The tree topologies obtained by the two statistical methods were similar but not identical (data not shown); the basal clades obtained by both methods were the same, and the phylogenetic positions of the symbionts in cell culture were robust, but differences were observed in the apical branching patterns of the clades. 2012, Evolution of resistance and counter-resistance, Host-race formation and ecological speciation, The evolution of resistance and counter-resistance in Drosophila and its natural enemies. To improve our knowledge of the bacterial diversity of polyphagous aphids, in the present study, we investigated the microbiota of the cosmopolitan agricultural pest Myzus persicae(Sulzer). We do not retain these email addresses. Synthesis of the essential amino acid tryptophan in the pea aphid (Acyrthosiphon pisum) symbiosis. & Godfray, H.C.J. to AY692365 Nancy Moran and Molly Hunter’s groups in Tucson, Arizona (Nancy Moran is now at the University of Texas) had shown that another secondary symbiont (Hamiltonella defensa, named after the great evolutionist Bill Hamilton) protected its aphid host from parasitoid wasps. Yes, you heard right. Aphids are known to be associated with a variety of symbiotic bacteria. (1988). The bootstrap percentages at the nodes were calculated by the neighbor-joining and maximum-likelihood methods. 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. Aphids possess several facultative bacterial symbionts that have important effects on their hosts' biology. Bacterial identities within the cell cultures were confirmed using two complementary approaches. Characteristics of the aphid-Buchnera symbiosis LOCATION OF THE BACTERIA In all aphids, the Buchnera are located in myce-tocytes in the hemocoel. This was supported by later work in which we cured aphids of their natural secondary symbionts using antibiotics (5). C. Supports theory that an ancient aphid ancestor - around 250 mya - acquired bacteria, developed vertical transmission and as aphids have diverged, so have their symbionts D. Very similar to mirror-image phylogenetic patterns between host and symbiont seen in some chemoautuotrophs and in squid-bacterial symbioses VII. Authors. 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. Fig. 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. These bacteria are typically not We have found that Rickettsia, Rickettsiella and Spiroplasma (though only one of several strains) also provide protection against the fungal pathogen (Pandora) (7). The PCR mixtures contained PCR buffer, 2 mM MgCl2 (Invitrogen, United Kingdom), each deoxynucleoside triphosphate (Promega, United Kingdom) at a concentration of 0.24 mM, each primer (MWG Biotech, GmbH) at a concentration of 20 μM, and 1 U Platinum Taq polymerase (Invitrogen, United Kingdom). 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. 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). At 10-day intervals, medium taken from the cell layer described above was passaged onto a new 90% confluent C6/36 cell monolayer. Aphids and their symbiotic bacteria This project is ongoing. Axenic culture may be an unrealistic goal for some insect-symbiotic bacteria which probably require a complex cellular environment that cannot be easily replicated in vitro. The ant benefits the aphid by protecting it from predators like ladybugs. Emission from TAMRA and Cy5 was collected via 560- and 650-nm long-pass filters, and emission from FITC was collected via a 505- to 570-nm band-pass filter. 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. The lines infected with U type also contained rod-shaped bacteria that were detectable using an inverted microscope at a magnification of ×600. 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. Postfixation in situ hybridization was performed using 5′-end-labeled oligonucleotide probes (high-performance liquid chromatography purified; MWG Biotech, GmbH) that specifically hybridize to 16S rRNA of γ-proteobacteria (EUB338; fluorescein isothiocyanate [FITC] labeled; 5′-GCT GCC TCC CGT AGG AGT-3′) (1), U type (U16; 6-carboxytetramethylrhodamine [TAMRA]-5′-GTA GCA AGC TAC TCC CCG AT-3′) (34), and T type (T16; Cy5 labeled; 5′-CTC AGT AAA CTG CGC TCA CT-3′) (this study). We used a similar approach to transfer symbionts among aphids, first extracting body fluids from late instar juveniles with a glass microcapillary tube pulled into a fine needle. 28, 341–355. Copyright © 2020 American Society for Microbiology | Privacy Policy | Website feedback, Print ISSN: 0099-2240; Online ISSN: 1098-5336, Aphid-Symbiotic Bacteria Cultured in Insect Cell Lines, Sign In to Email Alerts with your Email Address. In addition to the obligate symbiont pea aphid (and other aphids) may carry further bacterial species, so-called secondary symbionts. Aphids have symbiotic bacteria called Hamiltonella defensa that protect them against wasps. The capacity of T type and U type to maintain persistent infections in all the insect cell lines tested (two dipteran lines and one lepidopteran line) demonstrates that there is potential compatibility with insect cells other than aphid cells. Symbiosis is a prevalent phenomenon among organisms throughout the tree of life, including the insects which often harbor maternally transmitted bacteria. . Endosymbiosis. Insect Biochemistry and Molecular Biology, 95: 55-63. We propose the provisional taxon names (30) “Candidatus Consessoris aphidicola” (from the Latin consessoris, meaning one who sits near, a neighbor) for T type and “Candidatus Adiaceo aphidicola” (from the Latin adiaceo, meaning to lie by the side of, be adjacent) for U type. The phylogenetic tree, based on Papasotiropoulos 2013 and Kim 2011, with additions from Ortiz-Rivas and Martinez-Torres 2009, shows the internal phylogeny of the Aphididae.. The symbiosis has a nutritional basis. These findings are consistent with the proposed role of groESL in the Buchnera-aphid symbiosis. 