INSECTÐSYMBIONT INTERACTIONS Two Species of Symbiotic Present in the Soybean (Hemiptera: Aphididae)

1 2 B. D. WILLE AND G. L. HARTMAN

Environ. Entomol. 38(1): 110Ð115 (2009) ABSTRACT , which feed solely on plant phloem sap, have developed symbiotic associations with bacteria that provide them with the amino acids that are lacking in phloem. Three soybean aphid (Aphis glycines Mat samura) populations were screened for the presence of Buchnera aphidicola and three common species of secondary aphid symbionts (Serratia symbiotica, , and Regiella insecticola). Diagnostic polymerase chain reaction and subsequent DNA sequencing showed the presence of two species of symbiotic bacteria present in all three soybean aphid populations tested: B. aphidicola and Arsenophonus sp. Although Buchnera is commonly found in aphids, Arsenophonus is most commonly found in whiteßies (Hemiptera: Aleyrodidae), making the soybean aphid unique among aphids that have been tested for the presence of Arsenophonus.

KEY WORDS soybean aphid, bacterial symbiont, Buchnera aphidicola, Arsenophonus

Plant phloem sap is used as a sole food source by In addition to Buchnera, many aphids harbor addi- insects in the order Hemiptera (Douglas 2006). tional bacterial symbionts, known as secondary sym- Aphids (Hemiptera: Aphidoidea) need essential bionts. Unlike Buchnera, secondary symbionts are not amino acids for growth and fecundity but are only able found in all aphid species and are thought to undergo to synthesize 9 of the 20 essential amino acids. The low horizontal as well as vertical transmission (Moran and ratio of essential:nonessential amino acids in phloem Telang 1998, Gil et al. 2002, Russell et al. 2003). There is insufÞcient to support the growth rate observed in are three main species of secondary symbionts found aphids. To survive on an amino acidÐdeÞcient diet, in aphids: Serratia symbiotica (R-type), Hamiltonella nearly all aphids rely on an obligate symbiotic rela- defensa (T-type), and Regiella insecticola (U-type) tionship with bacteria in the genus Buchnera, which (Moran et al. 2005). In addition, Rickettsia, Spiro- synthesize essential amino acids that are lacking in plasma, and Wohlbachia species have also been found phloem (Douglas 1998, Russell et al. 2003). in aphids (Gomez-Valero et al. 2004, Sakurai et al. Buchnera are maternally transmitted ␥-proteobac- 2005). teria housed along the aphid digestive tract in spe- The soybean aphid, Aphis glycines Mat samura cialized cells called bacteriocytes. Phylogenetic anal- (Hemiptera: Aphididae), was Þrst introduced to the ysis of the relationship between aphids and Buchnera United States in the summer of 2000, where it has since shows an initial infection followed by strict vertical become an economically important pest of soybean, transmission dating back 150Ð250 million years (Mo- Glycine max L. Merr. (Hartman et al. 2001, Venette ran et al. 1993). The coevolutionary process between and Ragsdale 2004). In this paper, two species of sym- aphids and Buchnera has lead to the deletion of many biotic bacteria, B. aphidicola and Arsenophonus sp. (a of the genes necessary for Buchnera to survive outside genus closely related to Hamiltonella), present in the of its host and to the ampliÞcation of genes required soybean aphid are described. Partial 16S rDNA se- to synthesize amino acids lacking in the aphid diet (Gil quences were obtained for both species of bacteria, et al. 2002). and BlastN was used to determine the similarity be- Buchnera are considered to be the primary symbiont tween symbionts of the soybean aphid and other present in aphids, making up Ͼ90% of the symbiotic known aphid symbionts. This is the Þrst report of the bacteria present in bacteriocytes (Haynes et al. 2003). presence of bacterial symbionts in the soybean aphid.

