Insights into the bacterial community composition of farmed Caulerpa lentillifera: A comparison between contrasting health states.

The bacterial communities of Caulerpa lentillifera were studied during an outbreak of an unknown disease in a sea grape farm from Vietnam. Clear differences between healthy and diseased cases were observed at the order, genus, and Operational Taxonomic Unit (OTU) level. A richer diversity was detected in the diseased thalli of C. lentillifera, as well as the dominance of the orders Flavobacteriales (phylum Bacteroidetes) and Phycisphaerales (Planctomycetes). Aquibacter, Winogradskyella, and other OTUs of the family Flavobacteriaceae were hypothesized as detrimental bacteria, this family comprises some well-known seaweed pathogens. Phycisphaera together with other Planctomycetes and Woeseia were probably saprophytes of C. lentillifera. The Rhodobacteraceae and Rhodovulum dominated the bacterial community composition of healthy C. lentillifera. The likely beneficial role of Bradyrhizobium, Paracoccus, and Brevundimonas strains on nutrient cycling and phytohormone production was discussed. The bleaching of diseased C. lentillifera might not only be associated with pathogens but also with an oxidative response. This study offers pioneering insights on the co-occurrence of C. lentillifera-attached bacteria, potential detrimental or beneficial microbes, and a baseline for understanding the C. lentillifera holobiont. Further applied and basic research is urgently needed on C. lentillifera microbiome, shotgun metagenomic, metatranscriptomic, and metabolomic studies as well as bioactivity assays are recommended.

High-throughput sequencing revealed differences of microbial community structure and diversity between healthy and diseased Caulerpa lentillifera.

BACKGROUND: Caulerpa lentillifera is one of the most important economic green macroalgae in the world. Increasing demand for consumption has led to the commercial cultivation of C. lentillifera in Japan and Vietnam in recent decades. Concomitant with the increase of C. lentillifera cultivation is a rise in disease. We hypothesise that epiphytes or other microorganisms outbreak at the C. lentillifera farm may be an important factor contributing to disease in C. lentillifera. The main aims are obtaining differences in the microbial community structure and diversity between healthy and diseased C. lentillifera and key epiphytes and other microorganisms affecting the differences through the results of high-throughput sequencing and bioinformatics analysis in the present study. RESULTS: A total of 14,050, 2479, and 941 operational taxonomic units (OTUs) were obtained from all samples using 16S rDNA, 18S rDNA, and internal transcribed spacer (ITS) high-throughput sequencing, respectively. 16S rDNA sequencing and 18S rDNA sequencing showed that microbial community diversity was higher in diseased C. lentillifera than in healthy C. lentillifera. Both PCoA results and UPGMA results indicated that the healthy and diseased algae samples have characteristically different microbial communities. The predominant prokaryotic phyla were Proteobacteria, Planctomycetes, Bacteroidetes, Cyanobacteria, Acidobacteria, Acidobacteria and Parcubacteria in all sequences. Chlorophyta was the most abundant eukaryotic phylum followed by Bacillariophyta based on 18S rDNA sequencing. Ascomycota was the dominant fungal phylum detected in healthy C. lentillifera based on ITS sequencing, whereas fungi was rare in diseased C. lentillifera, suggesting that Ascomycota was probably fungal endosymbiont in healthy C. lentillifera. There was a significantly higher abundance of Bacteroidetes, Cyanobacteria, Bacillariophyta, Ulvales and Tetraselmis in diseased C. lentillifera than in healthy C. lentillifera. Disease outbreaks significantly change carbohydrate metabolism, environmental information processing and genetic information processing of prokaryotic communities in C. lentillifera through predicted functional analyses using the Tax4Fun tool. CONCLUSIONS: Bacteroidetes, Cyanobacteria, Bacillariophyta, Ulvales and Tetraselmis outbreak at the C. lentillifera farm sites was an important factor contributing to disease in C. lentillifera.

Spatial distribution of the culturable bacterial community associated with the invasive alga Caulerpa cylindracea in the Mediterranean Sea.

