Identification and Location of Symbionts Associated with Potato Psyllid () Lifestages Author(s): Daymon Hail, Scot E. Dowd, and Blake Bextine Source: Environmental Entomology, 41(1):98-107. 2012. Published By: Entomological Society of America DOI: http://dx.doi.org/10.1603/EN11198 URL: http://www.bioone.org/doi/full/10.1603/EN11198

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BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. -SYMBIONT INTERACTIONS Identification And Location Of Symbionts Associated With Potato Psyllid (Bactericera cockerelli) Lifestages

1,2 3 1 DAYMON HAIL, SCOT E. DOWD, AND BLAKE BEXTINE

Environ. Entomol. 41(1): 98Ð107 (2012); DOI: http://dx.doi.org/10.1603/EN11198 ABSTRACT The potato psyllid (Bactericera cockerelli, Sulc) is an invasive pest of solenaceous plants including potatoes (Solanum tuberosum L.)and tomatoes (Solanum lycopersicum L.). The insect transmits the phytopathogen Candidatus Liberibacter solanacearum, which has been identiÞed as the causal agent of Zebra Chip in potatoes. The microbiome of the potato psyllid provides knowledge of the insectÕs bacterial makeup which enables researchers to develop targeted biological control strategies. In this study, the microbes associated with four B. cockerelli life stages were evaluated by 16S bTEFAP pyrosequencing. The sequences were compared with a 16S-rDNA database derived from NCBIÕs GenBank. Some bacteria identiÞed are initial discoveries. Species of Wolbachia, Rhizobium, Gordonia, Mycobacterium, Xanthomonas and others were also detected and an assessment of the microbiome associated with B. cockerelli was established.

KEY WORDS pyrosequencing, psyllids, microbiome, symbionts, massively parallel bacterial tag- encoded FLX-Titanium amplicon pyrosequencing

Psyllids are jumping plant lice (Order: ) of dollars (Secor and Rivera-Varas 2004, CNAS 2006, and approximately forty species (19 genera) are Hernandez-Garcia et al. 2006, Salas-Marina et al. known to be of economic importance. These 2006); in some cases entire Þelds have been aban- are pests of a wide array of plants including pistachio doned (Flores et al. 2004). (Agonoscena targionii, Lichtenstein); avocado (Trioza The U.S. potato industry has suffered from a severe aguacate, Hollis & Martin); pear (Cacopsylla pyricola, loss in plant longevity and crop quality because of Fo¨rster), berry (Trioza tripunctata, Fitch); and many Zebra Chip. B. cockerelli have long been associated ornamentals (Percy 2000). Potato psyllids (Bactericera with the disease (Goolsby et al. 2007, Munyaneza et al. cockerelli) are phloem feeding pests of solanaceous 2007), which was thought to have been caused by a plants, such as potatoes, tomatoes, peppers, and have bacteria, virus, or toxigenic effects of the insectÕs sa- become a major limiting factor in the production of liva. A recent transmission electron microscopy and potatoes in both Central and North America (CNAS 16S rDNA sequencing study by Liefting et al. (2009) 2006). Historically, potato psyllid infestations were has now implicated Candidatus Liberibacter so- transient and sporadic (Eyer and Crawford 1933) but lanacearum as the putative cause of Zebra Chip. As the in recent years have become much more persistent potato psyllid has spread throughout Mexico, the with year-long and multiyear infestations becoming southwestern United States, and northward, insect the norm. related crop losses have followed and are likely to Zebra Chip, an emerging disease of the potato (So- continue. lanum tuberosum L.) (Abad et al. 2009), originally was To counter a diet largely deÞcient in amino acids identiÞed in 1994 in Þelds around Saltillo, Mexico (Sandstro¨m and Moran 1999), phloem-feeding insects where the disease was called “papa manchada” or have adapted by the incorporation of symbiotic bac- stained potato. Above ground symptoms include aerial teria. In exchange for permanent residence in special- tubers and yellowing and curling of leaves. Alternating ized bundles of cells called bacteriomes (Baumann et light and dark bands can be seen in freshly cut tubers al. 1995) bacteria like Candidatus Carsonella ruddii and result from starches being converted to soluble (Thao et al. 2001) supplement missing nutrients. Se- sugars in the medullary rays (Gao et al. 2009). Frying quencing analysis of Candidatus Carsonella ruddii caramelizes the bands and gives the chips a charac- conducted by Nakabachi et al. (2006) identiÞed genes teristic unpalatable taste. These symptoms reduce the related to amino acid biosynthesis, whereas other value of potato crops and result in losses in the millions genes considered essential for life were missing. Wolbachia, a well known insect endosymbiont and 1 University of Texas, Tyler 3900 University Blvd., Tyler, TX 75701- potent inßuencer of sex ratios, has been used as a 6699. genetic marker to differentiate between potato psyl- 2 Corresponding author, e-mail: [email protected]. 3 Research and Testing Laboratory, 4321 Marsha Sharp Fwy. Lub- lids from different locations (Liu et al. 2006). It is bock, TX 79407. probable that more symbionts are exploited by the