Trade and manufacturersÕ names are necessary to report factually on available data; however, the USDA neither guarantees nor war- Materials and Methods rants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may Aphids and DNA Preparation. Two clones of A. also be suitable. glycines from Champaign and Brown Counties in Il- 1 Department of Entomology, University of Maryland, College linois were initially collected and maintained by Curt Park, MD 20742. Hill (University of Illinois, Urbana, IL). The clones 2 Corresponding author: USDA-ARS and Department of Crop Sci- ences, National Soybean Research Center, University of Illinois, Ur- were established from single Þrst-instar nymphs bana, IL 61801 (e-mail: [email protected]). reared on a continuous supply of Williams 82 soybean February 2009 WILLE AND HARTMAN:SYMBIOTIC BACTERIA IN SOYBEAN APHID 111

fragment, spanning the 16S and 23S rDNA genes, along with the intergenic region, yields a product of char- acteristic length for each of the three secondary sym- bionts. All PCR reactions were performed in 25-␮l vol- umes with 1ϫ polymerase chain reaction (PCR) buffer (Eppendorf/Qiagen, Qiagen Inc., Valencia,

CA), 2 mM Mg(OAc)2 (Eppendorf/Qiagen), 0.2 mM of each dNTP, 0.5 pmol/ml of each primer, 0.5 ng/ml genomic DNA, and 2 U of TaqDNA polymerase (Ep- pendorf/Qiagen). To prevent false-positive or -nega- tive results, positive control reactions consisting of Fig. 1. Diagnostic PCR analysis of soybean aphid sym- genomic DNA from A. pisum clones known to harbor bionts. Results for all three soybean aphid clones tested were the relative symbiont (obtained from Nancy Moran, the same. Data shown are from the Urbana, IL, clone. Lane University of Arizona) and negative controls consist- 1 contains a 1-kb DNA ladder, lanes 2Ð4 contain products ing of water instead of DNA were used in all PCR from BS16S and 16S4, lanes 5Ð7 contain products from R1279 reactions. The running conditions for all PCR reac- F and 480R, lanes 8Ð10 contain products from T1279 F and tions were 94ЊC for 3 min, followed by 30 cycles of 480R, and lanes 11Ð13 contain products from U1279 F and Њ Њ Њ 480R. Lanes 2, 5, 8, and 11 contained no DNA (negative 94 C for 15 s, 60 C for 30 s, and 72 C for 1 min. PCR control), lanes 3, 6, 9, and 12 contained soybean aphid DNA, products were separated on an 0.8% agarose gel for 2 h and lanes 4, 7, 10, and 13 contained DNA from A. pisum at 125 V, stained with ethidium bromide, and visual- known to harbor the relative symbiont. ized with UV light. Diagnostic PCR testing was done one time per month for 8 consecutive mo for the two Illinois aphid plants in plant growth chambers, with each population clones. The Ohio soybean aphid clone was subjected maintained in a separate growth chamber. The growth Њ to diagnostic PCR testing three times. chambers were kept at 22 C, 70% RH, and continuous Sequencing and Phylogenetic Analysis of Symbiont 300 ␮mol/m2/s PAR radiation. In addition to the two 16S rDNA. Samples that yielded a distinct band in Illinois soybean aphid clones, a soybean aphid popu- the diagnostic screening tests were puriÞed using a lation from Ohio (Kim et al. 2008) was established by QIAquick PCR puriÞcation kit (Qiagen) and se- placing a soybean leaf with multiple aphids into a pot quenced directly. Sequencing reactions were per- containing four Williams 82 soybean plants in a growth formed at the University of Illinois W.M. Keck Center chamber as previously described. To simplify data for Comparative and Functional Genomics. BlastN reporting, the Ohio soybean aphid population will be described as a clone in this paper. was used to compare the sequences of symbiont 16S Total aphid DNA was extracted using the CTAB/Na rDNA from A. glycines to other known aphid symbi- method as previously described (Keim and Shoe- onts in GenBank. Samples were considered positive maker 1988). Each DNA sample was made from a for a speciÞc symbiont only if the diagnostic PCR random sample of 20 individual apterous adults from reaction yielded a band of the expected length and if each aphid clone. Total aphid DNA samples from four the sequenced 16S rDNA was at least 95% similar to A. pisum clones known to harbor Buchnera, S. symbi- entries for the relevant symbiont using BlastN. Sam- otica, H. defensa, and R. insecticola were obtained from ples were considered to be negative for a speciÞc Nancy Moran (University of Arizona) to use as pos- symbiont if they did not amplify with the diagnostic itive controls. primers, yielded a PCR product of the incorrect Ͻ Diagnostic 16S rDNA Amplification. To detect the length, or if the partial 16S rDNA sequence was 95% presence of Buchnera, primers BS16S and 16S4 were similar to other previously described symbionts. used as previously described (Nakabachi and Ish- CLUSTAL W (Thompson et al. 1994) was used to ikawa 1999). These primers were designed to spe- align the experimental sequences with the sequences ciÞcally amplify Buchnera 16S rDNA, yielding a obtained from the BlastN search. Bacterial species 984-bp product. and accession numbers used in this study are as The presence of secondary symbionts was deter- follows: Arsenophonus sp. isolated from Dialeurodes mined with a forward diagnostic primer (R1279, hongkongensis (AY264667.1), Glycaspis brimblecombei T1279, and U1279 for S. symbiotica, H. defensa, and R. (AF263561.1), Pentastiridius sp. (DQ834351.1), and insecticola, respectively) and a universal reverse Triatoma rubrofasciata (DQ508185.1); B. aphidicola primer (480R) as previously described (Russell et al. isolated from Acyrthosiphon pisum (AB033776.1), 2003). In secondary symbionts, the 16S rDNA and 23S Aphis fabae fabae (AY518294.1), Myzus persicae rDNA genes were separated by an intergenic region of (AY849937.1), Pterocomma populeum (AJ296747.1), varying length (Fig. 1). The universal reverse primer Rhopalosiphum maidis (M63247.1), Rhopalosiphum binds to a region in the 23S rDNA gene that is con- padi (M63246.1), Schizaphis graminum (L18927.1), served across all three secondary symbiont species. and Uroleucon sonchi (M63250.1); E. coli (V00331); The diagnostic forward primers bind to species-spe- and Hamiltonella defensa isolated from A. pisum ciÞc regions in the 16S rDNA gene. The resulting (AY296733.1). 112 ENVIRONMENTAL ENTOMOLOGY Vol. 38, no. 1

