Impact of sample preservation and manipulation on insect gut microbiome profiling : a test case with fruit flies (Diptera, Tephritidae)

High-throughput sequencing (HTS) techniques are of great value for the investigation of microbial communities, and have been extensively used to study the gut microbiome. While most studies focus on the human gut, many others have investigated insects. However, because of the rapid spread of HTS techniques, a lot of variation exists in the protocols for sample preparation. In the present study, we investigated the impact of two widely adopted sample-processing procedures preceding library preparation, i.e., preservation of insect tissue in 70% ethanol (EtOH) and sample dissection. We used the fruit fly Ceratitis capitata (Diptera: Tephritidae) as a model organism and set up two experiments, one comparing the effects of sample manipulation and preservation across life stages and the other across fruit samples from different sources. The results of this study showed no major effects of dissection on the outcome of HTS. However, EtOH preservation did have effects on the recovered gut microbiome, the main effect being a significant reduction of the dominant genus, Providencia, in EtOH-preserved samples. Less abundant bacterial groups were also affected resulting in altered microbial profiles obtained from samples preserved in 70% EtOH. These results have important implications for the planning of future studies and when comparing studies that used different sample preparation protocols

Manipulation of insect gut microbiota towards the improvement of <i>Bactrocera oleae</i> artificial rearing

Bactrocera oleae (Rossi) (Diptera: Tephritidae) is the main pest of olive trees (Olea europaeaL.), causing major damages in olive crops. Improvement of mass rearing is a prerequisite for the successful development of large-scale sterile insect technique (SIT) applications. This can be achieved through the enrichment of artificial diets with gut bacteria isolates. We assessed the efficiency of three gut bacteria previously isolated fromCeratitis capitata(Wiedemann), and four isolated fromB. oleae, as larval diet additives in both live and inactivated/dead forms. Our results showed that deadEnterobactersp. AA26 increased pupal weight, whereas both live and dead cells increased pupal and adult production and reduced immature developmental time, indicating that its bacterial cells serve as a direct nutrient source. LiveProvidenciasp. AA31 improved pupal and adult production, enhanced male survival under stress conditions, and delayed immature development. DeadProvidenciasp. AA31, however, did not affect production rates, indicating that live bacteria can colonize the insect gut and biosynthesize nutrients essential for larval development. Live and deadBacillussp. 139 increased pupal weight, accelerated immature development, and increased adult survival under stress. Moreover, liveBacillussp. 139 improved adult production, indicating thatBacilluscells are a direct source of nutrients. DeadSerratiasp. 49 increased pupal and adult production and decreased male survival under stress conditions whereas live cells decreased insect production, indicating that the live strain is entomopathogenic, but its dead cells can be utilized as nutrient source.Klebsiella oxytoca,Enterobactersp. 23, andProvidenciasp. 22 decreased pupal and subsequent adult production and were harmful forB. oleae. Our findings indicate that deadEnterobactersp. AA26 is the most promising bacterial isolate for the improvement ofB. oleaemass rearing in support of future SIT or related population suppression programs

The impact of fruit fly gut bacteria on the rearing of the parasitic wasp <i>Diachasmimorpha longicaudata</i>

Area-wide integrated pest management strategies against tephritid fruit flies include the release of fruit fly parasitic wasps in the target area. Mass rearing of parasitic wasps is essential for the efficient application of biological control strategies. Enhancement of fruit fly host fitness through manipulation of their gut-associated symbionts might also enhance the fitness of the produced parasitic wasps and improve the parasitoid rearing system. In the current study, we added three gut bacterial isolates originating from Ceratitis capitata (Wiedemann) and four originating from Bactrocera oleae (Rossi) (both Diptera: Tephritidae) to the larval diet of C. capitata and used the bacteria-fed larvae as hosts for the development of the parasitic wasp Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae). We evaluated the effect of the bacteria on wasp life-history traits and assessed their potential use for the improvement of D. longicaudata rearing. Enterobactersp. AA26 increased fecundity and parasitism rate and accelerated parasitoid emergence. Providencia sp. AA31 led to faster emergence of both male and female parasitoids, whereas Providencia sp. 22 increased the production of female progeny. Bacillus sp. 139 increased parasitoid fecundity, parasitism rate, and production of female progeny. Serratia sp. 49 accelerated parasitoid emergence for both males and females and increased production of female progeny. Klebsiella oxytoca delayed parasitoid emergence and Enterobacter sp. 23 decreased parasitoid fecundity and parasitism rate. Our findings demonstrate a wide range of effects of fruit fly gut symbionts on parasitoid production and reveal a great potential of bacteria use towards enhancement of parasitic wasp rearing

The effects of geographic origin and antibiotic treatment on the gut symbiotic communities of <i>Bactrocera oleae</i> populations

The olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), is the major insect pest of olive orchards (Olea europaea L.), causing extensive damages on cultivated olive crops worldwide. Due to its economic importance, it has been the target species for a variety of population control approaches including the sterile insect technique (SIT). However, the inefficiency of the current mass-rearing techniques impedes the successful application of area-wide integrated pest management programs with an SIT component. It has been shown that insect mass rearing and quality of sterile insects can be improved by the manipulation of the insect gut microbiota and probiotic applications. In order to exploit the gut bacteria, it is important to investigate the structure of the gut microbial community. In the current study, we characterized the gut bacterial profile of two wild olive fruit fly populations introduced in laboratory conditions using next generation sequencing of two regions of the 16S rRNA gene. We compared the microbiota profiles regarding the geographic origin of the samples. Additionally, we investigated potential changes in the gut bacteria community before and after the first exposure of the wild adult flies to artificial adult diet with and without antibiotics. Various genera - such as Erwinia, Providencia, Enterobacter, and Klebsiella - were detected for the first time in B. oleae. The most dominant species was Candidatus Erwinia dacicola Capuzzo et al. and it was not affected by the antibiotics in the artificial adult diet used in the first generation of laboratory rearing. Geographic origin affected the overall structure of the gut community of the olive fruit fly, but antibiotic treatment in the first generation did not significantly alter the gut microbiota community

Exploring the potential of computer vision analysis of pupae size dimorphism for adaptive sex sorting systems of various vector mosquito species

Several mosquito population suppression strategies based on the rearing and release of sterile males have provided promising results. However, the lack of an efficient male selection method has hampered the expansion of these approaches into large-scale operational programmes. Currently, most of these programmes targeting Aedes mosquitoes rely on sorting methods based on the sexual size dimorphism (SSD) at the pupal stage. The currently available sorting methods have not been developed based on biometric analysis, and there is therefore potential for improvement. We applied an automated pupal size estimator developed by Grupo Tragsa with laboratory samples of Anopheles arabiensis, Aedes albopictus, Ae. polynesiensis, and three strains of Ae. aegypti. The frequency distribution of the pupal size was analyzed. We propose a general model for the analysis of the frequency distribution of mosquito pupae in the context of SSD-sorting methods, which is based on a Gaussian mixture distribution functions, thus making possible the analysis of performance (% males recovery) and purity (% males on the sorted sample).Veterinari

Symbionts commonly provide broad spectrum resistance to viruses in insects: a comparative analysis of Wolbachia strains.

In the last decade, bacterial symbionts have been shown to play an important role in protecting hosts against pathogens. Wolbachia, a widespread symbiont in arthropods, can protect Drosophila and mosquito species against viral infections. We have investigated antiviral protection in 19 Wolbachia strains originating from 16 Drosophila species after transfer into the same genotype of Drosophila simulans. We found that approximately half of the strains protected against two RNA viruses. Given that 40% of terrestrial arthropod species are estimated to harbour Wolbachia, as many as a fifth of all arthropods species may benefit from Wolbachia-mediated protection. The level of protection against two distantly related RNA viruses--DCV and FHV--was strongly genetically correlated, which suggests that there is a single mechanism of protection with broad specificity. Furthermore, Wolbachia is making flies resistant to viruses, as increases in survival can be largely explained by reductions in viral titer. Variation in the level of antiviral protection provided by different Wolbachia strains is strongly genetically correlated to the density of the bacteria strains in host tissues. We found no support for two previously proposed mechanisms of Wolbachia-mediated protection--activation of the immune system and upregulation of the methyltransferase Dnmt2. The large variation in Wolbachia's antiviral properties highlights the need to carefully select Wolbachia strains introduced into mosquito populations to prevent the transmission of arboviruses.This is the final version published by PLoS in PLoS Pathogens here: http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1004369

Symbionts do not affect the mating incompatibility between the Brazilian-1 and Peruvian morphotypes of the Anastrepha fraterculus cryptic species complex