0046-225X/12/0098Ð0107$04.00/0 ᭧ 2012 Entomological Society of America February 2012 HAIL ET AL.: 16S BTEFAP IN POTATO PSYLLIDS LIFESTAGES 99 potato psyllid to perform various metabolic activities Table 1. Samples Collected for the 16S Microbiome Study and an in depth knowledge of these interactions could expand current strategies and methods of psyllid pest Potato psyllid samples management. Sample Life stage Note The identiÞcation of large varieties of bacterial sym- 1 Adult Not surface sterilized bionts is impractical when using culture- or biochem- 2 Adult Surface sterilized ical reaction-based methods because many types of 3 Fifth instar Not surface sterilized 4 Fifth instar Surface sterilized reagents are required for characterization. Cultures 5 Early instarsa Not surface sterilized are rarely able to accurately represent the true rich- 6 Early instars Surface sterilized ness in environmental samples (Rondon et al. 2000) 7 Eggs Not surface sterilized because the growth of some organisms can limit others 8 Eggs Surface sterilized 9 Psyllid sugars Excrement from leaf surface rendering them undetectable. Biochemical classiÞca- 10 Water Used to water the plants tion methods are also less reliable than polymerase chain reaction (PCR) or sequencing because their The surface sterilized samples were prepared with 3 washes in 70% results are sometimes variable and preconceived no- EtOH, 1 wash in 10% bleach, then 3 washes in nanopure water. tions deÞning a taxon can be misleading (Francino et Unsterilized samples were immediately submerged in sterile PBS buffer. al. 2006), especially for microbes that are not well a Here, Љearly instarsЉ means all instars except the Þfth. characterized. In this study, bacterial tag-encoded FLX amplicon 4- pyrosequencing (bTEFAP) (Dowd et al. 2008a,b,c; the pyrophosphate ion (PPi; P2O7 ), which is released Wolcott et al. 2009a,b; Bailey et al. 2010) was used to as DNA polymerase incorporates the nucleotides survey the bacterial constituents of four lifestages of B. present into the growing chain. Enzymatic processes 4- cockerelli. Pyrosequencing using the bTEFAP meth- convert P2O7 into ATP and then to a light signalÑthe odology is a more representative, faster, and more strength of which is proportional to the number of cost-effective way to survey microbiomes. nucleotides consecutively incorporated (Ronaghi 2001). 454 Pyrosequencing. Massively Parallel bacterial Materials and Methods tag-encoded FLX-Titanium amplicon pyrosequencing Insect Colonies. Potato psyllids initially were pro- (bTEFAP) Titanium was performed as described pre- vided by Drs. Tong-Xian Liu and Xiangbing Yang viously (Dowd et al. 2008a,b,c; Wolcott et al. 2009a,b; (Texas AgriLife Research) and were used to set up a Bailey et al. 2010). Brießy, the samples were lysed in colony maintained on potato plants (Solanum tubero- RLT/␤-mercaptoethanol buffer, a 5-mm steel bead sum L.) of various ages grown from tubers at 25ЊC, 40% (Qiagen, Valencia, CA) and 500 ␮l of sterile 0.1-mm humidity and a photoperiod of 12:12 (L:D) h. Adult, glass beads (ScientiÞc Industries, Inc., Bohemia, NY). Þfth-instar, and early-instar (Þrst-fourth) psyllids DNA was recovered from a DNA spin column by were collected from the leaves by attaching a 1,000-␮l following the QIAamp DNA Mini Kit - Tissue Protocol Þlter pipette tip to the end of a 25-ml serological (Qiagen, Valencia, CA) beginning with step 5. Sam- pipette connected to a vacuum line. These were ples were quantiÞed using a Nanodrop spectropho- placed in 1.5-ml microcentrifuge tubes and stored at tometer (Nyxor Biotech, Paris, France) then diluted Ϫ80ЊC. Potato psyllid eggs were removed by hand to 100 ng/␮l with sterile nuclease free water. using Þne point tweezers under a dissecting scope. One hundred ng (1 ␮l) of each DNA sample was Sample Preparation. All psyllid lifestages were col- used in a 50-␮l PCR reaction by using 16S universal lected and the psyllids that were surface sterilized Eubacterial primers 530 F (5Ј-GTG CCA GCM GCN (Table 1) were prepared by three washes with 70% GCG G) and 1100R (5Ј-GGG TTN CGN TCG TTG) ethanol, one wash with 10% bleach, and then three for ampliÞcation of a 600-bp region of the 16S gene. washes with Nanopure water. B. cockerelli then were HotStarTaq Plus Master Mix Kit (Qiagen, Valencia, resuspended in sterile 1X phosphate buffered saline. CA) was used under these conditions: 94ЊC for 3 min; Psyllid sugars, bead-like waxy excrement, were col- 32 cycles of 94ЊC for 30 s, 60ЊC for 40 s, and 72ЊC for lected from the adaxial surface of the leaf and also 1 min; and a Þnal extension at 72ЊC for 5 min. Another suspended in 1X PBS buffer. A sample also was taken PCR was performed for FLX (Roche, Nutley, NJ) from the faucet used to water the plants to check for amplicon sequencing under the same conditions but water borne contaminants and all the samples were with special fusion primers (LinkerA-Tags-530 F and shipped to the Research and Testing Lab in Lubbock, LinkerB-1100R), which prevent ampliÞcation of any Texas for pyrosequencing. bias potentially caused by tag and linker inclusions Although sequence analysis, speciÞcally Sanger se- during initial ampliÞcation. All secondary PCR ampli- quencing, is more reliable and faster, its limitation cons were mixed in equal concentrations and puriÞed comes from its inability to produce de novo sequence with Agencourt Ampure beads (Agencourt Biosci- information from complex mixtures of DNA. Newer ence Corporation, MA). generations DNA sequencing technology continually DNA size (bp) and concentration were measured are increasing in capability while cost continues to by using DNA chips under a Bio-Rad Experion Auto- drop. Massively parallel pyrosequencing is based on mated Electrophoresis Station (Bio-Rad Laboratories, sequencing by synthesis and relies on the detection of CA) and a TBS-380 Fluorometer (Turner Biosystems, 100 ENVIRONMENTAL ENTOMOLOGY Vol. 41, no. 1

