Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Activities: The goal of our project has been to use molecular methods to characterize and localize the bacterial endosymbionts of Adelgidae (Hemiptera: Sternorrhyncha), with emphasis on the devastating silvicultural pest, Adelges tsugae (woolly hemlock adelgid, HWA). One of our goals was to pinpoint the population in Japan from which HWA introduction in the eastern U.S. occurred. We obtained samples of several species (including Adelges abietis, A. cooleyi, A. japonicus, A. laricis, A. lariciatus, A. tsugae, Pineus pini, P. coloradensis, and P. similis) with the help of contacts and collaborators. These included multiple samples of HWA from eastern and western U.S. and Japan. To identify the symbiotic bacteria, genomic DNA was extracted from these samples, and 16S rRNA genes were PCR-amplified with general eubacterial primers. PCR products were subcloned with TA cloning kits to separate 16S molecules that derived from different species/strains of bacteria. Subclones were
sequenced and compared to each other and to known bacterial 16S with the BLAST search in GenBank. After this initial assessment of sequence diversity, specific primers were designed to test for the presence of each sequence in each host species. All unique sequences were compiled into an alignment along with 16S sequences of known, selected bacteria. Phylogenetic analyses (maximum parsimony and Bayesian inference) were conducted to determine the evolutionary relationships of bacteria in adelgids. To investigate whether endosymbionts had likely co-speciated with adelgid hosts, adelgid genes (cytochrome oxidase II and partial 12S/16S rRNA) were amplified and sequenced. A phylogeny of host genes was reconstructed and compared to the bacterial phylogeny. To determine that bacteria were in fact housed within the bodies (cells and/or organs) of adelgids, and where, we prepared thin sections of preserved, whole adelgids, and probed them with fluorescence-labeled primers specific to the
amplified 16S genes (FISH experiments). Localization of the probes on adelgid sections was visualized with a laser-scanning confocal microscope. Products: We house a physical collection of Adelgidae samples collected from the field, preserved in ethanol and stored at -70oC. In addition, we have a collection of genomic DNA extracted from each sample and stored at -70oC. We have compiled a database of 16S bacterial sequences from endosymbionts of each tested species, including 2-5 sequences from each host. We also have a database of adelgid host genes. All gene sequences will be submitted to the public database, GenBank, upon publication. We have amassed a large collection of electronic images of probed adelgid sections portraying the locality of endosymbionts within adelgid bodies. Dissemination: three manuscripts on this work are being prepared for journal publications. In addition, results of the investigations on Adelges tsugae will be presented at the 4th Annual Hemlock Woolly
Adelgid Symposium in Hartford, CT in February 2008.
PARTICIPANTS: Dr. Robert Foottit, Agriculture and Agri-Food Canada, Ottawa. Dr. Foottit directs the Sternorrhycha/Thysanoptera unit of the Canadian National Collection of Insects; he contributed several Adelges and Pineus samples. Dr. Kathleen Shields, USDA Forest Service, Northeastern Research Station, Hamden, CT. Dr. Shields arranged for a Cooperative Agreement with the Forest Service for additional funding for the Hemlock Woolly Adelgid work. In addition, she contributed many HWA samples, as well as transmission electron microscopy images. Dr. Nathan Havill, Postdoctoral Associate,Yale University and Visiting Scientist, USDA Forest Service Northern Research Station. Dr. Havill contributed many HWA samples and information on HWA phylogenetics. Usha Spaulding, former USU Biology graduate student, was supported as a technician on the project. She received training in fluorescent in situ hybridization methods, cryosectioning techniques, and operation of a laser-scanning confocal
microscope.
TARGET AUDIENCES: Target audiences include scientists working on basic biology, evolution, and genomics of bacterial endosymbionts, researchers studying potential applied aspects of bacterial endosymbionts in pest insects, and scientists working on biocontrol of HWA and other economically important adelgids.
