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      Medfly Gut Microbiota and Enhancement of the Sterile Insect Technique: Similarities and Differences of Klebsiella oxytoca and Enterobacter sp. AA26 Probiotics during the Larval and Adult Stages of the VIENNA 8 D53+ Genetic Sexing Strain

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          Abstract

          The Mediterranean fruit fly, Ceratitis capitata, is a major agricultural pest worldwide. The development of genetic sexing strains (GSSs) for this species that allows male-only sterile insects releases has boosted the effectiveness of the environmental friendly pest control method known as the sterile insect technique. The last generation of these strains, the VIENNA 7 and VIENNA 8, are currently used in all mass rearing facilities worldwide and are considered as models for such pest control applications. The sterile insect technique depends on the rearing of sufficient numbers of adequate “biological quality” laboratory flies to be released in the field. Currently, there is an increasing amount of studies focusing on the characterization of the symbiotic communities and development of probiotic diets. In our study, two bacterial isolates, an Enterobacter sp. (strain AA26) and a Klebsiella oxytoca strain, were used as probiotics in larval and adult diet. These strains have been shown to be beneficial, affecting several aspects related to the rearing efficiency and biological quality of the medfly VIENNA 8 D53+ GSS. Our results demonstrate the effect of K. oxytoca on the developmental duration of the immature stages and, to some extent, on flight ability. On the other hand, our study does not support the presence of any beneficial effect of (a) K. oxytoca on pupal and adult recovery and adults’ survival under stress conditions when provided as a larval diet supplement and (b) K. oxytoca and Enterobacter sp. AA26 on mating competitiveness when provided as adult diet supplements. Possible explanations for inconsistencies with previous studies and the need for universalizing protocols are discussed. Our findings, combined with previous studies can support the sterile insect technique, through the improvement of different aspects of mass rearing and biological properties of laboratory reared insect pests.

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          Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA.

          Using a set of synthetic oligonucleotides homologous to broadly conserved sequences in-vitro amplification via the polymerase chain reaction followed by direct sequencing results in almost complete nucleotide determination of a gene coding for 16S ribosomal RNA. As a model system the nucleotide sequence of the 16S rRNA gene of M.kansasii was determined and found to be 98.7% homologous to that of M.bovis BCG. This is the first report on a contiguous sequence information of an entire amplified gene spanning 1.5 kb without any subcloning procedures.
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            The probiotic paradox: live and dead cells are biological response modifiers.

            Probiotics are usually defined as products which contain viable non-pathogenic micro-organisms able to confer health benefits to the host. There are specific gastrointestinal effects of probiotics such as alleviating inflammatory bowel disease, reducing acute diarrhoea in children, inhibiting Salmonella and Helicobacter pylori, removing cholesterol, secreting enzymes and bacteriocins and immunomodulation. However, many of the effects obtained from viable cells of probiotics are also obtained from populations of dead cells. Heat-killed cells of Enterococcus faecalis stimulate the gastrointestinal immune system in chicks. Dead bifidobacteria induce significant increases in TNF-alpha production. Administration of heat-killed E. faecalis to healthy dogs increases neutrophil phagocytes. The probiotic paradox is that both live and dead cells in probiotic products can generate beneficial biological responses. The action of probiotics could be a dual one. Live probiotic cells influence both the gastrointestinal microflora and the immune response whilst the components of dead cells exert an anti-inflammatory response in the gastrointestinal tract. This is quite analogous to a proposed mode of action of antimicrobial growth promoters in animal production. This has several implications for the production and application of probiotics, as it will be difficult to assess the relative proportions of live and dead cells in a probiotic culture. Variable amounts of dead cells might contribute to the variation in response often seen with live probiotic cultures. However, the use of dead probiotics as biological response modifiers has several attractive advantages; such products would be very safe and have a long shelf-life.
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              Manipulation of the microbiota of mass-reared Mediterranean fruit flies Ceratitis capitata (Diptera: Tephritidae) improves sterile male sexual performance.

              The sterile insect technique (SIT) is a method of biological control whereby millions of factory reared sterile male insects are released into the field. This technique is commonly used to combat the Mediterranean fruit fly (Ceratitis capitata, Diptera: Tephritidae). Sterile medfly males are less competent in attracting and mating with wild females, a property commonly linked to the irradiation process responsible for the sterilization. As bacteria are important partners in the fly's life cycle, we used molecular analytical methods to study the community structure of the gut microbiota in irradiated male medflies. We find that the sterilizing irradiation procedure affects the gut bacterial community structure of the Mediterranean fruit fly. Although the Enterobacteriaceae family remains the dominant bacterial group present in the gut, the levels of Klebsiella species decreases significantly in the days after sterilization. In addition, we detected substantial differences in some bacterial species between the mass rearing strain Vienna 8 and the wild strain. Most notable among these are the increased levels of the potentially pathogenic species Pseudomonas in the industrial strain. Testing the hypothesis that regenerating the original microbiota community could result in enhanced competitiveness of the sterile flies, we found that the addition of the bacterial species Klebsiella oxytoca to the postirradiation diet enables colonization of these bacteria in the gut while resulting in decreased levels of the Pseudomonas sp. Feeding on diets containing bacteria significantly improved sterile male performance in copulatory tests. Further studies will determine the feasibility of bacterial amelioration in SIT operations.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                27 October 2017
                2017
                : 8
                : 2064
                Affiliations
                Insect Pest Control Laboratory, Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture , Vienna, Austria
                Author notes

                Edited by: Robert Brucker, Rowland Institute at Harvard, United States

                Reviewed by: Polychronis Rempoulakis, Macquarie University, Australia; Yuval Gottlieb, Hebrew University of Jerusalem, Israel; Clara G. De Los Reyes-Gavilan, Consejo Superior de Investigaciones Científicas (CSIC), Spain

                *Correspondence: Kostas Bourtzis, k.bourtzis@ 123456iaea.org

                Present address: Georgios A. Kyritsis, Laboratory of Entomology and Agricultural Zoology, Department of Agriculture Crop Production and Rural Environment, University of Thessaly, Volos, Greece

                These authors have contributed equally to this work.

                This article was submitted to Microbial Symbioses, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2017.02064
                5663728
                29163379
                1ef9d8b8-f96c-4e38-99d9-605342798218
                Copyright © 2017 Kyritsis, Augustinos, Cáceres and Bourtzis.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 05 July 2017
                : 09 October 2017
                Page count
                Figures: 4, Tables: 0, Equations: 0, References: 29, Pages: 10, Words: 0
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                ceratitis capitata,tephritids,insect symbiosis
                Microbiology & Virology
                ceratitis capitata, tephritids, insect symbiosis

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