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      Protocol to establish a genetically tractable synthetic symbiosis between Sodalis praecaptivus and grain weevils by insect egg microinjection

      research-article
      1 , 2 , 1 , 2 , 1 , 2 , 3 ,
      STAR Protocols
      Elsevier
      Evolutionary Biology, Microbiology, Model Organisms

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          Summary

          We present a protocol to establish a synthetic symbiosis between the mCherry-expressing Sodalis praecaptivus and the grain weevil host, Sitophilus zeamais. We describe steps to isolate grain weevil eggs, followed by microinjecting the bacterial symbiont into insect eggs using a modified Drosophila injection protocol, which leads to localization of bacteria in female insect ovaries. We then detail larval transplantation and visualization of bacteria in live insects using a fluorescence dissection microscope to assess the transgenerational transmission to offspring in weevils.

          For complete details on the use and execution of this protocol, please refer to Su et al. (2022). 1

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          Highlights

          • Protocol for the establishment of synthetic symbiosis in grain weevils

          • Procedures to inject fluorescently labeled bacterial cells into grain weevil eggs

          • Steps for larval transplantation and visualization of bacteria in live insects

          • Provides a platform to study insect-bacterial interactions

          Abstract

          Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

          Abstract

          We present a protocol to establish a synthetic symbiosis between the mCherry-expressing Sodalis praecaptivus and the grain weevil host, Sitophilus zeamais. We describe steps to isolate grain weevil eggs, followed by microinjecting the bacterial symbiont into insect eggs using a modified Drosophila injection protocol, which leads to localization of bacteria in female insect ovaries. We then detail larval transplantation and visualization of bacteria in live insects using a fluorescence dissection microscope to assess the transgenerational transmission to offspring in weevils.

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          Most cited references6

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          Genetic transformation of Drosophila with transposable element vectors.

          Exogenous DNA sequences were introduced into the Drosophila germ line. A rosy transposon (ry1), constructed by inserting a chromosomal DNA fragment containing the wild-type rosy gene into a P transposable element, transformed germ line cells in 20 to 50 percent of the injected rosy mutant embryos. Transformants contained one or two copies of chromosomally integrated, intact ry1 that were stably inherited in subsequent generations. These transformed flies had wild-type eye color indicating that the visible genetic defect in the host strain could be fully and permanently corrected by the transferred gene. To demonstrate the generality of this approach, a DNA segment that does not confer a recognizable phenotype on recipients was also transferred into germ line chromosomes.
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            Genome Degeneration and Adaptation in a Nascent Stage of Symbiosis

            Symbiotic associations between animals and microbes are ubiquitous in nature, with an estimated 15% of all insect species harboring intracellular bacterial symbionts. Most bacterial symbionts share many genomic features including small genomes, nucleotide composition bias, high coding density, and a paucity of mobile DNA, consistent with long-term host association. In this study, we focus on the early stages of genome degeneration in a recently derived insect-bacterial mutualistic intracellular association. We present the complete genome sequence and annotation of Sitophilus oryzae primary endosymbiont (SOPE). We also present the finished genome sequence and annotation of strain HS, a close free-living relative of SOPE and other insect symbionts of the Sodalis-allied clade, whose gene inventory is expected to closely resemble the putative ancestor of this group. Structural, functional, and evolutionary analyses indicate that SOPE has undergone extensive adaptation toward an insect-associated lifestyle in a very short time period. The genome of SOPE is large in size when compared with many ancient bacterial symbionts; however, almost half of the protein-coding genes in SOPE are pseudogenes. There is also evidence for relaxed selection on the remaining intact protein-coding genes. Comparative analyses of the whole-genome sequence of strain HS and SOPE highlight numerous genomic rearrangements, duplications, and deletions facilitated by a recent expansion of insertions sequence elements, some of which appear to have catalyzed adaptive changes. Functional metabolic predictions suggest that SOPE has lost the ability to synthesize several essential amino acids and vitamins. Analyses of the bacterial cell envelope and genes encoding secretion systems suggest that these structures and elements have become simplified in the transition to a mutualistic association.
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              Development and Life History ofSitophilus zeamais(Coleoptera: Curculionidae) on Cereal Crops

              The maize weevil,Sitophilus zeamaisMotschulsky (Coleoptera: Curculionidae), is one of the most destructive pests of stored cereals. Knowledge of the life history and biology is important to the development of an integrated pest management program. Investigation was carried out on developmental biology ofS. zeamaison four main cereal crops, maize, rice, sorghum, and millet, under laboratory conditions. Egg incubation, oviposition periods, and larval instar development were not different significantly among the food hosts. Number of eggs laid varied significantly among the cereal grains; mean fecundity was highest on maize ( 67.2 ± 3.16 ) and lowest on millet ( 53.8 ± 0.17 ). Number of immature (larva and pupa) and adult stages varied significantly among the cereal grains. There exist four larval instars with a varied mean head capsule width, with a mean total instar larval developmental period of 23.1, 22.2, 22.2, and 21.6 d on maize, rice, sorghum, and millet, respectively. There was linear relationship and significant correlation between the stages of larval development and head capsule width. The mean developmental period from egg to adult varied, being highest on maize (34.7 d) and lowest on sorghum (33.5 d).
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                Author and article information

                Contributors
                Journal
                STAR Protoc
                STAR Protoc
                STAR Protocols
                Elsevier
                2666-1667
                13 March 2023
                16 June 2023
                13 March 2023
                : 4
                : 2
                : 102156
                Affiliations
                [1 ]School of Biological Sciences, University of Utah, Salt Lake City, UT 84112, USA
                Author notes
                []Corresponding author colin.dale@ 123456utah.edu
                [2]

                Technical contact: crystal.su@utah.edu; adam.h.lin@utah.edu; colin.dale@utah.edu

                [3]

                Lead contact

                Article
                S2666-1667(23)00114-4 102156
                10.1016/j.xpro.2023.102156
                10025269
                36917608
                33577e23-a780-4366-aa65-9f614bdd1833
                © 2023 The Author(s)

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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                Protocol

                evolutionary biology,microbiology,model organisms
                evolutionary biology, microbiology, model organisms

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