Impacts
This research is the first ever to characterize bacterial endosymbionts in Adelgidae with molecular techniques. Our focus on Hemlock Woolly Adelgid is relevant to understanding the virulence of this pest because of the roles endosymbionts play in host fitness. Our work on host adelgid and endosymbiont phylogeny has suggested a pattern of evolutionary gains and losses of symbionts. From our phylogenetic studies, we conclude that the original (primary) endosymbiont infection in Adelgidae was a gamma-Proteobacteria closely related to the primary endosymbiont of Aphididae, Buchnera aphidicola. This symbiont is found, however, only in Pineus spp. and A. tsugae. We believe that the symbiont was lost in the ancestor of remaining Adelges and replaced by a different symbiont from the beta-Proteobacteria, related to Burkholderia. The beta-Proteobacteria is present in all Adelges, including A. tsugae, and exhibits co-speciation with its hosts. The beta-symbiont may have been a
secondary symbiont acquired after the divergence of Adelges and Pineus, which then replaced the primary Buchnera-type symbiont after its extinction. In addition, we found sequences from other "secondary" symbionts. Most Adelges contained one additional symbiont from the gamma-Proteobacteria. In A. cooleyi, this bacterium was closely related to Serratia spp. In A. laricis, A. japonicus, and A. abietis the symbiont was closest to Rahnella aquatilis. Pineus spp. also contained a gamma-proteobacterial endosymbiont that was related to Providencia rettgeri. Pineus strobii contained a sequence from the symbiont, Hamiltonella defensa. A. tsugae contained up to 3 additional bacterial types depending on the population. The first was a beta-Proteobacteria related to Janthinobacter. The others were two types of gamma-Proteobacteria, one related to Pseudomonas aeruginosa and the other to Serratia symbiotica, a symbiont of aphids that enhances resistance to parasitoids. FISH experiments confirmed
that most symbionts were housed within cells of the adelgid's bacteriome. Exceptions were found in A. tsugae: one gamma symbiont was in the haemocoel and two beta symbionts were found only in salivary gland. The Buchnera-like symbiont was found in both salivary glands and bacteriomes in early instars. To our knowledge this is the first report of sternorrhynchan endosymbionts in salivary glands. We also found population-and stage-specific variation in symbionts of A. tsugae. In western U.S. (WNA) samples, the Serratia-type is absent, but was present in eastern U.S. (ENA) populations and in some southern Honshu populations. Sequence comparisons suggested that Niryo, Osaka was the source population for HWA in ENA. In support of this, the Burkholderia beta symbiont was present in several Japanese populations, but absent in Niryo, ENA, and WNA. Both beta symbionts were found only in the 1st-instar/crawler stages of some Japanese populations. Changes in symbiont composition can affect host
fitness. The Serratia symbiont of aphids can be transferred horizontally between hosts; possibly a novel infection of the Serratia-type symbiont in the source population facilitated the establishment of HWA in the US.
Publications
- Havill, N.P., R.G. Foottit, & C.D. von Dohlen 2007. History of life cycle evolution and host plant associations of the Adelgidae (Insecta: Hemiptera) inferred from molecular phylogenetics. Molecular Phylogenetics and Evolution 44:357-370.
|
Progress 07/01/01 to 06/30/07
Outputs
OUTPUTS: Activities: The goal of our project has been to use molecular methods to characterize and localize the bacterial endosymbionts of Adelgidae (Hemiptera: Sternorrhyncha), with emphasis on the devastating silvicultural pest, Adelges tsugae (woolly hemlock adelgid, HWA). One of our goals was to pinpoint the population in Japan from which HWA introduction in the eastern U.S. occurred. We obtained samples of several species (including Adelges abietis, A. cooleyi, A. japonicus, A. laricis, A. lariciatus, A. tsugae, Pineus pini, P. coloradensis, and P. similis) with the help of contacts and collaborators. These included multiple samples of HWA from eastern and western U.S. and Japan. To identify the symbiotic bacteria, genomic DNA was extracted from these samples, and 16S rRNA genes were PCR-amplified with general eubacterial primers. PCR products were subcloned with TA cloning kits to separate 16S molecules that derived from different species/strains of bacteria. Subclones were
sequenced and compared to each other and to known bacterial 16S with the BLAST search in GenBank. After this initial assessment of sequence diversity, specific primers were designed to test for the presence of each sequence in each host species. All unique sequences were compiled into an alignment along with 16S sequences of known, selected bacteria. Phylogenetic analyses (maximum parsimony and Bayesian inference) were conducted to determine the evolutionary relationships of bacteria in adelgids. To investigate whether endosymbionts had likely co-speciated with adelgid hosts, adelgid genes (cytochrome oxidase II and partial 12S/16S rRNA) were amplified and sequenced. A phylogeny of host genes was reconstructed and compared to the bacterial phylogeny. To determine that bacteria were in fact housed within the bodies (cells and/or organs) of adelgids, and where, we prepared thin sections of preserved, whole adelgids, and probed them with fluorescence-labeled primers specific to the
amplified 16S genes (FISH experiments). Localization of the probes on adelgid sections was visualized with a laser-scanning confocal microscope. Products: We house a physical collection of Adelgidae samples collected from the field, preserved in ethanol and stored at -70oC. In addition, we have a collection of genomic DNA extracted from each sample and stored at -70oC. We have compiled a database of 16S bacterial sequences from endosymbionts of each tested species, including 2-5 sequences from each host. We also have a database of adelgid host genes. All gene sequences will be submitted to the public database, GenBank, upon publication. We have amassed a large collection of electronic images of probed adelgid sections portraying the locality of endosymbionts within adelgid bodies. Dissemination: three manuscripts on this work are being prepared for journal publications. In addition, results of the investigations on Adelges tsugae will be presented at the 4th Annual Hemlock Woolly
Adelgid Symposium in Hartford, CT in February 2008.