0.01045 0.00121 BuchBMp1 0.01185 0.00208 BuchBUs 0.0089 0.00364 BuchBAp1 0.0112 BuchBPp 0.00686 BuchIL2 0.022882151 0.01288 0.00903 BuchIL1 0.00682 BuchOH 5.4E-4 0.00317 BuchBAf 0.00818 3.5E-4 BuchBRp 0.00134 0.00552 BuchBSg2 0.00335 BuchBRm E.coli7

Scale: 0.01 Fig. 2. Neighbor-joining phylogenetic tree for B. aphidicola found in three clones of the soybean aphid (BuchIL1, BuchIL2, and BuchOH). Strains and accession numbers used are as follows: B. aphidicola isolated from Acyrthosiphon pisum (BuchAp1, AB033776.1), Aphis fabae fabae (BuchAf, AY518294.1), Myzus persicae (BuchMp1, AY849937.1), Pterocomma populeum (BuchPp, AJ296747.1), Rhopalosiphum maidis (BuchRm, M63247.1), Rhopalosiphum padi (BuchRp, M63246.1), Schizaphis graminum (BuchSg2, L18927.1), Uroleucon sonchi (BuchUs, M63250.1), and E. coli (V00331).

Phylip version 3.6 (Felsenstein 1989) was used to results further conÞrmed the absence of H. defensa in create distance matrix calculations using the Kimura the soybean aphid clones, because BlastN searches two-parameter method. These calculations were used with the partial 16S rDNA sequences obtained from to create neighbor-joining trees for the endosymbiotic the diagnostic PCR reactions were Ͻ95% similar to bacteria detected in the soybean aphid, with E. coli known H. defensa 16S rDNA sequences. Instead, used as an outgroup for each tree. BlastN searches indicated that a different ␥-pro- teobacterium, Arsenophonus, was present in the soy- bean aphid clones tested. Phylogenetic analysis placed Results the secondary symbiont found in the soybean aphid Diagnostic PCR and Sequencing of Symbiont 16S closest to Arsenophonus found in Glycaspis brimble- rDNA. AmpliÞcation of Buchnera 16S rDNA with the combei (Fig. 3). Sequencing results were submitted to speciÞc diagnostic primers BS16S and 16S4 yielded a GenBank. product of Ϸ985 bp in the three aphid clones (Fig. 1). The two Illinois soybean aphid clones were tested Subsequent puriÞcation and sequencing of the PCR for the presence of Buchnera, S. symbiotica, H. defensa, products (submitted to GenBank) conÞrmed the pres- and R. insecticola monthly over the course of 8 mo. The ence of Buchnera in the three soybean aphid clones. species of bacteria present in each aphid clone did not The partial Buchnera 16S rDNA sequences of the soy- change throughout the testing period, providing evi- bean aphid clones tested were at least 98% similar to dence that the 16S rDNA sequences obtained were each other, and BlastN searches conÞrmed that the not the result of bacterial contamination, as could be obtained sequences were at least 95% similar to Buch- the case if a clone was found to gain a new symbiont nera 16S rDNA sequences from other aphid species. during the test period. Likewise, the Ohio soybean The neighbor-joining phylogenetic tree showed that aphid clone was tested for the presence of symbiotic the Buchnera strains found in the soybean aphid are bacteria three times. Results for all three tests were the most closely related to Buchnera found in A. fabae same. fabae (Fig. 2). AmpliÞcation of S. symbiotica secondary symbiont Discussion 16S rDNA with the diagnostic forward primer R1279 F and the universal reverse primer 480R did not yield Many aphid species are known to harbor multiple a product in the soybean aphid populations tested bacterial symbionts, with most studies focusing on the (Fig. 1), indicating the absence of S. symbiotica. Di- symbiosis between aphids, Buchnera, and the second- agnostic PCR tests for R. insecticola using the primers ary symbionts S. symbiotica, H. defensa, and R. insec- U1279 F and 480R did not yield a product in any of the ticola. In addition to the aforementioned species, aphid clones tested (Fig. 1), indicating the absence of Wohlbachia, Spiroplasma, Erwinia, and Staphylococcus R. insecticola. spp. have been reported in C. cedri and A. pisum PCR tests using the diagnostic primers T1279 F and (Haynes et al. 2003, Gomez-Valero et al. 2004). These 480R yielded a product of Ϸ1,000 bp for the three additional species of bacteria are considered to be soybean aphid clones (Fig. 1). This product was longer facultative, because they are found in insect orders than the PCR product obtained in the positive control other than Hemiptera and in some instances decrease reaction using A. pisum genomic DNA known to con- host Þtness. In total, secondary symbionts of 10 sep- tain H. defensa, indicating that the bacterium detected arate bacterial groups have been discovered in aphids, was not H. defensa. Subsequent sequencing and BlastN and it is possible that there are additional undiscov- February 2009 WILLE AND HARTMAN:SYMBIOTIC BACTERIA IN SOYBEAN APHID 113

1.41332 0.58999 ArsIL2 1.94858 ArsOH 0.76362 0.93447 0.53885 ArsDh 0.54495 0.82066 1.62363 0.36985087 ArsGb 2.25805 ArsIL1 1.97553 0.46208 ArsP1 0.98207 ArsTr 1.50325 HamAp1 1.7382991 E. coli