The South American fruit fly, Anastrepha fraterculus, is clearly undergoing a speciation process. Among others, two of their morphotypes, the Brazilian-1 and Peruvian, have accumulated differences in pre- and post-zygotic mechanisms resulting in a degree of reproductive isolation. Both harbor a different strain of Wolbachia, which is a widespread endosymbiotic bacterium among many invertebrates producing a range of reproductive effects. In this paper, we studied the role of this bacterium as one of the factors involved in such isolation process. Infected and cured laboratory colonies were used to test pre- and post-zygotic effects, with special emphasis in uni- and bi-directional cytoplasmic incompatibility (CI). We showed that Wolbachia is the only known reproductive symbiont present in these morphotypes. Wolbachia reduced the ability for embryonic development in crosses involving cured females and infected males within each morphotype (uni-directional CI). This inhibition showed to be more effective in the Peruvian morphotype. Bi-directional CI was not evidenced, suggesting the presence of compatible Wolbachia strains. We conclude that Wolbachia is not directly involved in the speciation process of these morphotypes. Other mechanisms rather than CI should be explored in order to explain the reduced mating compatibility between the Brazilian-1 and Peruvian morphotypes.Instituto de BiotecnologíaFil: Devescovi, Francisco. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética. Laboratorio de Genética de Insectos de Importancia Económica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Conte, Claudia Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética. Laboratorio de Genética de Insectos de Importancia Económica; ArgentinaFil: Augustinos, Antonios A. Vienna International Centre. Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture. Insect Pest Control Laboratory; AustriaFil: Cancio Martinez, Elena I. Vienna International Centre. Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture. Insect Pest Control Laboratory; AustriaFil: Segura, Diego Fernando. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética. Laboratorio de Genética de Insectos de Importancia Económica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Caceres, Carlos. Vienna International Centre. Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture. Insect Pest Control Laboratory; AustriaFil: Lanzavecchia, Silvia Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética "Ewald A. Favret"; ArgentinaFil: Bourtzis, Kostas. Vienna International Centre. Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture. Insect Pest Control Laboratory; Austri

New criteria for selecting the origin of DNA replication in Wolbachia and closely related bacteria

© 2007 Ioannidis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The definitive version was published in BMC Genomics 8 (2007): 182, doi:10.1186/1471-2164-8-182.Background: The annotated genomes of two closely related strains of the intracellular bacterium Wolbachia pipientis have been reported without the identifications of the putative origin of replication (ori). Identifying the ori of these bacteria and related alpha-Proteobacteria as well as their patterns of sequence evolution will aid studies of cell replication and cell density, as well as the potential genetic manipulation of these widespread intracellular bacteria. Results: Using features that have been previously experimentally verified in the alpha-Proteobacterium Caulobacter crescentus, the origin of DNA replication (ori) regions were identified in silico for Wolbachia strains and eleven other related bacteria belonging to Ehrlichia, Anaplasma, and Rickettsia genera. These features include DnaA-, CtrA- and IHF-binding sites as well as the flanking genes in C. crescentus. The Wolbachia ori boundary genes were found to be hemE and COG1253 protein (CBS domain protein). Comparisons of the putative ori region among related Wolbachia strains showed higher conservation of bases within binding sites. Conclusion: The sequences of the ori regions described here are only similar among closely related bacteria while fundamental characteristics like presence of DnaA and IHF binding sites as well as the boundary genes are more widely conserved. The relative paucity of CtrA binding sites in the ori regions, as well as the absence of key enzymes associated with DNA replication in the respective genomes, suggest that several of these obligate intracellular bacteria may have altered replication mechanisms. Based on these analyses, criteria are set forth for identifying the ori region in genome sequencing projects.PI, PS, SS, GT and KB acknowledge support of their work from intramural funding from the University of Ioannina. SB, JDH, LB and JW acknowledge support of their work from the U.S. National Science Foundation grant EF-0328363. SB also acknowledges the support from the NASA Astrobiology Institute (NNA04CC04A

De novo assembly of the olive fruit fly (Bactrocera oleae) genome with linked-reads and long-read technologies minimizes gaps and provides exceptional Y chromosome assembly

Background: The olive fruit fly, Bactrocera oleae, is the most important pest in the olive fruit agribusiness industry. This is because female flies lay their eggs in the unripe fruits and upon hatching the larvae feed on the fruits thus destroying them. The lack of a high-quality genome and other genomic and transcriptomic data has hindered progress in understanding the fly’s biology and proposing alternative control methods to pesticide use. Results: Genomic DNA was sequenced from male and female Demokritos strain flies, maintained in the laboratory for over 45 years. We used short-, mate-pair-, and long-read sequencing technologies to generate a combined male-female genome assembly (GenBank accession GCA_001188975.2). Genomic DNA sequencing from male insects using 10x Genomics linked-reads technology followed by mate-pair and long-read scaffolding and gap-closing generated a highly contiguous 489 Mb genome with a scaffold N50 of 4.69 Mb and L50 of 30 scaffolds (GenBank accession GCA_001188975.4). RNA-seq data generated from 12 tissues and/or developmental stages allowed for genome annotation. Short reads from both males and females and the chromosome quotient method enabled identification of Y-chromosome scaffolds which were extensively validated by PCR. Conclusions: The high-quality genome generated represents a critical tool in olive fruit fly research. We provide an extensive RNA-seq data set, and genome annotation, critical towards gaining an insight into the biology of the olive fruit fly. In addition, elucidation of Y-chromosome sequences will advance our understanding of the Y-chromosome’s organization, function and evolution and is poised to provide avenues for sterile insect technique approaches