Understanding the mechanisms underlying the complex seaweed-bacteria associations in nature may provide information on the fitness of an invasive host. This may require the use of different approaches. In this study, we employed, for the first time, the Biolog system-Ecoplates™ to analyze the functional diversity of the culturable fraction of the bacterial assemblages associated with the surface of Caulerpa cylindracea, the invasive seaweed of the Mediterranean Sea. Seaweed samples were collected at five sites across the basin. A high similarity in the bacterial activity, expressed as Average Well Color Development (AWCD), among the study sites was observed. Culturable heterotrophic bacteria at 22 °C showed mean values ranging from 1.4 × 105 CFU g-1 at Porto Cesareo (Ionian Sea, Italy) to 5.8 × 106 CFU g-1 at Othonoi, Diapontine Island (Ionian Sea, Greece). The analysis of the DNA sequences on isolated bacteria demonstrated that the genera Shewanella, Marinobacter, Vibrio, Granulosicoccus and the family Rhodobacteraceae are consistently present on C. cylindracea, irrespective of its geographical origin. The present study provided new insights into the complex association between bacteria and this algal species, suggesting a specific composition and function of the associated culturable bacteria across the basin.

Entangled fates of holobiont genomes during invasion: nested bacterial and host diversities in Caulerpa taxifolia.

Successful prevention and mitigation of biological invasions requires retracing the initial steps of introduction, as well as understanding key elements enhancing the adaptability of invasive species. We studied the genetic diversity of the green alga Caulerpa taxifolia and its associated bacterial communities in several areas around the world. The striking congruence of α and ß diversity of the algal genome and endophytic communities reveals a tight association, supporting the holobiont concept as best describing the unit of spreading and invasion. Both genomic compartments support the hypotheses of a unique accidental introduction in the Mediterranean and of multiple invasion events in southern Australia. In addition to helping with tracing the origin of invasion, bacterial communities exhibit metabolic functions that can potentially enhance adaptability and competitiveness of the consortium they form with their host. We thus hypothesize that low genetic diversities of both host and symbiont communities may contribute to the recent regression in the Mediterranean, in contrast with the persistence of highly diverse assemblages in southern Australia. This study supports the importance of scaling up from the host to the holobiont for a comprehensive understanding of invasions.

Hologenome theory supported by cooccurrence networks of species-specific bacterial communities in siphonous algae (Caulerpa).

The siphonous algae of the Caulerpa genus harbor internal microbial communities hypothesized to play important roles in development, defense and metabolic activities of the host. Here, we characterize the endophytic bacterial community of four Caulerpa taxa in the Mediterranean Sea, through 16S rRNA amplicon sequencing. Results reveal a striking alpha diversity of the bacterial communities, similar to levels found in sponges and coral holobionts. These comprise (1) a very small core community shared across all hosts (< 1% of the total community), (2) a variable portion (ca. 25%) shared by some Caulerpa taxa but not by all, which might represent environmentally acquired bacteria and (3) a large (>70%) species-specific fraction of the community, forming very specific clusters revealed by modularity in networks of cooccurrence, even in areas where distinct Caulerpa taxa occurred in sympatry. Indirect inferences based on sequence homology suggest that these communities may play an important role in the metabolism of their host, in particular on their ability to grow on anoxic sediment. These findings support the hologenome theory and the need for a holistic framework in ecological and evolutionary studies of these holobionts that frequently become invasive.

Invasion is a community affair: Clandestine followers in the bacterial community associated to green algae, Caulerpa racemosa, track the invasion source.

Biological invasions rank amongst the most deleterious components of global change inducing alterations from genes to ecosystems. The genetic characteristics of introduced pools of individuals greatly influence the capacity of introduced species to establish and expand. The recently demonstrated heritability of microbial communities associated to individual genotypes of primary producers makes them a potentially essential element of the evolution and adaptability of their hosts. Here, we characterized the bacterial communities associated to native and non-native populations of the marine green macroalga Caulerparacemosa through pyrosequencing, and explored their potential role on the strikingly invasive trajectory of their host in the Mediterranean. The similarity of endophytic bacterial communities from the native Australian range and several Mediterranean locations confirmed the origin of invasion and revealed distinct communities associated to a second Mediterranean variety of C. racemosa long reported in the Mediterranean. Comparative analysis of these two groups demonstrated the stability of the composition of bacterial communities through the successive steps of introduction and invasion and suggested the vertical transmission of some major bacterial OTUs. Indirect inferences on the taxonomic identity and associated metabolism of bacterial lineages showed a striking consistency with sediment upheaval conditions associated to the expansion of their invasive host and to the decline of native species. These results demonstrate that bacterial communities can be an effective tracer of the origin of invasion and support their potential role in their eukaryotic host's adaptation to new environments. They put forward the critical need to consider the 'meta-organism' encompassing both the host and associated micro-organisms, to unravel the origins, causes and mechanisms underlying biological invasions.