CA) in preparation for FLX sequencing (Roche, Nut- Table 2. Primers Designed for Genera Overlap Analysis ley, NJ). DNA with a concentration of 9.6 by 106 molecules/␮l and an average size of 625 bp was com- Primers bined with 9.6 million DNA capture beads and am- Achromobacter F1-TAGCGGCGGACGGGTGAGT pliÞed by emulsion PCR. After recovery and enrich- Achromobacter R1-TTCCCCACTGCTGCCTCCCG Product size: 265 ment, the bead-attached DNAs were denatured with Achromobacter F2-GCGGCGGACGGGTGAGTAAT NaOH and primers for sequencing were annealed. A Achromobacter R2-TTCCCCACTGCTGCCTCCCG two-region 454 sequencing run was performed on a 70 Product size: 263 by 75 GS PicoTiterPlate (PTP) using a Genome Se- Bacteroides F1-GGTCCAAACTCCTACGGGAGGC Bacteroides R1-ACGACCCATAGAGCCTTCATCCTTCA quencer FLX System according to the manufacturers Product size: 100 protocol (Roche, Nutley, NJ). Methylobacterium F1-TGGCTCAGAGCGAACGCTGG Data Analysis. After sequencing, failed reads, low Methylobacterium R1-ATCAGACGCGGGCCGATCCT quality ends and tags, and nonbacterial ribosome se- Product size: 197 Methylobacterium F2-ACGCTGGCGGCAGGCTTAAC quences were removed (Dowd et al. 2008a). The re- Methylobacterium R2-CCCGTAGGAGTCTGGGCCGT maining sequences were queried against 16S rDNA Product size: 288 bacterial sequences derived from NCBI (Cole et al. Microbacterium F1-AACGCTGGCGGCGTGCTTA 2009) by using a distributed BLASTn.NET algorithm Microbacterium R1-GTCGACGCCTTGGTGAGCCA Product size: 248 (Dowd et al. 2005). BLASTn results were compiled Microbacterium F2-GCTCGCGGCCTATCAGCTTGT and validated as described previously (Dowd et al. Microbacterium R2-CCCCACTGCTGCCTCCCGTA 2008a,b; Wolcott et al. 2009) and rarefaction estimates Product size: 131 of maximum diversity were performed according to Niastella F1-TCAGCCACACGGGCACTGAG Niastella R1-TCCTTCACGCGGCATGGCTG Acosta-Martinez et al. 2008. BLASTn results were as- Product size: 103 signed to the appropriate taxonomic classiÞcation Niastella F2-ACACGGGCACTGAGACACGG based on their identity scores to known 16S sequences. Niastella R2-TCCTTCACGCGGCATGGCTG Those with Ͻ3% divergence were resolved at the Product size: 97 Nocardioides F1-CTCAGGACGAACGCTGGCGG species level, those divergent between 3 and 5% to the Nocardioides R1-TCAGTCCCAGTGTGGCCGGT genus level, between 5 and 10% to the family level, and Product size: 298 those divergent between 10 and 20% to the order level. Nocardioides F2-GGTACTCGAGCGGCGAACGG A principal component analysis, PCA, was per- Nocardioides R2-CCCCACTGCTGCCTCCCGTA Product size: 273 formed using PC-ORD 5.0 software (http://home. Rhodoferax F1-AGACCTCGCGCGATTGGAGC centurytel.net/Ϸmjm/) to compare the bacterial gen- Rhodoferax R1-ATCCTGCACGCGGCATTGCT era present in the surface sterilized and nonsterile Product size: 200 lifestage insect samples. Three