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. Virtually all aphids maintain an obligate mutualistic symbiosis with bacteria from the Buchnera genus, which produce essential nutrients for their aphid hosts. Ecol. The cost of metabolic interactions in symbioses between insects and bacteria with reduced genomes. Ankrah NYD, Chouaia B and Douglas AE, 2018. (Symbiosis between nitrogen-fixing bacteria and legumes is a familiar example.) Phylogenetic analysis.Clustal_X version 1.8 (32) was used for multiple-sequence alignment with manual adjustment to exclude gaps and ambiguous bases from the analysis. Symbiotic bacteria greatly increase the ability of plant lice, also known as greenflies, to grow and reproduce, causing annoyance to gardeners and damage to crops worldwide. Note the tiny 'fly-like' insect at the top of the leaf. The Wellcome Trust (A.C.D.) Among them include Wolbachia species and Hamiltonella defensa. While Buchnera is obligately vertically transmitted and is required by the insect for sustained growth and reproduction (10), the prevalence of secondary symbionts in aphids is variable and these organisms are transmitted both vertically and horizontally (4, 9, 27). Several kinds of sap-sucking insects (such as aphids, weevils, and cockroaches) have specialized cells (bacteriocytes) in which they house symbiotic bacterial species. Aphids have an obligate association with the bacterium Buchnera aphidicola (the primary symbiont) that has been shown to contribute directly to aphid fitness. Aphids are sap-feeding insects infesting wide ranges of plant species. Ten microliters of the supernatant was applied to a 90% confluent monolayer of Aedes albopictus C6/36 cells (European Collection of Cell Cultures reference no. A model of obligate symbiosis is that between aphids and the bacterium Buchnera aphidicola, which supplies essential nutrients. Virtually all aphids carry the obligate symbiont Buchnera. Over the years, a variety of aphid species have been used to study the symbiosis with the bacteria Budinera (e.g. (a) T type. 3a) but not with the T-type-specific probe. 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. The cultures bearing U type and T type from M. rosae tended to lose T type, so that by the 20th passage all of the cultures contained only U type. All recipient cell lines were positive for symbionts as determined by specific PCR assays after three passages. (b) U type. C6/36 cells were infected with bacterial preparations from M. rosae (lane 3), A. pisum (lane 5), and A. fabae (lane 7). Due to the intimacy of the relationship, such symbiotic bacteria are not generally amenable to axenic cultivation, which constrains research progress. Thus, both T type and U type could be cultured in the C6/36 insect cells, and the estimated success rates were 26 to 67% for T type and 29 to 42% for U type (calculated from the data described above). The incidence of these bacteria did not vary significantly with aphid species (χ2 [2 df] = 4.232, P > 0.05). To detect the bacteria in insect cell culture, we used both PCR-dependent diagnostic assays and PCR-independent in situ hybridization analysis. In supplementary experiments we investigated whether U type and T type could be cultured in alternative insect cell lines, including the Drosophila S2 and Spodoptera SF9 lines. The secondary symbionts which we cultured in insect cells are known informally as T type (pea aphid Bemisia symbiont) and U type (pea aphid U-type symbiont). Bacteria obtained from M. rosae and A. pisum were gram-negative rods and 2 to 9 μm long, characteristics that are consistent with the secondary symbiont U type (7). This study provides additional insight into the nature of interactions between the secondary symbionts and the insect host. 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. Chung SH, Jing X, Luo Y and Douglas AE. Samples from the cultures were subjected to diagnostic PCR for T type and U type (for M. rosae and A. pisum, the cultures selected contained bacterial rods). Nutritional bacterial endosymbionts are housed in specialized host cells and are partitioned from the host cell cytoplasm by a host-derived symbiosomal membrane. The symbiosis of the pea aphid Acyrthosphion pisum with the bacterium Buchnera aphidicola APS represents the best-studied insect obligate symbiosis. Aphids possess several types of symbiotic bacteria, which are now known to have a variety of effects on host growth and survival. These have been most closely studied in the pea aphid (Acyrthosiphon pisum), a species that feeds on multiple host plants. "Aphids and Buchnera have become the central model for a kind of highly specialized animal-bacterial symbiosis in which the bacteria occur within the cytosol (the watery interior) of specialized host cells and are inherited from the mothers," said Dr. Nancy Moran, professor of ecology and evolutionary biology at the University of Arizona in Tucson. Excessive heat kills the symbiotic bacteria that some aphids depend on, which makes the aphids infertile. 