Enterobacter sp. AA26 gut symbiont as a protein source for Mediterranean fruit fly mass-rearing and sterile insect technique applications

Background

The interaction between gut bacterial symbionts and Tephritidae became the focus of several studies that showed that bacteria contributed to the nutritional status and the reproductive potential of its fruit fly hosts. Anastrepha fraterculus is an economically important fruit pest in South America. This pest is currently controlled by insecticides, which prompt the development of environmentally friendly methods such as the sterile insect technique (SIT). For SIT to be effective, a deep understanding of the biology and sexual behavior of the target species is needed. Although many studies have contributed in this direction, little is known about the composition and role of A. fraterculus symbiotic bacteria. In this study we tested the hypothesis that gut bacteria contribute to nutritional status and reproductive success of A. fraterculus males.

Results

AB affected the bacterial community of the digestive tract of A. fraterculus, in particular bacteria belonging to the Enterobacteriaceae family, which was the dominant bacterial group in the control flies (i.e., non-treated with AB). AB negatively affected parameters directly related to the mating success of laboratory males and their nutritional status. AB also affected males’ survival under starvation conditions. The effect of AB on the behaviour and nutritional status of the males depended on two additional factors: the origin of the males and the presence of a proteinaceous source in the diet.

Conclusions

Our results suggest that A. fraterculus males gut contain symbiotic organisms that are able to exert a positive contribution on A. fraterculus males’ fitness, although the physiological mechanisms still need further studies.

     

Biochemical and nutritional characterization of the medfly gut symbiont Enterobacter sp. AA26 for its use as probiotics in sterile insect technique applications

Background

Symbiotic bacteria contribute to a multitude of important biological functions such as nutrition and reproduction and affect multiple physiological factors like fitness and longevity in their insect hosts. The melon fly, Zeugodacus cucurbitae (Coquillett), is an important agricultural pest that affects a variety of cultivated plants belonging mostly to the Cucurbitaceae family. It is considered invasive and widespread in many parts of the world. Several approaches are currently being considered for the management of its populations including the environmentally friendly and effective sterile insect technique (SIT), as a component of an integrated pest management (IPM) strategy. In the present study, we examined the effect of diet and radiation on the bacterial symbiome of Z. cucurbitae flies with the use of Next Generation Sequencing technologies.

Results

Melon flies were reared on two diets at the larval stage, an artificial bran-based diet and on sweet gourd, which affected significantly the development of the bacterial profiles. Significant differentiation was also observed based on gender. The effect of radiation was mostly diet dependent, with irradiated melon flies reared on the bran diet exhibiting a significant reduction in species diversity and richness compared to their non-irradiated controls. Changes in the bacterial symbiome of the irradiated melon flies included a drastic reduction in the number of sequences affiliated with members of Citrobacter, Raoultella, and Enterobacteriaceae. At the same time, an increase was observed for members of Enterobacter, Providencia and Morganella. Interestingly, the irradiated male melon flies reared on sweet gourd showed a clear differentiation compared to their non-irradiated controls, namely a significant reduction in species richness and minor differences in the relative abundance for members of Enterobacter and Providencia.

Conclusions

The two diets in conjunction with the irradiation affected significantly the formation of the bacterial symbiome. Melon flies reared on the bran-based artificial diet displayed significant changes in the bacterial symbiome upon irradiation, in all aspects, including species richness, diversity and composition. When reared on sweet gourd, significant changes occurred to male samples due to radiation, only in terms of species richness.

     

A modified static respiration assay and its relationship with an enzymatic test to assess compost stability and maturity

Despite the numerous compost stability and maturity tests, no universally accepted compost stability or maturity index exists. The fluorescein di-acetate (FDA) enzymatic assay, originating from soil studies, is examined here as a potential new compost stability test, and is compared to microbial respiration and phytotoxicity indices. Thirteen composts were used in the study from different source materials. Static microbial respiration activity indices calculated were the cumulative O₂ consumptions, O₂ consumption rates, total C-CO₂ production, the respiratory quotient and the bio C/N ratio. Compost phytotoxicity was quantified via a 7-day tomato seed germination assay. Results showed that the net fluorescein release rates correlated with all stability indices. The germination index marginally correlated with the fluorescein release rates, but not with any of the other stability indices. New limits to classify composts regarding their stability were proposed.     