PARTICIPANTS: Dr. Robert Foottit, Agriculture and Agri-Food Canada, Ottawa. Dr. Foottit directs the Sternorrhycha/Thysanoptera unit of the Canadian National Collection of Insects; he contributed several Adelges and Pineus samples. Dr. Kathleen Shields, USDA Forest Service, Northeastern Research Station, Hamden, CT. Dr. Shields arranged for a Cooperative Agreement with the Forest Service for additional funding for the Hemlock Woolly Adelgid work. In addition, she contributed many HWA samples, as well as transmission electron microscopy images. Dr. Nathan Havill, Postdoctoral Associate,Yale University and Visiting Scientist, USDA Forest Service Northern Research Station. Dr. Havill contributed many HWA samples and information on HWA phylogenetics. Usha Spaulding, former USU Biology graduate student, was supported as a technician on the project. She received training in fluorescent in situ hybridization methods, cryosectioning techniques, and operation of a laser-scanning confocal
microscope.
TARGET AUDIENCES: Target audiences include scientists working on basic biology, evolution, and genomics of bacterial endosymbionts, researchers studying potential applied aspects of bacterial endosymbionts in pest insects, and scientists working on biocontrol of HWA and other economically important adelgids.
Impacts
This research is the first ever to characterize bacterial endosymbionts in Adelgidae with molecular techniques. Our focus on Hemlock Woolly Adelgid is relevant to understanding the virulence of this pest because of the roles endosymbionts play in host fitness. Our work on host adelgid and endosymbiont phylogeny has suggested a pattern of evolutionary gains and losses of symbionts. From our phylogenetic studies, we conclude that the original (primary) endosymbiont infection in Adelgidae was a gamma-Proteobacteria closely related to the primary endosymbiont of Aphididae, Buchnera aphidicola. This symbiont is found, however, only in Pineus spp. and A. tsugae. We believe that the symbiont was lost in the ancestor of remaining Adelges and replaced by a different symbiont from the beta-Proteobacteria, related to Burkholderia. The beta-Proteobacteria is present in all Adelges, including A. tsugae, and exhibits co-speciation with its hosts. The beta-symbiont may have been a
secondary symbiont acquired after the divergence of Adelges and Pineus, which then replaced the primary Buchnera-type symbiont after its extinction. In addition, we found sequences from other "secondary" symbionts. Most Adelges contained one additional symbiont from the gamma-Proteobacteria. In A. cooleyi, this bacterium was closely related to Serratia spp. In A. laricis, A. japonicus, and A. abietis the symbiont was closest to Rahnella aquatilis. Pineus spp. also contained a gamma-proteobacterial endosymbiont that was related to Providencia rettgeri. Pineus strobii contained a sequence from the symbiont, Hamiltonella defensa. A. tsugae contained up to 3 additional bacterial types depending on the population. The first was a beta-Proteobacteria related to Janthinobacter. The others were two types of gamma-Proteobacteria, one related to Pseudomonas aeruginosa and the other to Serratia symbiotica, a symbiont of aphids that enhances resistance to parasitoids. FISH experiments confirmed
that most symbionts were housed within cells of the adelgid's bacteriome. Exceptions were found in A. tsugae: one gamma symbiont was in the haemocoel and two beta symbionts were found only in salivary gland. The Buchnera-like symbiont was found in both salivary glands and bacteriomes in early instars. To our knowledge this is the first report of sternorrhynchan endosymbionts in salivary glands. We also found population-and stage-specific variation in symbionts of A. tsugae. In western U.S. (WNA) samples, the Serratia-type is absent, but was present in eastern U.S. (ENA) populations and in some southern Honshu populations. Sequence comparisons suggested that Niryo, Osaka was the source population for HWA in ENA. In support of this, the Burkholderia beta symbiont was present in several Japanese populations, but absent in Niryo, ENA, and WNA. Both beta symbionts were found only in the 1st-instar/crawler stages of some Japanese populations. Changes in symbiont composition can affect host
fitness. The Serratia symbiont of aphids can be transferred horizontally between hosts; possibly a novel infection of the Serratia-type symbiont in the source population facilitated the establishment of HWA in the US.
