U type from A. pisum and T type from A. fabae persisted for at least 20 passages (the maximum number of passages tested). Abstract. The identities of the cultured bacteria were confirmed by PCR with sequencing of 16S rRNA gene fragments and fluorescence in situ hybridization. The bacteria are transmitted vertically via the aphid ovary, and the association is obligate for both partners: Bacteria-free aphids grow poorly and produce few or no offspring, and Buchnera are both unknown apart from aphids and apparently unculturable. The inoculated coverslip cultures were incubated for 16 h at 26.5°C to allow the uptake of bacteria into the cells, washed with PBS, fixed in 3% glutaraldehyde, postfixed in 1% osmium tetroxide, dehydrated in acetone, and embedded in araldite resin. They have been described previously on the basis of their 16S rRNA gene sequences (8, 27), and their morphology has been identified by in situ hybridization (8, 34). Chung SH, Jing X, Luo Y and Douglas AE. In cell lines infected with bacteria derived from aphids harboring both T type and U type, the U type persisted, while the T type was lost. line CRT01/43, containing the secondary symbiont T type; (ii) pea aphids, Acyrthosiphon pisum Harris line JF 99/14, containing U type; and (iii) rose aphids, Macrosiphum rosae L. line ACD03/01, containing both T- and U type. The secondary symbionts are phylogenetically and functionally distinct from the primary symbionts (the γ-proteobacterium Buchnera in most aphids). Secrete enzymes that dissolve part of plant material to penetrate 3. PCR assays and sequencing.For PCR-based detection and identification of secondary symbionts, total DNA was extracted from single aphids and 10-day-old insect cell cultures using a DNeasy tissue kit (QIAGEN, United Kingdom) by following the manufacturer's protocol for cultured animal cells, and this DNA was used as a template for amplification of 16S rRNA gene fragments. A. E. Douglas. All aphids were wingless summer parthenogenetic female morphs. The bootstrap percentages at the nodes were calculated by the neighbor-joining and maximum-likelihood methods. Such symbiotic bacteria are traditionally described as intractable to cultivation in vitro. A symbiosis is defined as an interaction between two different species that is beneficial to both. ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology. We have studied the development and evolution of aphid bacteriocytes, th … Developmental origin and evolution of bacteriocytes in the aphid-Buchnera symbiosis PLoS Biol. Incidence of secondary symbionts in aphids and cell cultures. We suggest that the two bacteria persist in aphids because competition between them is limited by differences in tropism for insect tissues or cell types. Douglas AE (1998) Nutritional interactions in insect-microbial symbioses: aphids and their symbiotic bacteria Buchnera. Structure and expression of the dnaKJ operon of Buchnera, an intracellular symbiotic bacteria of aphid. The optimum model for the 1,480-nt 16S rRNA gene fragment data was found to be a HKY85+G+I model, and the optimum model for the 635-nt 16S rRNA gene fragment data set was found to be a HKY+G model (16). Each symbiont-cell line combination was replicated three times and maintained as described above for the C6/36 cell cultures. In order to fit into various types of environmental niches, many species have established mutualistic relationships with endosymbiotic bacteria for nutritional, digestive or other benefits. Such symbiotic bacteria are traditionally described as intractable to cultivation in vitro. This is in contrast to the existence of T- and U-type coinfections in field aphids (17, 29) and in long-term laboratory aphid cultures (12). The insect cell cultures were also tested routinely for microorganisms cultivable on 5% sheep blood agar plates incubated at 26.5°C, and they were inspected daily for 10 days at a magnification of ×600 for microbial growth using an inverted M100 microscope (Swift-Microtec, Oxford, United Kingdom). Besides Buchnera, other bacteria have also been found in aphids either living within bacteriocytes or not . Virtually all aphids carry the obligate symbiont Buchnera. Phylogenetic trees of 16S rRNA gene sequences of the bacteria cultured in insect cells in this study (underlined). Facilitated by their ancient association with intracellular symbiotic bacteria that synthesize essential amino