Scale: 0.1 Fig. 3. Neighbor-joining phylogenetic tree for Arsenophonus sp. found in three clones of the soybean aphid (ArsIL1, ArsIL2, and ArsOH). Strains and accession numbers used are as follows: Arsenophonus sp. isolated from Dialeurodes hongkongensis (ArsDH, AY264667.1), Glycaspis brimblecombei (ArsGp, AF263561.1), Pentastiridius sp. (ArsP1, DQ834351.1), and Triatoma rubrofasciata (ArsTr, DQ508185.1); E. coli (V00331) and Hamiltonella defensa isolated from A. pisum (HamAp1, AY296733.1). ered associations between aphids and bacterial sym- that the Buchnera in soybean aphids would be most bionts (Russell et al. 2003). closely related to Buchnera in other members of the This study determined that two species of symbiotic Aphidini tribe. bacteria are present in soybean aphids: Buchnera and The diagnostic primers used in this study to detect Arsenophonus. Evidence presented in this study H. defensa (Russell et al. 2003) yielded a PCR product strongly suggests that the partial 16S rDNA sequences that, when sequenced, corresponded to Arsenophonus obtained from soybean aphid total genomic DNA are instead of H. defensa. Arsenophonus is a ␥-proteobac- from symbiotic bacteria and not from bacterial con- terium known to establish secondary symbiotic rela- tamination. The three soybean aphid populations tionships with whiteßies, psyllids, and aphids in the tested were reared in isolated growth chambers over tribe Aphidini (Thao and Baumann 2004, Dale et al. many generations, and the two bacteria species were 2006). Blood-feeding Diptera from Streblidae (bat found to be consistently present in the aphid clones ßies) and Hippoboscidae (louse ßies), which rely on throughout the study. Aphid clones did not acquire or symbiotic bacteria to supplement an amino acidÐde- lose symbionts, as would be expected if the bacteria Þcient diet, also harbor Arsenophonus (Trowbridge et species reported were contaminants. In addition, di- al. 2006). Unlike the primary symbionts of aphids, agnostic PCR reactions showed very strong bands for Arsenophonus has been cultured in insect cell lines both Buchnera and Arsenophonus. If either of these (Dale et al. 2006). bacteria was a contaminant instead of a symbiont, it The16S rDNA and 23S rDNA genes of Arsenophonus would not likely be present in very large quantities and and H. defensa are separated by an intergenic spacer. therefore would not amplify strongly in diagnostic Primers used to detect H. defensa in this study in- testing. Finally, the reported partial 16S rDNA se- cluded a universal reverse primer, which binds to a quences obtained from the soybean aphid clones region of the 23S rDNA, and a diagnostic forward tested were similar (Ͼ95%) to other known symbi- primer, which binds to a region in the 16S rDNA. The onts. The 16S rDNA sequences of bacteria are very resulting PCR fragment, spanning the intergenic highly conserved, and it is therefore unlikely that the spacer, yields a product of Ϸ900 bp. This PCR product reported sequences do not correspond to Buchnera or length is considered to be diagnostic for H. defensa, Arsenophonus. and previous aphid symbiont studies have discarded The Hemipteran subfamily Aphidinae, which in- PCR products that were not of the correct length cludes aphids, is composed of multiple tribes. The (Sandstrom et al. 2001, Russell et al. 2003). When soybean aphid is a member of the Aphidini tribe, soybean aphid populations were tested for the pres- which contains a number of important agricultural ence of H. defensa, the resulting PCR product was pests. The partial Buchnera 16S rDNA sequences ob- Ϸ1,000 bp long, indicating that the bacterial 16S rDNA tained from soybean aphids in this study were closest being ampliÞed was not that of H. defensa, because the to the Buchnera 16S rDNA sequences in