Publications
- Havill, N.P., R.G. Foottit, & C.D. von Dohlen 2007. History of life cycle evolution and host plant associations of the Adelgidae (Insecta: Hemiptera) inferred from molecular phylogenetics. Molecular Phylogenetics and Evolution 44:357-370.
- Gruwell, M.E., C.D. von Dohlen, K. Patch, and B.B. Normark 2005. Preliminary PCR survey of bacteria associated with scale insects (Hemiptera: Coccoidea). L. Erkili, M. B. Kaydan (Eds.), Proceedings of the Tenth International Symposium on Scale Insect Studies, Adana, Turkey. 19-23 April.
- Kistie B. Patch 2003. Identification and Molecular Characterization of the Bacterial Endosymbionts of Adelgidae (Hemiptera: Aphidoidea). M.S. Thesis.
- von Dohlen, C.D., Teulon, D.A.J. 2003. Phylogeny and historical biogeography of New Zealand indigenous Aphidini aphids (Hemiptera: Aphididae): A hypothesis. Annals of the Entomological Society of America 96:107-116.
- Spaulding, A.W., von Dohlen, C.D. 2001. Psyllid endosymbionts exhibit patterns of co-speciation with hosts and destabilizing substitutions in ribosomal RNA. Insect Molecular Biology 10(1):57-67.
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Progress 01/01/06 to 12/31/06
Outputs
Our project focus has been characterizing and localizing the bacterial endosymbionts of Adelges tsugae, the woolly hemlock adelgid. This pest has decimated eastern hemlock (Tsuga canadensis and Tsuga caroliniana) stands in the eastern United States, but does not cause a problem in the western U.S. on T. heterophylla and T. mertensiana. Our work in 2004 and 2005 on samples from eastern U.S., western U.S. and Japan suggested that this species has up to five endosymbionts, from both gamma-Proteobacteria and beta-Proteobacteria subdivisions. Small subunit ribosomal RNA genes (16S rRNA) from endosymbionts were PCR-amplified and sequenced, and then localized to thin sections of the insect with in situ hybridization using fluorescent, specific probes (FISH). Three endosymbionts were found to be members of the gamma-Proteobacteria subdivision: gamma-A, gamma-B, and gamma-C. Two endosymbionts were members of the beta-Proteobacteria: beta-A and beta-B. FISH experiments with
probes specific to each of the five bacteria confirmed the locations of most of the putative endosymbionts. The locations of beta-Proteobacteria were less clear, and our work in 2006 concentrated on clarifying the anatomical positions, as well as geographical extent, of these endosymbionts, among other tasks. Several new samples from Japan were obtained and screened with PCR for symbionts. These results determined that gamma-B is found only in Eastern North American (ENA) populations and in some southern Honshu populations on Tsuga sieboldii. This suggested that the source for the ENA infection lies in Southern Honshu. Several PCRs were done with various primers and different protocols to test for the presence of beta-A in WNA and ENA populations. None yielded any positive results. BetaA could thus be absent in ENA and WNA populations. Previous years' FISH results suggested the presence of Gamma-C, Beta-A and Beta-B in the salivary glands of T.sieboldii populations from Southern
Honshu. We performed several FISH experiments on different stages (crawlers through adult), where available, from different populations except Chinese and Taiwanese. Early stages from several populations showed the presence of Gamma-C in both the salivary glands and the bacteriomes. Later stages showed Gamma-C only in the bacteriomes in all populations. Beta-A probing occurred in the salivary glands and only in the youngest stages of only T. sieboldii populations. We also have evidence of Gamma-A and Gamma-C in the eggs of T. diversifolia populations. Beta-B probing yielded positive results only in the1st instar stage of T. sieboldii populations. No probing anywhere was observed in other stages or other populations.