acids, aphids feed on phloem (sap). Whether secondary … This study provides additional insight into the nature of interactions between the secondary symbionts and the insect host. Confocal microscopy was performed with a Zeiss LSM 510 Meta attached to a Zeiss Axiovert 200 M fitted with a Plan-Apochromat ×63 oil immersion lens (N.A. Authors. Cell lines bearing secondary symbionts from M. rosae and A. pisum known to contain U type and cell lines bearing secondary symbionts from A. fabae known to contain T type (as determined by diagnostic PCR) were examined. Plasmid DNA was purified using a QIAprep miniprep kit (QIAGEN) according to manufacturer's instructions, and consensus sequences were derived from the sequences of three clones, determined in both directions. We thank P. O'Toole and T. Tsuchida for advice concerning the FISH analysis and J. Ferrari and C. R. Tosh for use of aphid lineages JF 99/14 and CRT01/43, respectively. The lab of Nancy Moran is seeking a reliable volunteer undergraduate research assistant to help with experimental studies involving aphids and their symbiotic bacteria. Symbiotic bacteria in insects are generally regarded as nonpathogenic and include both mutualists (i.e., bacteria advantageous to the insect) and commensals (bacteria that have no apparent significance to the insect). Virtually all aphids maintain an obligate mutualistic symbiosis with bacteria from the Buchnera genus, which produce essential nutrients for their aphid hosts. The symbiosis has a nutritional basis. Bacteria were detected in all inoculated cultures tested by FISH, and many of them appeared to be cytoplasmic. PCR products were purified with a QIAquick PCR purification kit (QIAGEN, United Kingdom), ligated into a pGEM-T vector (Promega, United Kingdom), and transformed into Escherichia coli strain DH5α (Promega, United Kingdom) according to the manufacturer's instructions, except that the competent bacteria were pretreated with 0.1% (vol/vol) β-mercaptoethanol before transformation. Living inside the cytoplasm of an insect’s bacteriocytes, these bacteria produce … Ehrhardt, 1969; Mittler, 1971; Hinde, 1971; Campbell & Nes, 1983). Pea aphids, Acyrthosiphon pisum, harbour a range of facultative accessory bacteria (secondary symbionts), including those informally known as PASS (R‐type), … In the last 20 years it has become increasingly apparent than many aspects of the life history of insects are influenced by symbiotic bacteria. In this study we focused on two secondary symbionts, T type and U type. In addition to the obligate symbiont pea aphid (and other aphids) may carry further bacterial species, so-called secondary symbionts. Structure and function of an essential component of the outer membrane protein assembly machine. Salmonella [7, 23, 34]. The growth and reproduction of phloem sap-feeding insects requires the sustained function of intracellular bacteria localized in specialized cells known as bacteriocytes, giving the potential to target the bacterial symbiosis as a novel strategy for controlling sap-feeding insect pests. The lines infected with U type also contained rod-shaped bacteria that were detectable using an inverted microscope at a magnification of ×600. The three control treatments used in all experiments were (i) a probe-free control for autofluorescence; (ii) an RNA-free control (treatment, prior to hybridization, with an RNase solution containing 150 mM NaCl, 10 mM Tris-HCl [pH 7.5], 1 mM EDTA, and 20 μg RNase [QIAGEN] ml−1 at 37°C for 30 min); and (iii) competitive suppression (addition of 4.2 nmol unlabeled probe to the hybridization solution) (data not shown) (3). Insect material.The following three sources of secondary symbionts were used as inocula: (i) black bean aphids, Aphis fabae Scop. In M. rosae cultures, rod-shaped bacteria were detected in 50% of the 100 insect cells examined, and each infected cell contained 1 to 20 bacteria that were up to 15 μm long and hybridized with both the general eubacterial probe and the U-type-specific probe (Fig. It is unlikely that their infective range is restricted by limited access to alternative host species for two reasons. For the insect cells in which T-type bacteria were detected by diagnostic PCR, about 25% of the 100 insect cells examined yielded a signal with the FISH probe specific for T type, and the readily detectable hybridizations were hybridizations of coccoid structures that were