separate PCA tests Rhodoferax F2-CGGAACGTGCCCAGTCGTGG Rhodoferax R2-ATCCTGCACGCGGCATTGCT were performedÑthe Correlation Cross-Product Ma- Product size: 285 trix, the Non-Centered Cross-Product Matrix, and the Pseudomonas F1-AAGCGCGCGTAGGTGGTTCG Variance-Covariance Cross-Product Matrix. All dis- Pseudomonas R1-GTCCAGGTGGTCGCCTTCGC tance-based biplots included a randomization test for Product size: 172 resampling. Sequences recovered from sterile and non-sterile sample sets were Genera Overlap. When reviewing the bTEFAP se- aligned using 10 iterations of the MUSCLE alignment tool in Ge- quence data, concern was raised that some of the neious. From these consensus sequences, primers were designed to bacteria identiÞed could have been artifacts of artiÞ- determine the presence of the genera in wild populations from Texas cial rearing or contaminants from the lab acquired by and Washington State. the insects. Attention was focused on overlaps in the sequence data (genera) between the surface steril- cording to the manufacturerÕs protocol, using the Am- ized and nonsurface sterilized samples. Eight such plitaq Gold 360 PCR kit and genera presence per genera (Achromobacter, Bacteroides, Methylobacte- location was noted. rium, Microbacterium, Niastella, Nocardioides, Rhod- oferax, and Pseudomonas) were selected. Sequences Results from both sets of samples were aligned using 10 iter- ations of the MUSCLE alignment tool in Geneious In total, just over 20,000 sequences were recovered (Drummond et al. 2010, v5.1.6). From these consensus from the various potato psyllid lifestage samplesÑ sequences, primers (Table 2) were designed (Ge- 13,046 from the nonsterile group and 7,081 from the neious, Primer3) to investigate the presence of these sterilized group. Potato psyllid excrement yielded 203 genera in previously extracted potato psyllid DNA sequences and a water sample, taken from the source from wild populations in Texas and Washington State, used for the plants, resulted in 108 sequences. Across with the assumption that a positive identiÞcation in all the samples, the average number of sequences was both locations implied a positive identiÞcation in lo- 2044 (min Ð 98, max Ð 4,155). In the nonsterile sample, cations in between. From each location, DNA was Wolbachia and Staphylococcus were recovered from extracted from individual Þeld trapped potato psyllids all lifestages. In the sterilized samples, Ralstonia and by using the DNeasy Kit (Qiagen) according the man- Staphylococcus were recovered from all lifestages. ufacturerÕs protocol; Þve samples from each location The eggs had the highest richness of bacteria. Sev- were combined. AmpliÞcation was performed, ac- eral prominent genera of bacteria including Mycobac- February 2012 HAIL ET AL.: 16S BTEFAP IN POTATO PSYLLIDS LIFESTAGES 101