Impacts
Sternorrhynchan insects comprise many of the most destructive agricultural pests. Because bacterial endosymbionts appear essential to host nutrition, and may be manipulated more easily than their eukaryotic hosts, they are potential targets of biological control. In addition, because endosymbionts are involved in host nutrition and parasitoid susceptibility, changes in the complement of endosymbionts or their relative abundances possibly could be correlated with successful establishment of introduced pests. Although our studies are not yet complete, we have evidence so far that the pest populations of Hemlock Wooly Adelgid (HWA) in the eastern US share a type of endosymbiont with populations in Japan from which this pest originated. This endosymbiont is absent in other Japanese populations and those in the western US. The endosymbiont is closely related to the R-type secondary endosymbiont (PASS) of aphids (Aphididae), close relatives of adelgids. This endosymbiont is
known to be transferred horizontally between different species of aphids. Thus, an intriguing possibility is that a novel infection of this endosymbiont in the source Japanese population had something to do with the success (and virulence) of the HWA introduction in the US.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
Our project focus has continued to be characterizing and localizing the bacterial endosymbionts of Adelges tsugae, the wooly hemlock adelgid. This pest has decimated eastern hemlock (Tsuga canadensis and Tsuga caroliniana) stands in the eastern United States, but appears not to be a problem in the western U.S. on T. heterophylla and T. mertensiana. Our work in 2004 on samples from eastern U.S., western U.S. and Japan suggested that this species had two endosymbionts, both gamma-Proteobacteria (gamma-A and gamma-B). However, in situ hybridization experiments with probes specific to these bacteria left large regions of the bacteriome unprobed, suggesting the presence of other bacteria for which we had not amplified sequences. In 2005, we continued to survey clones derived from whole-DNA extracts of acetone-preserved individuals, PCR-amplified with universal, eubacterial 16S ribosomal RNA primers. From this survey we discovered evidence of three additional bacterial
sequences that were parts of chimeric PCR products amplified from one Japanese sample. We designed PCR primers specific to these additional sequences and were able to amplify nearly complete 16S sequences from all. These new bacteria were another gamma-Proteobacteria (gamma-C) and two beta-Proteobacteria (beta-A and beta-B). In situ hybridization experiments with probes specific to each of the five bacteria have confirmed the locations of most of the putative endosymbionts. In eastern U.S. samples, the gamma-C bacteria are the dominant forms in the bacteriome and occupy most of the cells in that organ. Gamma-B cells are less numerous, appear to be larger and less densely packed, and occupy either smaller cells or portions of bacteriocytes that are toward the center of the bacteriome. Gamma-A endosymbionts are numerous and free-living in the hemocoel (mostly near bacteriocytes). In western U.S. samples, gamma-A and gamma-C are in the same locations, but gamma-B is absent. In Japanese
samples, all gamma-Proteobacteria were localized to the same areas as in the eastern U.S. populations. The locations of beta-Proeobacteria are less clear. In Japanese samples, the beta-B form appears to reside in the salivary glands, at least in some stages. We have tentative evidence of the beta-A form in the bacteriome in the first-instar stage of the insect, but cannot detect this form in later stages. In western U.S. samples we have evidence that the beta-B form is also present in the salivary glands. In eastern U.S. populations we have not yet detected either beta-proteobacterial form. However, we believe they are both present because we have performed specific PCR experiments with primers specific to these bacteria, which have yielded positive results.
Impacts
Sternorrhynchan insects comprise many of the most destructive agricultural pests. Because bacterial endosymbionts appear essential to host nutrition, and may be manipulated more easily than their eukaryotic hosts, they are potential targets of biological control. In addition, because endosymbionts are involved in host nutrition and parasitoid susceptibility, changes in the complement of endosymbionts or their relative abundances possibly could be correlated with successful establishment of introduced pests. Although our studies are not yet complete, we have evidence so far that the pest populations of Hemlock Wooly Adelgid (HWA) in the eastern US share a type of endosymbiont with populations in Japan from which this pest originated. This endosymbiont is absent in other Japanese populations and those in the western US. The endosymbiont is closely related to the R-type secondary endosymbiont (PASS) of aphids (Aphididae), close relatives of adelgids. This endosymbiont is
known to be transferred horizontally between different species of aphids. Thus, an intriguing possibility is that a novel infection of this endosymbiont in the source Japanese population had something to do with the success (and virulence) of the HWA introduction in the US.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
We have focused much of the work this year on Adelges tsugae, the wooly hemlock adelgid. This introduced pest from Asia has decimated eastern hemlock (Tsuga canadensis and Tsuga caroliniana) stands in the eastern United States, but appears not to be a problem in the western US on T. heterophylla and T. mertensiana. Our previous work on A. tsugae (only one sample from Virginia, USA) suggested that this species had two endosymbionts, but they were both gamma-Proteobacteria. We obtained many more samples from several geographic regions, including several sites on the east coast of the US, northwestern US, China, and Japan. From whole-DNA extracts of acetone-preserved individuals we amplified bacterial 16S ribosomal DNA with universal, eubacterial primers and cloned the products. At least ten clones from each species were sequenced with a single primer to determine the unique variants. All samples from the eastern US yielded sequences of two different gamma-Proteobacteria
(gamma-A and gamma-B) but western US and Asian samples yielded sequences of gamma-A, only). We performed in-situ experiments with fluorescent-labeled, specific oligonucleotide probes on eastern and western US samples. Both gamma-A and gamma-B probes detected bacteria: gamma-B endosymbionts were located inside one type of bacteriocyte, while gamma-A endosymbionts were free-living in the hemocoel (mostly near bacteriocytes). Both probes failed to detect a third form of bacteria in additional bacteriocytes; yet, this third form was detected by a general, eubacterial probe. Recently, we have PCR-amplified a beta-Proteobacteria from one Chinese sample; we believe this may represent the third type of endosymbiont because its 16S ribosomal DNA sequence is similar to beta-proteobacterial sequences amplified from other Adelges species.