ca. First, there is strong phylogenetic and experimental evidence that the secondary symbionts are horizontally transmissible (5, 9, 27); and second, other insect-associated bacteria with mixed vertical and horizontal transmission patterns show broad host distributions (e.g., Wolbachia is estimated to occur in 20% of all insect species [38, 39]). All these genera have previously been described as genera that are associated with aphids, but they have not been described as symbiotic bacteria (15; C. Francois personal communication). Aphids engage in symbiotic interactions with several maternally transmitted bacteria, and many associate with microbes known as secondary symbionts. (b) U type. The filtrate, which contained extracellular bacterial cells, was added to a 90% confluent cell monolayer of the Drosophila melanogaster S2 cell line (Drosophila Genetic Resource Centre stock number 6) or the Spodoptera frugiperda SF9 cell line (European Collection of Cell Cultures reference number 89070101). Host plant effects on an aphid–bacteria symbiosis 3029 populations in the total aphid tissues and embryos, bacterial density was expressed per unit of aphid protein. Additionally, the possibility that U type possesses traits (e.g., bacteriocins) which are directly antagonistic to T type expressed in cell culture but not in the symbiosis cannot be excluded. The research project will consist of the maintenance of aphids and plants, using molecular biology and biochemistry to study the proteins underlying symbiosis, as well as engineering of aphid symbionts to develop genetic tools for interrogating symbiosis. Buchnera aphidicola is an obligate symbiotic bacterium that sustains the physiology of aphids by complementing their exclusive phloem sap diet. C6/36 cells were infected with bacterial preparations from M. rosae (lane 3), A. pisum (lane 5), and A. fabae (lane 7). 1. (a) T type. & Godfray H.C.J. 1.5 μm in diameter (Fig. Previous studies have reported the successful transfer of these bacteria between aphids by microinjection of hemolymph from an infected aphid (13, 27, 50). The incidence of these bacteria did not vary significantly with aphid species (χ2 [2 df] = 4.232, P > 0.05). (a and b) Fluorescence in situ hybridization of Cy5-labeled probes (red) to U type isolated from M. rosae (a) and to T type isolated from A. fabae (b). Symbiotic viruses exist in many insects; however, their functions in host insects are not well understood. 2018. Excessive heat kills the symbiotic bacteria that some aphids depend on, which makes the aphids infertile. & Godfray, H.C.J. These bacteria are the focus of considerable research interest as they may modulate ecologically important traits of aphids, including plant utilization characteristics (5, 12, 17, 33, 41), natural enemy resistance (12, 27, 29), and thermal tolerance (5). Host symbiont energetics Transmission electron microscopy.A monolayer of A. albopictus C6/36 cells was grown in MMI containing 20% FBS to 90% confluence on plastic Thermanox coverslips (Nalge Nunc International, United States) in 24-well culture plates. It has been suggested that the phylogeny of the aphid groups might be revealed by examining the phylogeny of their bacterial endosymbionts, especially the obligate endosymbiont Buchnera. The bacteria are restricted to large insect cells in the abdominal haemocoel called mycetocytes or bacteriocytes, and their population density, at approximately 10 7 bacteria per mg aphid weight, is appreciable. In the last 20 years it has become increasingly apparent than many aspects of the life history of insects are influenced by symbiotic bacteria. Ecol. The second approach used to establish the identities of secondary symbionts in the cell cultures was a microscopic analysis using fluorescence in situ hybridization (FISH) of a DNA probe to 16S rRNA of the bacteria, a method that is independent of PCR amplification. Over the years, a variety of aphid species have been used to study the symbiosis with the bacteria Budinera (e.g. This prompted us to inject Regiella into aphids that carried no symbionts to see if it protected them from fungus attack. The number of bacteria per insect cell and the number of cells infected varied with passage and culture. The milkweed aphids eat the plant and produce a sugary substance called 'honeydew' that the ants 'milk' from the aphid's anus. Excessive heat kills the symbiotic bacteria that