic; increase in contrast to reveal preys for larvae; or increase in predator behaviour of larvae. Green water produced with a mix of strains Isochrysis galbana and Chaetoceros muelleri (50:50, cells:cells) applied in four different ways was tested. The survival and the growth of American lobster (Homarus americanus) between stage I and stage IV post‐larvae were not affected by the addition of live algae. The lipid classes were not affected by the addition of algae and limited variation was observed in the fatty acids and bacterial profiles. Furthermore, the green water techniques had a limited effect on the behaviour of post‐larvae stage IV lobster at releasing. Behaviour was mostly affected by the age of post‐larvae. The bacteria Lewinella sp., Leucothrix sp. and Thiothrix sp. appeared to represent a common and core component of Stage IV lobster post‐larvae microflora. The results show that the algae do not increase either nutritional value or feed intake of the lobster larvae. Probiotic effect may be more important when larvae are raised in a close system where potential bacterial pathogens could have more chances to colonize the culture. Also, the dark green colour of the larval tank used in this study may have mimicked the effect of green water in the control group. Biochemical results suggest that dietary supplementation with phospholipids and DHA is needed in a lobster hatchery using frozen Artemia and our open formula Dry mix.     

Isolation and evaluation of putative probiotic strains from different teleost to prevent Pseudomonas aeruginosa infection in Cyprinus carpio

Probiotics renowned as valuable microbes serve as a potential alternative to control diseases in aquaculture and are considered as an efficient and environment‐friendly approach to reduce the use of antibiotics. The present study aims at the isolation of putative probiotic bacteria from the intestinal tract of different fish species from the Doaba region of Punjab, India. In this study, isolated bacterial strains were characterized based on their morphological, biochemical and molecular characterization by 16S rRNA gene sequencing, followed by in vitro evaluation of different selection parameters described in FAO/WHO guidelines. A total of 169 different bacterial strains were isolated from the gastrointestinal tract of 52 different fish species. After in vitro evaluation, out of 169 bacterial strains only five bacteria (S3, S7, BDK2', BDK7 and BDK9) identified as Enterococcus and Bacillus species showed antagonistic activity against the fish pathogen Pseudomonas aeruginosa (MTCC 4 673). These isolates were screened based on their response towards bile tolerance, pH tolerance, adhesion and drug susceptibility to different antibiotic discs. And, the in vivo results indicated improved growth and survival against the infection (P. aeruginosa) after oral administration of the probiotics. The observations of in vitro and in vivo evaluation indicate that these isolated probiotic strains serve as effective probiotics and can be used as a novel and safe treatment to cure current issues in aquaculture.     

The effect of density in larval rearing of the pullet carpet shell Venerupis corrugata (Gmelin, 1791) in a recirculating aquaculture system

The pullet carpet shell Venerupis corrugata is an economically valuable species in several European countries, however, nowadays stocks are under high fishing pressure. Hatchery production of juveniles for release is a major contributor to strengthen the stock and consequently improve the sustainability of the natural stocks. This study aimed to determine the feasibility of rearing V. corrugata larvae with different larval densities (10, 40 and 200 larvae per mL) in a recirculating aquaculture system (RAS), compared with the traditional larval rearing methodology (Batch). The mean survival, growth and metamorphic rate of V. corrugata larvae in RAS was higher (11.1%; 71.3 μm; 21.6% respectively) than in the Batch system, in all tested densities. The larval growth was not affected by the initial density until 40 larvae per mL, however, 200 larvae per mL decreased the larval growth in length nearly 54 μm. The larval rearing time was shortened in 2 days in the RAS system. The physical, chemical and microbiologic parameters suggested that the tested densities were not excessive to disturb the biofilter stability of RAS. The V. corrugata larval rearing performed at high larval stocking densities in RAS system present a reduction in the operating costs to produce this species.     

Revealing the compositions of the intestinal microbiota of three Anguillid eel species using 16S rDNA sequencing

Anguilla japonica, A. marmorata and A. bicolor pacifica are important eel species for aquaculture in Taiwan. In the past, seldom studies focused on the intestinal microbiome of them or the probiotics used in their cultivation. Probiotics are microbes that can improve the health of organisms, and the microbes isolated from the intestines of organisms might be the potential probiotics. Therefore, the aim of this study was to collect the intestinal mucus samples of these three eel species for investigating the autochthonous microbiome using 16S rDNA sequencing. The diversity, abundance of the intestinal microbiome and compositions of all the libraries were identified. Moreover, the composition of intestinal microbiome of eel was suggested that it would be affected by the waters and the characteristics of different eel species. Furthermore, most of the identified predominant genera of the intestinal microbiome of these three eel species, such as Cetobacterium, Clostridium, Shewanella, Acinetobacter and Bacteroides, seemed to have great potential as the probiotics. In the future, more studies shall be conducted to promote the practical application of these candidate probiotics for improving the techniques of eel farming.