Acaricidal action of destruxins produced by a marine-derived Beauveria felina on the bovine tick Rhipicephalus (Boophilus) microplus.

The increasing resistance of Rhipicephalus (Boophilus) microplus tick to commercial insecticides requires alternative methods for the control of this cattle plague. The enthomopathogenic fungus Beauveria felina produces destruxins in culture media, cyclic depsipeptides which display an array of biological activities. The present investigation aimed to evaluate the acaricide action of destruxins isolated from B. felina culture media on R. (B.) microplus engorged females. B. felina was grown in MF medium under 19 different growth conditions. HPLC-PDA analysis of chromatographic fractions obtained from the 19 different growth media extracts indicated the presence of destruxins in all lipophylic fractions. Such fractions were combined and subjected to separation by HPLC. Fractions containing distinct destruxins composition were tested against R. (B.) microplus. Two fractions, composed of destruxin Ed(1) and pseudodestruxin B and/or pseudodestruxin C (fraction P1) as well as by hydroxyhomodestruxin B and/or destruxin D and/or roseotoxin C (fraction P7), displayed 30% and 28.7% acaricidal efficacy, respectively. This activity profile in such low concentration is adequate to consider destruxins as potential leading compounds to be developed for tick biological control.

Use of experimental design for the optimization of the production of new secondary metabolites by two Penicillium species.

A fractional factorial design approach has been used to enhance secondary metabolite production by two Penicillium strains. The method was initially used to improve the production of bioactive extracts as a whole and subsequently to optimize the production of particular bioactive metabolites. Enhancements of over 500% in secondary metabolite production were observed for both P. oxalicum and P. citrinum. Two new alkaloids, citrinalins A (5) and B (6), were isolated and identified from P. citrinum cultures optimized for production of minor metabolites.

Evaluation of the petrifilm aerobic count plate for enumeration of aerobic marine bacteria from seawater and Caulerpa lentillifera.

The enumeration and evaluation of the activity of marine bacteria are important in the food industry. However, detection of marine bacteria in seawater or seafood has not been easy. The Petrifilm aerobic count plate (ACP) is a ready-to-use alternative to the traditional enumeration media used for bacteria associated with food. The purpose of this study was to evaluate the usefulness of a simple detection and enumeration method utilizing the Petrifilm ACP for enumeration of aerobic marine bacteria from seawater and an edible seaweed, Caulerpa lentillifera. The efficiency of enumeration of total aerobic marine bacteria on Petrifilm ACP was compared with that using the spread plate method on marine agar with 80 seawater and 64 C. lentillifera samples. With sterile seawater as the diluent, a close correlation was observed between the method utilizing Petrifilm ACP and that utilizing the conventional marine agar (r=0.98 for seawater and 0.91 for C. lentillifera). The Petrifilm ACP method was simpler and less time-consuming than the conventional method. These results indicate that Petrifilm ACP is a suitable alternative to conventional marine agar for enumeration of marine microorganisms in seawater and C. lentillifera samples.

[Investigation and culture of microbial contaminants of Caulerpa lentillifera (Sea Grape)].

Caulerpa lentillifera is a kind of edible seaweed, known as 'sea grape' or 'green caviar'. It is used in fresh salads. However, it is sensitive to low temperature and osmotic pressure, and is easily spoilt by storage in a refrigerator or washing with tap water. That is the reason why it is difficult to prevent food poisoning, especially due to Vibrio parahaemolyticus. In this study we investigated of marine bacteria and V. parahaemolyticus in C. lentillifera and cultured them in order to develop effective control of bacteria in commercial farms. The sixteen farms in the Okinawa Islands were investigated from August to September in 2006. A total of 176 samples were collected from eleven points during the cultivation processes and from the products. About 10(3) cfu/mL of marine bacteria were detected in the seawater used in the tank culture, but after cultivation of C. lentillifera the number had increased to about 10(6) cfu/mL. The number of marine bacteria in C. lentillifera did not change significantly through the process of planting to the final product (about 10(7) cfu/g). V. parahaemolyticus was detected in seawater from all processes and C. lentillifera was isolated from 56% of seawater, 25% of seed-stocks, and 18.8% of product samples, though but thermostable direct hemolysin gene was not detected from enrichment cultures or isolated V. parahaemolyticus strains. These results indicate that for prevention of food poisoning by V. parahaemolyticus in C. lentillifera, it is important to establish a suitable sterilization procedure for each process.