A. fabae fabae, 16S rDNA and intergenic spacer of this species are R. maidis, and S. graminum, all of which belong to the highly conserved. Sequencing of the PCR product Aphidini tribe. Soybean aphid 16S rDNA Buchnera instead indicated the presence of a different insect sequences were less consistent with the Buchnera spe- symbiont: Arsenophonus. cies present in the largest aphid tribe, Macrosiphini. Aphid symbiosis with bacteria from the genus Ar- This tribe includes A. pisum and Uroleucon species, senophonus has not been widely reported in the past. which has been the subject of most of the work on Tsuchida et al. (2002) tested 858 aphids from 43 Jap- aphid symbionts. Buchnera are known to have an an- anese locations and did not Þnd Arsenophonus. Aphis cient relationship with aphids (Moran et al. 1993), spiraecola, Wahlgreniella nervata, and Myzocallis spp. during which they have been transmitted vertically are known to contain Arsenophonus (Russell et al. through their aphid hosts. It therefore seems logical 2003, Dale et al. 2006). Previous restriction analysis 114 ENVIRONMENTAL ENTOMOLOGY Vol. 38, no. 1 studies have detected the presence of aphid secondary References Cited symbionts of unknown identiÞcation, and it is possible Broderick, N., K. Raffa, and J. Handelsman. 2006. Midgut that the unknown bacteria detected in these studies bacteria required for Bacillus thuringiensis insecticidal could be Arsenophonus (Sandstrom et al. 2001, Russell activity. Proc. Natl. Acad. Sci. U.S.A. 103: 15196Ð15199. et al. 2003). In these cases, diagnostic PCR fragments Dale, C., M. Beeton, C. Harbison, T. Jones, and M. Pontes. that yielded incorrect product lengths were discarded 2006. Isolations, pure culture, and characterization of without sequencing the product. Because of the ge- “Candidatus Arsenophonus arthropodicus,” an intracellu- netic similarity between Arsenophonus and H. defensa, lar secondary from the Hippoboscid louse it is possible that a diagnostic PCR reaction meant to ßy Pseudolynchia canariensis. Appl. Environ. Microbiol. detect H. defensa would also amplify the 16S rDNA 72: 2997Ð3004. gene of Arsenophonus. The intergenic spacer of these Douglas, A. E. 1998. Nutritional interactions in insect-mi- crobial symbioses: aphids and their symbiotic bacteria two species is different in length, so the aphid would Buchnera. Annu. Rev. Entomol. 43: 17Ð37. be determined to be negative for the presence H. Douglas, A. E. 2006. Phloem-sap feeding by animals: prob- defensa, and the PCR product would not be se- lems and solutions. J. Exp. Bot. 57: 747Ð754. quenced. This study suggests that Arsenophonus may Felsenstein, J. 1989. PHYLIP: phylogeny inference pack- be present in more aphids than previously thought and age. Clad 5: 164Ð166. that current diagnostic PCR screening methods for Gil, R., B. Sabater, A. Latorre, F. J. Silva, and A. Moya. 2002. aphid symbionts may be discarding essential data. Extreme genome reduction in Buchnera spp.: toward the The soybean aphid is an economically important minimal genome needed for symbiotic life. Proc. Nat. Acad. Sci. U.S.A. 99: 4454Ð4458. pest of soybeans, and the discovery of two types of Gomez-Valero, L., M. Soriano-Navarro, V. Perez-Brocal, A. symbiotic bacteria, Buchnera and Arsenophonus, in Heddi, A. Moya, J. M. Garcia-Verduga, and A. Latorre. this aphid species could aid in the development of 2004. Coexistence of Wohlbachia with Buchnera aphidi- future control strategies. Because this is the Þrst cola and a secondary symbiont in the aphid Cinara cedri. report of symbionts in the soybean aphid, little is J. Bacteriol. 