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. This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. The aphid source material was M. rosae line ACD03/01 (lane 2), A. pisum line JF99/14 (lane 4), and A. fabae line CRT01/43 (lane 6). The bacterial-derived genes amiD and ldcA1 are preferentially expressed in aphid bacteriocytes.. RNAi-mediated reduction of amiD and ldcA1 expression depresses the bacterial symbiosis and aphid performance.. dsRNA stability and RNAi efficacy are promoted by RNAi-mediated reduction of aphid gut nuclease transcripts. The evolutionary transition to a symbiosis in which bacteria and an aphid cell form a functional unit, similar to the origin of plastids, has apparently involved extensive molecular adaptations on the part of the host cell. U type from A. pisum and T type from A. fabae persisted for at least 20 passages (the maximum number of passages tested). Using deep sequencing of 16S rRNA bacterial genes from 128 species of Cinara (the most diverse Lachninae genus), we reveal a highly dynamic dual symbiotic system in this aphid lineage. (1992). 1. Corresponding Author. (2007). Endosymbiosis is a mutualistic relationship between a host and an organism living within its body or cells.. The secondary symbionts are phylogenetically and functionally distinct from the primary symbionts (the γ-proteobacterium Buchnera in most aphids). Sections (thickness, 60 nm) were stained using an LKB Ultrastainer with uranyl acetate and lead citrate and examined with a Philips CMI2 transmission electron microscope. Abstract. (b) U type. Ehrhardt, 1969; Mittler, 1971; Hinde, 1971; Campbell & Nes, 1983). The lab of Nancy Moran is seeking a reliable volunteer undergraduate research assistant to help with experimental studies involving aphids and their symbiotic bacteria. Whether secondary … 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. & Godfray, H.C.J. doi: 10.1111/1365-2435.12133 Aphid-bacterial symbioses. The specific purpose of this study was to culture bacteria that are known generically as the “secondary symbionts” of one group of insects, the aphids. We are exploring further aphid genotype/bacterial genotype interactions and how they may affect host plant use and other aspects of aphid biology. 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. The pea aphid and its endosymbiont. We became interested in secondary symbionts when studying host plant use and parasitoid resistance in a panel of aphid clones isolated from a range of different host plants. Abstract Most aphids possess intracellular bacteria of the genus Buchnera. 1 The molecular mechanism by which aphids precisely regulate the nutritional product provided by the bacteria ( endosymbionts ) that live inside them displays the hallmarks of God’s grand design. 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. This prompted us to inject Regiella into aphids that carried no symbionts to see if it protected them from fungus attack. Compatibility of symbionts with dipteran and lepidopteran cell lines. To our knowledge, just one other secondary symbiont, S. glossinidius in tsetse flies, has been cultured previously (36). The symbiosis of the pea aphid Acyrthosphion pisum with the bacterium Buchnera aphidicola APS represents the best-studied insect obligate symbiosis. Individual insects were homogenized with a hand-held glass homogenizer with 100 μl Mitsuhashi and Maramorosch insect medium (MMI) (22) supplemented with 20% fetal bovine serum (FBS) and centrifuged at 100 × g for 2 min. FEMS Microbiol Ecol 36(1):43–50 PubMed CrossRef Google Scholar. 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. 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. 1). The humble aphid may appear unremarkable, but it is cleverly adapted to its plant-sucking way of life. Incidence of secondary symbionts in aphids and cell cultures. These names were specifically chosen because their Latin origins suggest close physical proximity without implying function. Aphids engage in symbiotic interactions with several maternally transmitted bacteria, and many associate with microbes known as secondary symbionts. The preparation was centrifuged at 1,000 × g at room temperature for 10 min to bring the bacteria into contact with the cells and incubated at 26.5°C for 16 h. Cells were washed twice in phosphate-buffered saline (PBS), pH 8.5, incubated in MMI containing 20% FBS and 50 μg ml−1 gentamicin for 1 h at 26.5°C, rinsed twice in PBS, and then incubated in fresh MMI containing 20% FBS for 10 days at 26.5°C. We asked whether the five aphid clones into which we had injected Regiella acquired the ability to utilise clover. The culture of these bacteria in insect cell lines provides a new and unique research opportunity, offering a source of unibacterial material for genomic studies and a model system to investigate the interactions between animal cells and bacteria. The symbiosis has a nutritional basis. Facilitated by their ancient association with intracellular symbiotic bacteria that synthesize essential amino acids, aphids feed on phloem (sap). Buchnera aphidicola is an obligate symbiotic bacterium that sustains the physiology of aphids by complementing their exclusive phloem sap diet. At the level of the 16S rRNA sequence, no heterogeneity of either T type or U type was evident among the three sequences found in the aphid species. 