Fig. 1. Probable Location of 16S rDNA Sequences Recovered from Potato Psyllid Eggs. “Tissue” is determined by removing genera which were identiÞed in a) “Water” and b) “Psyllid Sugars”. “Surface” is determined by removing a) “Water” b) “Psyllid Sugars” and c) “Tissue” from NSS. terium (0.67% in “nonsterilized” and 0% in “steril- genera were common between the sterile and non- ized”), Wolbachia (41.65% in “nonsterilized” and sterile Þfth instars: Bradyrhizobium, Staphylococcus, 12.76% in “sterilized”), and Microbacterium (2.65% in and Wolbachia. “nonsterilized” and 0.82% in “sterilized”) showed a Many of the sequences recovered from the adult marked reduction. Sequences aligning to Rhizobium potato psyllids were identiÞed as a variety of Rhizo- (9.10%), Propionibacterium (18.91%), Gordonia bium-related genera. The sequences aligned to Rhi- (10.13%), and Xanthomonas (0.99%) were only recov- zobium (80.60% in “nonsterilized” and 0% in “steril- ered in “nonsterilized” but Ralstonia (19.75%) and ized”) and Mesorhizobium (0.12% in “nonsterilized” Legionella (7.82%) were only found in the sterilized and 0% in “sterilized”); Bradyrhizobium (0.11%) and sample. Bradyrhizobium had a modest increase from Sinorhizobium (0.08%) only were recovered from 1.19% in “nonsterilized” to 4.53% in “sterilized” and “sterile.” Sequences aligning to Ralstonia (0.06%) and Staphylococcus sequences (5.46% in “nonsterilized” Gordonia (0.03%) were recovered from “sterile” and and 30.86% in “sterilized”) increased with sterilization. Devosia (0.39%) was only found in “not sterile”. Two The survey of bacteria found in the eggs also indicated noteworthy genera Wolbachia (17.11% in “nonsteril- an overlap of genera between the surface sterilized ized” and 84.18% in “sterilized”) and Staphylococcus and nonsterilized samples: Achromobacter, Bradyrhi- (1.49% in “nonsterilized” and 10.69% in “sterilized”) zobium, Microbacter, Niastella, Staphylococcus, and increased in response to surface sterilization. Two Wolbachia. genera were common between the sterile and non- In the early-instar nymphs, sequences aligning to sterile adults: Staphylococcus and Wolbachia. Wolbachia only were recovered in “nonsterilized” Controls and Comparisons. Using a method of sub- (95.62% and 0% in “sterilized”). Geitlerinema and Gloeothece were found in Þfth instars, early instars, and tractive comparisons of the various samples, the pu- eggs (not sterile) and sequences of Staphylococcus tative locations of the bacteria were identiÞed in this increased (1.59% in “nonsterilized” and 14.29% in study. To identify surface bacteria on a particular “sterilized”). Ralstonia (30.61%) and Delftia (7.14%) lifestage, the genera identiÞed in the psyllid sugars and only were found in “sterile” and Mycobacterium the water were removed from those found in the (0.06%) was only found in “not sterile.” Five genera nonsterilized insects (Figs. 1 and 2, 3 and 4). Com- were common between surface sterilized and non- parisons of the surface bacteria of the various life- sterile early instars: Bacteroides, Methylobacterium, stages indicated that some genera were present in two Pseudomonas, Sinorhizobium, and Staphylococcus. (Bradyrhizobium, Corynebacterium); three (Gei- In the Þfth-instar nymphs Wolbachia accounted for tlerinema, Rhizobium); or all four (Wolbachia, Staph- Ͼ95% of the sequences recovered without or with ylococcus) stages. To isolate the bacteria located in the surface sterilization (3,155/3,164 in “nonsterilized” tissues but not in the gut, the genera from the excre- and 3,091/3,137 in “sterilized”). Staphylococcus (0.09% ment and the water were removed from those found in “nonsterilized” and 0.61% in “sterilized”) and Bra- in the surface sterilized insects (Figs. 1 and 2, 3 and 4). dyrhizobium (0.03% in “nonsterilized” and 0.29% in Comparison of these genera showed Tepidimicrobium, “sterilized”) increased slightly. Ralstonia (0.13%) and Methylobacterium, Sporocytophaga, Niastella, and Le- Legionella (0.03%) only were found in “sterile.” Three gionella were present in at least two lifestages. 102 ENVIRONMENTAL ENTOMOLOGY Vol. 41, no. 1