186: 6626Ð6633. known about their potential role in the insectÕs Hartman, G. L., L. L. Domier, L. M. Wax, C. G. Helm, D. W. biology. Arsenophonus has been cultured outside of Onstad, J. T. Shaw, L. F. Solter, D. J. Voegtlin, C.J.D’Arcy, its host in insect cells lines, allowing recombinant M. E. Gray, K. L. Steffey, S. A. Isard, and P. L. Orwick. DNA techniques to manipulate its genome (Dale et 2001. Occurrence and distribution of Aphis glycines on soybeans in Illinois in 2000 and its potential control. Plant al. 2006). Secondary symbionts are known to carry Health Progr. DOI: 10.1094/PHP-2001Ð0205-01-HN. genes that protect their aphid host from heat stress Haynes, S., A. C. Darby, T. J. Daniell, G. Webster, F.J.F. van or , so the potential ability to manipulate Veen, H.C.J. Godfray, J. I. Prosser, and A. E. Douglas. the Arsenophonus symbiont of the soybean aphid in 2003. Diversity of bacteria associated with natural aphid the laboratory could lead to innovative future con- populations. Appl. Environ. Microbiol. 69: 7216Ð7223. trol strategies. In addition, recently discovered soy- Hill, C. B., L. Yan, and G. L. Hartman. 2006. A single dom- bean aphid resistance genes to the soybean aphid inant gene for resistance to the soybean aphid in the (Hill et al. 2006) and aphid biotypes that overcome soybean cultivar Dowling. Crop Sci. 46: 1601Ð1605. this resistance (Kim et al. 2008) may be caused by Keim, P., and R. C. Shoemaker. 1988. Construction of a random recombinant DNA library that is primarily single some interaction with aphid symbionts. The role of copy sequence. Soybean. Genet. Newsl. 15: 147Ð148. symbionts in the soybean aphid has not been de- Kim, K. S., C. B. Hill, G. L. Hartman, M.A.R. Mian, and B. W. termined, but it has been shown that enteric bac- Diers. 2008. Discovery of soybean aphid biotypes. Crop teria are required for Bacillus thuringeinsis insecti- Sci. 48: 923Ð928. cidal activity (Broderick et al. 2006). More research Moran, N. A., and A. Telang. 1998. Bacteriocyte-associated on the soybean aphidÐsymbiont interactions may in insects. Bioscience 48: 295Ð304. show more about how symbionts inßuence aphid Moran, N. A., M. A. Munson, P. Baumann, and H. Ishikawa. behavior. 1993. A molecular clock in endosymbiotic bacteria is calibrated using the insect hosts. Proc. R. Soc. Lond. Ser. B. 253: 167Ð171. Moran, N. A., J. A. Russell, R. Koga, and T. Fukatsu. 2005. Acknowledgments Evolutionary relationships of three new species of En- terobacteriaceae living as symbionts of aphids and other This research was supported by the Illinois Soybean As- insects. Appl. Environ. Microbiol. 71: 3302Ð3310. sociation, North Central Soybean Research Program, and the Nakabachi, A., and H. Ishikawa. 1999. Provision of riboßavin United Soybean Board. We thank C. B. Hill and J. Hauden- to the host aphid, Acyrthosiphon pisum, by endosymbiotic shield (Department of Crop Sciences, University of Illinois at bacteria, Buchnera. J. Insect. Physiol. 45: 1Ð6. Urbana-Champaign), respectively, for suggestions about Russell, J. A., A. Latorre, B. Sabater-Munoz, A. Moya, and rearing aphid clones and for redrawing of Þgures; Dr. Moran N. A. Moran. 2003. Side-stepping secondary symbionts: (Department of Ecology and Evolutionary Biology, Univer- widespread horizontal transfer across and beyond the sity of Arizona) for aphid DNA samples to use as positive Aphidoida. Mol. Ecol. 12: 1061Ð1075. controls; and Drs. Berenbaum (Department of Entomology, Sakurai, M., R. Koga, T. Tsuchida, X. Y. Meng, and T. Fukatsu. University of Illinois at Urbana-Champaign) and Domier 2005. Rickettsia symbiont in the pea aphid Acyrthosiphon (Department of Crop Sciences, University of Illinois at Ur- pisum: novel cellular tropism, effect on host Þtness, and bana-Champaign) for comments and suggestions for this interaction with the essential symbiont Buchnera. Appl. research. Environ. Microbiol. 71: 4069Ð4075. February 2009 WILLE AND HARTMAN:SYMBIOTIC BACTERIA IN SOYBEAN APHID 115