Impacts
Sternorrhynchan insects comprise many of the worlds most destructive agricultural pests. Because bacterial endosymbionts appear essential to host nutrition, and may be manipulated more easily than their eukaryotic hosts, they are potential targets of biological control. First, however, basic information on their identity and biology must be gathered. This research is building the foundation of information on which future applied studies may be based.
Publications
- No publications reported this period
|
Progress 01/01/03 to 12/31/03
Outputs
We have continued the work on Adelgidae symbionts including more Adelges species and an additional Pineus species. With the addition of Pineus, our work covers the full generic diversity of the family. From whole DNA extracts of acetone-preserved individuals we amplified bacterial 16S ribosomal DNA with universal, eubacterial primers and cloned the products. At least ten clones from each species were sequenced with a single primer to determine the unique variants. Most Adelges species had at least one beta-proteobacterium and one gamma-proteobacterium (with an occasional presumed contaminant), but Pineus species contained only gamma-Proteobacteria. We will also repeat our PCR experiments with another 16S rDNA primer that should have a more universal amplification. We have prepared 16S rDNA alignments with these full sequences of the unique clones plus other endosymbiont and free-living bacterial sequences. Phylogenetic analysis of this alignment suggests that
beta-proteobacterial sequences represent the primary endosymbionts. Results with in situ hybridization studies on A. cooleyi and A. abietis also indicate that both beta- and gamma-proteobacteria reside inside host bacteriocytes.
Impacts
Sternorrhynchan insects comprise many of the worlds most destructive agricultural pests. Because bacterial endosymbionts appear essential to host nutrition, and may be manipulated more easily than their eukaryotic hosts, they are potential targets of biological control. First, however, basic information on their identity and biology must be gathered. This research is building the foundation of information on which future applied studies may be based.
Publications
- No publications reported this period
|
Progress 01/01/02 to 12/31/02
Outputs
We have continued the work on Adelgidae symbionts including more Adelges species and an additional Pineus species. With the addition of Pineus, our work covers the full generic diversity of the family. From whole DNA extracts of acetone-preserved individuals we amplified bacterial 16S ribosomal DNA with universal, eubacterial primers and cloned the products. At least ten clones from each species were sequenced with a single primer to determine the unique variants. Most Adelges species had at least one beta-proteobacterium and one gamma-proteobacterium (with an occasional presumed contaminant), but Pineus species contained only gamma-Proteobacteria. We will also repeat our PCR experiments with another 16S rDNA primer that should have a more universal amplification. We are in the process of preparing 16S rDNA alignments with these full sequences of the unique clones plus other endosymbiont and free-living bacterial sequences. Phylogenetic analysis of this alignment will
help to determine whether the gamma- or the beta-proteobacterial sequences are the primary endosymbionts. To address that question further, we have also performed in situ hybridization studies on A. cooleyi and A. abietis. Results indicate that both beta- and gamma-proteobacteria reside inside host bacteriocytes.
Impacts
Sternorrhynchan insects comprise many of the world's most destructive agricultural pests. Because bacterial endosymbionts appear essential to host nutrition, and may be manipulated more easily than their eukaryotic hosts, they are potential targets of biological control. First, however, basic information on their identity and biology must be gathered. This research is building the foundation of information on which future applied studies may be based.
Publications
- No publications reported this period
|
Progress 01/01/01 to 12/31/01
Outputs
We have extended the work on Adelgidae symbionts to five additional species, including four more Adelges and one Pineus obtained from a researcher in Canada. With the addition of Pineus, our work covers the full generic diversity of the family. From whole DNA extracts of acetone-preserved individuals we amplified bacterial 16S ribosomal DNA with universal, eubacterial primers and cloned the products. At least ten clones from each species were sequenced with a single primer to determine the unique variants. All species had at least one beta-proteobacterium and one gamma-proteobacterium, with an occasional presumed contaminant. We are in the process of preparing 16S rDNA alignments with these full sequences of the unique clones plus other endosymbiont and free-living bacterial sequences. Phylogenetic analysis of this alignment will help to determine whether the gamma- or the beta-proteobacterial sequences are the primary endosymbionts. To address that question further,
we have also begun in situ hybridization studies on A. cooleyi. Results so far indicate that both beta- and gamma-proteobacteria reside inside host bacteriocytes.