Fig. 2. Probable Location of 16S rDNA Sequences Re- covered from Early Instar Potato Psyllids. “Tissue” is deter- mined by removing genera which were identiÞed in a) Wa- “ Fig. 3. Probable Location of 16S rDNA Sequences Recov- ter” and b) “Psyllid Sugars”. “Surface” is determined by ered from Þfth Instar Potato Psyllids. “Tissue” is determined by removing a) “Water” b) “Psyllid Sugars” and c) “Tissue” from removing genera which were identiÞed in a) “Water” and b) NSS. “Hindgut” was determined by removing genera iden- “Psyllid Sugars”. “Surface” is determined by removing a) “Wa- tiÞed in “Water”. “Hindgut” here is less certain; most of the ter” b) “Psyllid Sugars” and c) “Tissue” from NSS. “Hindgut” was psyllid sugars collected were likely from Þfth instars and/or determined by removing genera identiÞed in “Water”. adults so the early instar ßora could be different.

A sample was taken from the water source used for genera data from the water and the psyllid sugars the plants to serve as a type of control with the as- indicated (Fig. 6) an overlap of two, Clostridium and sumption that the water may harbor bacteria trans- Pelonomas, leaving sequences of seven unique genera missible to insects. There were 12 genera identiÞed; from the excrement, many of which are commonly Ralstonia (32%), a common phytopathogen, was iden- isolated from guts. Sequences homologous to tiÞed in all life stages. In surface sterilized eggs, se- Pelomonas and Enterobacter were recovered from the quences homologous to Ralstonia were a signiÞcant surface sterilized Þfth instars and adults. Pelomonas fraction (53%) of those recovered but tapered off in was present in the water (23%), which could have adults (2%). Corynebacterium (19.4%), a widely com- carried through to contaminate the likeliest cause, mon bacterium was found only in the Þfth instars and residual excrement in the hindgut. Because Entero- Pelomonas (23.15%), was recovered from only non- bacter was not present in the water that brings the total sterilized samples. Nine of the genera were recovered to eight (Shigella, Escherichia, Parkia, Enterococcus, in single digits but were not notably represented in Ulvibacter, Citrobacter, Enterobacter, and Parvibacu- any of the insect samples. A single Ureibacillus se- lum). quence was recovered from the water but not from The hindgut contents also were compared with the the others and Gordonia, recovered six times from sterilized insects (Fig. 7) to check the surface steril- the water, was only found a single time in the adult ization procedure and nonsterilized lifestages (Fig. 8) surface sterilized samples. In total, eight genera to determine if the psyllids are surface contaminated present in the water also were present in various with fecal matter. Two genera from the excrement lifestages of surface sterilized insects leaving eggs were identiÞed in the surface sterilized lifestages, En- with 12 possibly unique genera, early instars with 11, terobacter (0.44%) from the adults and a single se- Þfth instars with 12, and adults with 22 (Fig. 5). quence of Pelomonas (3.19 ϫ 10Ϫ4 %) from the Þfth In an attempt to survey the microbial contents of the instars. Both of these genera were present in the water potato psyllid hindgut, a sample of psyllid sugars was and so cannot be discounted as contaminants. A single taken from the top surface of the leaves. Several gen- sequence of Escherichia and another single sequence era recovered from the excrement were found in the of Enterococcus, both recovered exclusively from the other nonsterile samples. Clostridium, possibly from feces, were found in the eggs and Þfth instars, respec- the water, was identiÞed in the nonsterilized adult tively. insects, and Escherichia was found in the adults and Genera Overlap. All of the bacterial genera tested Þfth-instar nymphs. Enterococcus (72.91%) also was (Achromobacter, Bacteroides, Methylobacterium, Mi- found in the adults and Þfth instars. Citrobacter crobacterium, Niastella, Nocardioides, Rhodoferax, and (12.81%) only was found in adults. Comparing the Pseudomonas) were present in DNA previously ex- February 2012 HAIL ET AL.: 16S BTEFAP IN POTATO PSYLLIDS LIFESTAGES 103