High quality draft genome sequence of Olivibacter sitiensis type strain (AW-6T), a diphenol degrader with genes involved in the catechol pathway

Olivibacter sitiensis Ntougias et al. 2007 is a member of the family Sphingobacteriaceae, phylum Bacteroidetes. Members of the genus Olivibacter are phylogenetically diverse and of significant interest. They occur in diverse habitats, such as rhizosphere and contaminated soils, viscous wastes, composts, biofilter clean-up facilities on contaminated sites and cave environments, and they are involved in the degradation of complex and toxic compounds. Here we describe the features of O. sitiensis AW-6^T, together with the permanent-draft genome sequence and annotation. The organism was sequenced under the Genomic Encyclopedia for Bacteria and Archaea (GEBA) project at the DOE Joint Genome Institute and is the first genome sequence of a species within the genus Olivibacter. The genome is 5,053,571 bp long and is comprised of 110 scaffolds with an average GC content of 44.61%. Of the 4,565 genes predicted, 4,501 were protein-coding genes and 64 were RNA genes. Most protein-coding genes (68.52%) were assigned to a putative function. The identification of 2-keto-4-pentenoate hydratase/2-oxohepta-3-ene-1,7-dioic acid hydratase-coding genes indicates involvement of this organism in the catechol catabolic pathway. In addition, genes encoding for β-1,4-xylanases and β-1,4-xylosidases reveal the xylanolytic action of O. sitiensis.     

Bacterial diversity in spent mushroom compost assessed by amplified rDNA restriction analysis and sequencing of cultivated isolates

Spent mushroom compost (SMC) is the residual by-product of commercial Agaricus spp. cultivation, and it is mainly composed of a thermally treated cereal straw/animal manure mixture colonized by the fungal biomass. Research on the valorization of this material is mainly focusing on its use as soil conditioner and plant fertilizer. An investigation of the bacterial diversity in SMC was performed using molecular techniques in order to reveal the origin of SMC microflora and its potential effect on soil microbial communities after incorporation into agricultural soils. The bacterial population was estimated by the plate count method to a mean of 2.7 10⁹ colony forming units (cfu) per g of dry weight, while the numbers of Gram-positive and Gram-negative bacteria were 1.9 10⁹ and 4.9 10⁸ cfu per g dw respectively as estimated by enumeration on semi-selective media. Fifty bacterial isolates were classified into 14 operational taxonomic units (OTUs) following ARDRA-PCR of the 16S rDNA gene. Sequencing of the 16S rDNA amplicon assigned 12 of the 14 OTUs to Gram-positive bacteria, associated with the genera Bacillus, Paenibacillus, Exiguobacterium, Staphylococcus, Desemzia, Carnobacterium, Brevibacterium, Arthrobacter and Microbacterium of the bacterial divisions Firmicutes and Actinobacteria. Two bacterial groups have phylogenetic links with the genera Comamonas and Sphingobacterium, which belong to β-Proteobacteria and Bacteroidetes respectively. Two potentially novel bacteria are reported, which are associated with the genera Bacillus and Microbacterium. Most of the bacteria identified are of environmental origin, while strains related to species usually isolated from insects, animal and clinical sources were also detected. It appears that bacterial diversity in SMC is greatly affected by the origin of the initial material, its thermal pasteurization treatment and the potential unintended colonization of the mushroom substrate during the cultivation process.     