Impacts
Sternorrhynchan insects comprise many of the world's most destructive agricultural pests. Because bacterial endosymbionts appear essential to host nutrition, and may be manipulated more easily than their eukaryotic hosts, they are potential targets of biological control. First, however, basic information on their identity and biology must be gathered. This research is building the foundation of information on which future applied studies may be based.
Publications
- von Dohlen, C. D., S. Kohler, S. T. Alsop, and W. R. McManus. 2001. Mealybug beta-proteobacterial endosymbionts contain gamma-proteobacterial symbionts. Nature 412:433-436.
- Spaulding, A. W. & C. D. von Dohlen. 2001. Psyllid endosymbionts exhibit patterns of co-speciation with hosts and destabilizing substitutions in ribosomal RNA. Insect Molecular Biology 10:57-67.
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Progress 01/01/00 to 12/31/00
Outputs
In this past year of work, we have concentrated on our objective to identify and characterize bacterial endosymbionts from members of the sternorrhynchan family Adelgidae. We collected weekly series of nymphs from the spruce (Picea) phase of Adelges cooleyi (Gillette) in Utah, USA. We also obtained a sample of the hemlock woolly adelgid (Adelges tsugae Annand) from Virginia, USA, and an unidentified Adelges from Michigan, USA. From whole DNA extracts of these species we amplified bacterial 16S ribosomal DNA with universal, eubacterial primers and cloned the products. Unique 16S clonal variants were identified by BLAST searches at the NCBI website. From A. cooleyi, two unique variants were identified, one a member of the beta-Proteobacteria (13 clones; 93% identical to Burkholderia cepacia and relatives) and the other a gamma-Proteobacteria (6 clones; similar to Klebsiella). From A. tsugae, three unique variants were identified, all members of the gamma-Proteobacteria;
one variant was 98% identical to the aphid S endosymbiont (4 clones), the second was 95% identical to Pseudomonas spp. (3 clones), and the third was 90% identical to the tsetse fly S endosymbiont and a psyllid S endosymbiont (1 clone). From the unidentified Michigan Adelges, two unique variants were identified, one a member of the beta-Proteobacteria (11 clones; 94% identical to Burkholderia thailandensis and relatives) and the other a gamma-Proteobacteria (2 clones; 95% identical to several Enterobacteriaceae). We have designed oligonucleotide probes unique to the 16S variants found in A. cooleyi, in preparation for in situ hybridization studies to determine whether these genes localize to the bacteriome organ of the insect.
Impacts
Sternorrhynchan insects comprise many of the world's most destructive agricultural pests. Because bacterial endosymbionts appear essential to host nutrition, and may be manipulated more easily than their eukaryotic hosts, they are potential targets of biological control. First, however, basic information on their identity and biology must be gathered. This research is building the foundation of information on which future applied studies may be based.
Publications
- Spaulding, A. W. & C. D. von Dohlen. 2000. Psyllid endosymbionts exhibit patterns of co-speciation with hosts and destabilizing substitutions in ribosomal RNA. Insect Molecular Biology 9(6) (in press).
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Progress 01/01/99 to 12/31/99
Outputs
We analyzed bacterial clones of putative endosymbionts from psyllids (Hemiptera:Psylloidea) and whiteflies (Hemiptera:Aleyrodoidea) to investigate the following questions: 1) does the sister relationship between primary (P) endosymbionts of psyllids and whiteflies (Aleyrodoidea) hold with the addition of new taxa? 2) Does the phylogeny of psyllid P endosymbionts indicate co-speciation with their insect hosts? 3) Do secondary (S) endosymboints of most psyllids exhibit AT-rich, highly substituted rRNA sequences? 4) Do the AT-rich, highly substituted rRNA sequences of all psyllid P endosymbionts show evidence of less stable secondary structure? We characterized 16S rRNA in endosymbionts of five new psyllid species in four genera, in addition to published sequences from four other species. Under maximum-likelihood analysis with a parameter-rich model, the sister relationship between psyllid and whitefly P endosymbionts is still only moderately supported. This is likely
due to the challenge of modeling substitution processes in taxa with such a wide range of AT content and substitution rates. The phylogeny of pyllid P endosymbionts shows major points of congruence with the published host phylogeny (from morphology), though the degree of variation in rRNA sequences may be too low to reconstruct all relationships with confidence. AT-rich, highly substituted rRNA sequences of S endosymbionts were found in four psyllid species, suggesting they may represent relatively old associations. Compared to the published findings on aphid P endosymbionts, the rRNA sequences of psyllid P endosymbionts have much lower free-energy estimates of secondary structure, indicating that their AT-rich ribosomal RNAs have become less stable during endosymbiosis.