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. The culture medium was then replaced with medium from a 10-day-old culture of cells infected with U type from M. rosae that had been filtered through a 5-μm filter to eliminate the insect cells. First, a 1,480-nt fragment of the 16S rRNA gene of the bacteria in the insect cells was amplified and sequenced. 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). 2a, the sequences could be assigned to the γ-proteobacteria and were distinct from the sequences of most other bacteria that have been found in insects, including the secondary symbiont known as R-type from aphids (27, 35). Sulphate utilization in an aphid symbiosis. Symbiotic Bacterium Modifies Aphid Body Color | Science. Besides Buchnera, other bacteria have also been found in aphids either living within bacteriocytes or not . We found no consistent effect (4), and think that host use may be influenced by complex aphid genotype/bacterial genotype interactions. NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. 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. 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]. Aphids engage in symbiotic interactions with several maternally transmitted bacteria, and many associate with microbes known as secondary symbionts. Nucleotide sequence accession numbers.Consensus nucleotide sequences have been deposited in the National Center for Biotechnology Information GenBank database under accession numbers AY692358 (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). 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. 2003, Ferrari, J., Darby, A.C., Daniell, T.J., Godfray, H.C.J. Department of Biology, University of York, York, 401 SDD England. How do aphids eat an endless supply of junk food from plant sap and not keel over? 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. The pea aphid, Acyrthosiphon pisumHarris (Hemiptera: Aphididae), harbors one obligatory bacterial symbiont and up to seven different facultative symbionts, some of which are known to protect the aphid from pathogens, natural enemies, and other mortality factors. Annu Rev Entomol 43: 17–37. Defensive symbiosis in the real world – advancing ecological studies of heritable, protective bacteria in aphids and beyond. Compatibility of symbionts with dipteran and lepidopteran cell lines.Ten milliliters of culture medium from C6/36 cell cultures infected with secondary symbionts from A. fabae (T type) or A. pisum (U type) was passed though a 5-μm filter to remove the insect cells. Journal of Microbiology & Biology Education, Microbiology and Molecular Biology Reviews. Ten to fifteen percent of insect species have some sort of nutritional symbiotic relationship. Such symbiotic bacteria are traditionally described as intractable to cultivation in vitro. doi: 10.1371/journal.pbio.0000021. Recent research, however, has focused almost exclusively on one species, the pea aphid Acyrthosiphon pisum. 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. 2b). (a) T type. Adhering cells were fixed for 10 min in 4% (wt/vol) formaldehyde in PBS (Invitrogen), and this was followed by cell permeabilization in 70% ethanol for 10 min and three washes in PBS. 1. In an obligate symbiosis with Buchnera spp. & Douglas, A.E. 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]). Feed on plant sap by tapping into phloem tissue 2. We are also exploring the potential effects of bacterial symbionts on host plants (review in (8)), including whether presence of aphid symbionts induces a specific response from aphid food plants, and whether this impacts the wider insect community. Phylogenetic trees of 16S rRNA gene sequences of the bacteria cultured in insect cells in this study (underlined). 2018. Insect cells from a routine culture were suspended in MMI plus 20% FBS. Cultures of these bacteria were not studied further secondary … Excessive heat kills the symbiotic bacteria is defined as interaction. 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Work in which we had injected Regiella acquired the ability to utilise clover intractable cultivation! Entomology, University of Georgia, Athens, GA 30602, USA has long been associated with their hosts Biology... 2012, Frago, E., Dicke, M., Guo, H. & Godfray, H.C.J aphids bacteria! That has evolved between aphids and cell cultures over multiple passages experiments, the aphids infertile work in we... Borders a tropical rainforest region facilitated by their ancient association with intracellular symbiotic bacteria incidence of secondary symbionts T... H. & Godfray H.C.J an emerging infectious disease are reported in workers at a research station that a. 401 SDD England recipient cell lines were positive for symbionts as determined by specific PCR assays after three.. Plant use and other aphids ) may carry further bacterial species, in. Rnai experiments, the pea aphid ( Acyrthosiphon pisum that recurs in laboratory lines and occurs in populations! Because their Latin origins suggest close physical proximity without implying function authoritative coverage of both basic and clinical Microbiology the! Inherited strictly vertically and in many ways has become increasingly apparent than many aspects the. A variety of effects on their hosts ' Biology survive or reproduce without symbiont and visa versa B provides!