Fig. 4. Probable Location of 16S rDNA Sequences Recovered from Adult Potato Psyllids. “Tissue” is determined by removing genera which were identiÞed in a) “Water” and b) “Psyllid Sugars”. “Surface” is determined by removing a) “Water” b) “Psyllid Sugars” and c) “Tissue” from NSS. “Hindgut” was determined by removing genera identiÞed in “Water”. tracted from potato psyllids in both Texas and Wash- the genera in question, it is unlikely that this suite of ington State potato Þelds. Assuming that psyllids at all bacteria was introduced during colony rearing in the points in between also would have tested positive for lab.

Fig. 5. Comparison of Genera IdentiÞed in Water Control as Compared with Those in Surface Sterilized Lifestages. Com- parison showed eight genera in the surface sterilized lifestages that were also in the water. Because of this, Ralstonia, Curvibacter, Comamonas, Achromobacter, Acidovorax, Pseudomonas, and Gordonia are possible carry-over contaminants. This leaves eggs with 12, early instars with 11, Þfth instars with 12, and adults with 22 possibly unique genera. (Online Þgure in color.) 104 ENVIRONMENTAL ENTOMOLOGY Vol. 41, no. 1

Fig. 6. Comparison of Genera IdentiÞed in Water Control as Compared with Those in Psyllid Sugars. Comparison showed two genera in the excrement that were also in the water. Because of this, Clostridium and Pelomonas are possible carry-over contaminants. Several genera identiÞed in the excrement (Escherichia, Enterobacter, Citrobacter, Enterococcus, Shigella, etc.) are common ßora in animal guts. (Online Þgure in color.)

Discussion were very dissimilar on the whole but adults and Þfth instars were the most alike. In the Non-Centered In a principal component analysis performed on the surface sterilized and nonsterile insects, there Cross-Product Matrix, the sterilized adults and Þfth was unexpected grouping of samples with respect to instars (sterilized and nonsterile) clustered the closest sterilization and lifestage when using Correlation and the sterilized eggs and early instars grouped to- Cross-Product Matrix- , Non-Centered Cross-Prod- gether but away from the others. In the Variance- uct Matrix- or Variance-Covariance Cross-Product Covariance Cross-Product Matrix, sterilized adults Matrix- based biplots. In the Correlation Cross- and Þfth instars grouped with nonsterilized Þfth in- Product Matrix, the sample grouping indicates 1) stars and early instars and, as the Non-Centered Cross- surface sterilized insects are similar to nonsterilized Product Matrix, the sterilized eggs and early instars insects but are more similar to one another, 2) among grouped together but away from the others. the surface sterilized insects, the early and Þfth instar In the Non-Centered and Variance-Covariance lifestages are more similar to each other than to the analyses, there was a consistent grouping of the Þfth- adult and egg pair, and 3) the nonsterilized insects instar nymphs, the nonsterile early instars, and the

Fig. 7. Comparison of Genera IdentiÞed in the Surface Sterilized Lifestages as Compared with Those in Psyllid Sugars. Comparison showed two genera in the excrement that were also in the surface sterilized lifestages. Because of this, Enterobacter and Pelomonas are possible carry-over contaminants; the points of overlap in the Þfth instar and the adult were (0.44%) and (3.19 ϫ 10Ϫ4 %) respectively, also testing the sterilization protocol. (Online Þgure in color.) February 2012 HAIL ET AL.: 16S BTEFAP IN POTATO PSYLLIDS LIFESTAGES 105