Phylogeny and ecophysiological features of prokaryotes isolated from temporary saline tidal pools

Although hypersaline environments have been extensively examined, only a limited number of microbial community studies have been performed in saline tide pools. We have studied a temporary salt-saturated tide pool and isolated prokaryotes from the water. Chlorinity measurements revealed that the tide pool brine could be characterized as one of the most hypersaline ecosystems on earth. Enumeration of microorganisms at different salinities showed that the tide pool was dominated by moderate halophiles. Based on 16S rRNA gene sequence analysis, the prokaryotic strains isolated were related to the bacterial genera Rhodovibrio, Halovibrio, Aquisalimonas, Bacillus and Staphylococcus and to the haloarchaeal species Haloferax alexandrinus. Four bacterial isolates were distantly related to their closest validly described species Aquisalimonas asiatica (96.5 % similarity), representing a novel phylogenetic linkage. Ecophysiological analysis also revealed distinct phenotypic profiles for the prokaryotic strains analyzed. The herbicide 2,4-dichlorophenoxyacetate could be effectively utilized by selected strains as the sole carbon source, but phenolic compounds could not be utilized by any of the halophilic isolates examined. None of the halophilic strains were able to grow without the presence of sea salt or seawater. Based on these results, we conclude that moderate halophilic bacteria rather than extremely halophilic archaea dominate in such a hypersaline environment.     

Bacterial and β-proteobacterial diversity in Olea europaea var. mastoidis- and O. europaea var. koroneiki-generated olive mill wastewaters: influence of cultivation and harvesting practice on bacterial community structure

Despite the fact that several approaches have been applied for the bioremediation of olive mill wastewaters, little information is available on bacteria inhabiting these agro-industrial effluents. In the present study, 16S rRNA gene clone libraries were constructed to identify bacterial diversity in olive-oil mill wastewaters generated by two olive varieties, Olea europaea var. mastoidis and O. europaea var. koroneiki. Due to chloroplast excess in wastewater produced from the processing of O. europaea var. koroneiki, a clone library using specific PCR primers for β-Proteobacteria was further constructed. The bacterial diversity in O. europaea var. mastoidis-generated olive mill wastewaters consisted mainly of members of Acetobacteriaceae, Prevotellaceae and Lactobacillaceae, while the majority of β-proteobacteria identified in O. europaea var. koroneiki-generated olive mill wastewaters were placed within the families Comamonadaceae, Oxalobacteraceae, Hydrogenophilaceae and Rhodocyclaceae. At least 17 novel phylogenetic linkages among Bacteria were identified. Olive-oil mill wastewaters microbiota appears to have originated from soil and freshwater environments, while the cultivation and harvesting practice highly influenced the bacterial community structure in olive mill wastewaters. The presence of fecal bacteria in O. europaea var. mastoidis-generated olive mill wastewaters, due to the long harvesting period, should be of concern.     

Continuous biohydrogen production from fruit wastewater at low pH conditions

Biohydrogen production from a simulated fruit wastewater (soluble COD = 3.17 ± 0.10 g L^?1) was carried out in a continuous stirred tank reactor (CSTR) of 2 L operational volume without biomass inoculation, heat pre-treatment or pH adjustment, resulting in a low operational pH (3.75 ± 0.09). The hydraulic retention time (HRT) varied from 15 to 5 h. A strong negative correlation (p < 0.01) between the biogas production rate and the HRT was observed. Biogas production rates were higher at 30 °C than at 25 °C (p < 0.01), when the CSTR was operated under the same HRT. The biogas hydrogen content was estimated as high as 55.8 ± 2.3 % and 55.4 ± 2.5 % at 25 and 30 °C, respectively. The main fermentation end products were acetic and butyric acids, followed by ethanol. Significant differences (p < 0.01) during the operation of the CSTR at 25 or 30 °C were identified for butyric acid at almost all HRTs examined. Simulation of the acidogenesis process in the CSTR (based on COD and carbon balances) indicated the possible metabolic compounds produced at 25 and 30 °C reactions and provided an adequate fit of the experimental data.