some aphids depend on, which makes the aphids infertile. Insect Biochemistry and Molecular Biology, 95: 55-63. Virtually all aphids carry the obligate symbiont Buchnera which provides essential nutrients missing in the aphid diet. Aphid-bacterial symbioses. Buchnera is believed to have had a free-living, Gram-negative ancestor similar to a modern Enterobacterales, such as Escherichia coli. To explore the dynamics of secondary symbiont associations in aphids, we characterized bacteria infecting 15 species of macrosiphine aphids using DNA sequencing, diagnostic polymerase chain reaction (PCR), diagnostic restriction digests, phylogenetic analyses, and electron microscopy to examine aphids from nature and from laboratory colonies. DAPI was excited with the 405-nm diode laser line, and emission was collected via a 420- to 428-nm band-pass filter. How do aphids eat an endless supply of junk food from plant sap and not keel over? The answer is a special relationship that has evolved between aphids and bacteria. Phylogenetic trees were generated with PAUP* 4.0b2 (31). 1.4). GenBank accession numbers, bacterial species or “informal” names, and (in parentheses) the relevant insect hosts (where relevant) are indicated. 2003, Ferrari, J., Darby, A.C., Daniell, T.J., Godfray, H.C.J. To prevent potential bleed-through problems under the experimental conditions, images were acquired sequentially. When samples of all the cell cultures were tested for microorganisms cultivable on 5% sheep blood plates, only the cultures with the rapidly growing bacteria yielded bacterial colonies, including Micrococcus, Acinetobacter, and Staphylococcus colonies, as identified by sequencing of PCR-amplified 16S rRNA gene fragments, and colonies with the color and morphology of Serratia colonies (which were not studied further). Microbial processes and activities Biosynthesis of essential amino acids. 2011, Ferrari J., West J.A., Via S. & Godfray H.C.J. For the cultures examined at a magnification of × 600 at the third passage, rod-shaped bacteria were evident in 29% (19 of 65) and 42% (27 of 64) of the replicate cultures initiated from M. rosae and A. pisum, respectively, but no bacteria were detected in cultures initiated from A. fabae (n = 58). This was supported by later work in which we cured aphids of their natural secondary symbionts using antibiotics (5). The cells of T type are generally <2 μm long (8), and they would not be readily detectable in association with living cells using an inverted microscope at a magnification of ×600. For RNAi experiments, the aphids were co-administered with dsRNA against an aphid nuclease nuc1, protecting the dsRNA against non-specific degradation. (Symbiosis between nitrogen-fixing bacteria and legumes is a familiar example.) For example, secondary symbionts may be able to invade the aphid across the gut wall (9) (during horizontal acquisition by ingestion of bacterial cells released from other aphids in honeydew) or the ovarial sheath (during vertical transmission) but may not be able to invade other insect taxa by these routes; or initial infections may be detected and eliminated by the humoral defense systems of insects other than aphids. Symbiotic relationships between animals and microorganisms are common in nature, yet the factors controlling the abundance and distributions of symbionts are mostly unknown. Three species have been characterised in detail (from studies of pea aphid) and have been shown to have a range of effects on aphid biology including substituting for Buchnera after heat shock and improving resistance to parasitoids. Shorter products (630 nt) were generated using the γ-proteobacterium-specific primers 16SC1 Forward (5′-GAA TTC TAG GTG TAG CGG TGA) and 16SD1 Reverse (5′-GCG ATT CCG ACT TCG TGG A-3′) (this study). For example, we (and other groups) found clones collected on clover (Trifolium) nearly always carried the symbiont Regiella insecticola (1, 2) [incidentally, Regiella is named after the well-known insect physiologist, Reg Chapman]. The persistence of secondary symbionts from the three aphid species was scored in insect cell cultures over multiple passages. Aphids were surface sterilized in 70% ethanol, rinsed in sterile water, air dried on sterile blotting paper, and then subjected to UV radiation (CL-1000 UV cross-linker; UVP, Cambridge, United Kingdom) for 10 min. 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