Sandstrom, J. P., J. A. Russell, J. P. White, and N. A. Moran. and -type bacteria from adult Hippobosci- 2001. Independent origins and horizontal transfer of bac- dae and Streblidae (Hippoboscoidea). J. Invertebr. terial symbionts of aphids. Mol. Ecol. 10: 217Ð228. Pathol. 91: 64Ð68. Thao, M. L., and P. Baumann. 2004. Evidence for multiple Tsuchida, T., R. Koga, H. Shibao, T. Matsumoto, and T. acquisition of Arsenophonus by whiteßy species (Sternor- Fukatsu. 2002. Diversity and geographic distribution of rhyncha:Aleyrodidae). Curr. Microbiol. 48: 140Ð144. secondary endosymbiotic bacteria in natural populations Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. of the pea aphid, Acyrthosiphon pisum. Mol. Ecol. 11: CLUSTAL W: improving the sensitivity of progressive 2123Ð2135. multiple sequence alignments through sequence weight- Venette, R. C., and D. W. Ragsdale. 2004. Assessing the ing, position speciÞc gap penalties and weight matrix invasion by soybean aphid (Homoptera: Aphididae): choice. Nucleic Acids Res. 22: 4693Ð4680. where will it end?. Ann. Entomol. Soc. Am. 97: 219Ð226. Trowbridge, R. E., K. Dittmar, and M. F. Whiting. 2006. IdentiÞcation and phylogenetic analysis of Arsenophonus- Received 3 July 2008; accepted 29 October 2008.