Impacts
Sternorrhynchan insects comprise many of the world's most destructive agricultural pests. Because bacterial endosymbionts appear essential to host nutrition, and may be manipulated more easily than their eukaryotic hosts, they are potential targets of biological control. First, however, basic information on their identity and biology must be gathered. This research is building the foundation of information on which future, applied studies may be based.
Publications
- No publications reported this period
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Progress 01/01/98 to 12/31/98
Outputs
We investigated the diversity of primary and secondary endosymbionts in additional Psylloidea taxa, and in an univestigated whitefly subfamily, Aleurodicinae. Using PCR amplification of bacterial 16S ribosomal DNA, cloning, and sequencing, we have characterized symbionts in five new psyllid species in four genera, representing four major clades in a published morphological psyllid phylogeny. For each host species we analyzed 5-11 clones by sequencing 400-600 base pairs from the 5' end. Searches for sequence similarity in genetic databases (GenBank, Ribosomal Database) indicated that we had cloned a primary (P) endosymbiont for each psyllid species and a secondary (S) symbiont for most host species. Three species of the genus Pachypsylla apparently harbored no S endosymbionts, a condition that may be related to their galling habit. Full-length sequences were obtained for one clone each of P and S endosymbionts (where found) in each host taxon. P endosymbiont sequences
in the new host taxa were A+T rich and highly substituted as we had found in taxa previously investigated. S endosymbiont sequences appeared to represent independent bacterial lineages in the gamma subdivision of Proteobacteria. In the whitefly, we found clones similar to the published report of Aleyrodinae P endosymbionts and also clones of another, unrelated gamma Proteobacteria, possibly an S endosymbiont. The new sequences will be included in an alignment and phylogenetic analyses to test the hypotheses that P endosymbionts have co-speciated with their hosts, that S endosymbionts represent independent infections, and that psyllid and whitefly P endosymbionts share a most recent common ancestor.
Impacts
(N/A)
Publications
- Spaulding, A. W. & C. D. von Dohlen. 1998. Phylogenetic characterization and molecular evolution of bacterial endosymbionts in psyllids (Hemiptera: Sternorrhyncha). Molecular Biology and Evolution 15:1506-1513.
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Progress 01/01/97 to 12/31/97
Outputs
We have characterized bacterial endosymbionts in Psylloidea and Adelgidae through PCR amplification, cloning, and sequencing of bacterial ribosomal (16S) genes. 16S genes were amplified from four psyllid species. Two distinct clones were found in two species and one distinct clone was found in two other species. Primary clones from three different species showed high A+T bias (approx. 64%); secondary clones from two of the same species and the single clone from the fourth species showed A+T content similar to free-living bacteria (approx. 45%). The highly A+T-biased clones also exhibited 3X-4X greater relative substitution rates compared to free-living relatives. These base-compositional biases and substitution rates are substantially greater than those reported for other insect endosymbionts. In phylogenetic analyses using parsimony, distance, or likelihood methods, all sequences were placed with high bootstrap support within the gamma subdivision of Proteobacteria,
along with aphid, whitefly, and other insect endosymbionts. Non-A+T-biased clones usually clustered with weevil symbionts and the secondary tsetse fly symbiont. The position of highly A+T-biased sequences within the gamma subdivision was difficult to determine using parsimony methods, probably because of their base-compositional biases and elevated substitution rates. Results using distance and likelihood methods were also conflicting, but a maximum-likelihood analysis using the GTR+gamma+ invariant model (chosen through evaluation of several models) placed these divergent sequences in a clade with the primary endosymbionts of whiteflies, with high bootstrap support. The high A+T bias and elevated substitution rates in three clones suggest a longer intracellular history and we tentatively suggest that these may belong to primary, bacteriocyte-dwelling endosymbionts. Sequences that were more similar to free-living bacteria may represent more recently acquired, secondary or accessory
endosymbionts. We have also obtained electron micrographs of the bacteriome-associated endosymbionts, which appear to be round or oval shaped. Bacterial products amplified from two adelgid species were 97-99.3% similar to several Pseudomonas and Burkholderia species that are commonly found in the insect environment and therefore may have been derived from contaminants, not endosymbionts.
Impacts
(N/A)
Publications
- No publications reported this period
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