Three washes in 70% ethanol, one wash in 10% bleach, then three washes in nanopure water were used to surface sterilize the insects in this study. Al- though some surface contaminants were certainly lysed by the 70% ethanol and washed away by the nanopure water, junctions of body segments, wing, and leg attachments and overlapping segments of the abdomen are possible sites of persistent contamina- tion with either bacteria or DNA. Contamination also could have resulted from exposure of samples to air while transferring solutions between washes, how- ever, Fig. 8 seems to indicate this did not occur. In a study by Davidson et al. (1994) silverleaf white- ßy (Bemisia argentifolii) pupae and eggs were surface- sterilized using soapy water, a water rinse, a 70% eth- anol dip, and then 2-min agitation in 20% household bleach solution. Although this treatment may be suf- Þcient for soft-bodied life stages like eggs and pupae, there is no uniformly accepted method for surface sterilization of all insect life stages. That said, the two Fig. 8. Comparison of Genera IdentiÞed in the Non- most commonly used methods of surface sterilization Sterilized (NSS) Lifestages as Compared with Those in Psyl- are ethanol and bleach (Connell 1981), the latter be- lid Sugars. Comparison showed two genera in the excrement ing used only when development, life stage viability, that were also in the unsterilized lifestages. Because of this, or both are not a concern (Vail et al. 1968, Leppla et Enterococcus and Escherichia are possible carry-over contam- al. 1974). inants; the points of overlap in the Þfth instar and the egg To acquire as much information as possible as to the were from single sequences, and determined if the psyllids constituents of the potato psyllid microbiome, many were surface contaminated with fecal matter. Escherichia and insect samples were taken. Sampling all lifestages and Enterococcus were both recovered exclusively from the feces. using overlapping controls takes great advantage of (Online Þgure in color.) the large data set from even a single sequencing run. Analysis of the outer surface, inner tissues, and gut ßora is possible by comparing the bacterial sequences sterilized adults. The Þfth instars and the nonsterile identiÞed in 1) a water sample to those in the feces, 2) early instars had high percentages of Wolbachia but the feces to those in the surface sterilized insects, 3) the sterilized adults did as well. After the total removal the surface sterilized insect to those in the nonsurface of Rhizobium (80.60% in “nonsterilized” and 0% in sterilized, and 4) the water to those in the surface “sterilized”), the percentage of Wolbachia increased sterilized insects. A clear understanding of the bacte- nearly Þve-fold (17.11% in “nonsterilized” and 84.18% rial ßora of the 1) inside of the insect, excluding the in “sterilized”). gut; and 2) the gut, excluding other organs, identiÞed Across all the PCA biplots, the samples that grouped insect-speciÞc pathogens, and commensal or mutual- the closest were the Þfth-instar nymphs. This result is istic bacteria, excellent targets for reducing potato most likely because of the overwhelming presence psyllid Þtness. (Ϸ98%) of sequences homologous to Wolbachia. After The genera chosen for this experiment are known or sterilization, sequences identiÞed as Wolbachia were suspected to associate with insects and are of interest reduced, i.e., the non-Wolbachia sequences increased, as these organisms could 1) serve as infectious agents, by only 1.19%. In early-instar nymphs, Wolbachia was 2) allow infectious agents to ßourish in their absence, removed completely by surface sterilization (95.62% or 3) serve as candidates for delivering designer mol- and 0% in “sterilized”) and therefore signiÞcantly ecules. skewed the sample groupings. Surface sterilization Although not insects, various species of spiders na- removed 23 genera from the eggs and contributed to tive to Japan were found to have species of Nocar- their failure to group. Although grouped closely in the dioides in their webs, cuticles, and egg sacs (Iwai Correlation Cross-Product Matrix Distance-Based Bi- 2009). Although there are no other reports of Rhod- plot, the adult samples were widely separated in the oferax ferrireducens being present in any insect spe- other analyses. cies, the completion of its genome sequence showed As indicated by the PCA data, surface sterilization a sequence homologous to a portion of the carotenoid appears to be necessary for determining the speciÞc biosynthesis pathway in insects. Similarly, Niastella locations of bacteria present in or on the various life- has not been reported in insects but was recovered stages of the potato psyllid. For example, although the from the lab reared and wild psyllids. egg material is very soft and moist, it is protected by Although associated with the water, Achromobacter, a more rigid shell. Bacteria growing on the shells were known (Poinar 1967) to infect the Greater Wax Moth washed away by surface sterilization to reveal fewer [Galleria mellonella (L.)], was chosen because of its total genera. association with the surface sterilized eggs and pre- 106 ENVIRONMENTAL ENTOMOLOGY Vol. 41, no. 1 vious association with the Asian Citrus Psyllid (Dia- could provide important insights into designing novel phorina citri), a sister species of the potato psyllid. targeted management strategies for the insect. Anaerobic species in the genus Bacteroides have been identiÞed as constituents of the gut ßora (Breznak 1982) of the eastern subterranean termite [Reticu- References Cited litermes flavipes (Kollar)]. If truly anaerobic in the potato psyllid, Bacteroides also may be performing a Abad, J., M. Bandla, R. D. French-Monar, L. W. Liefting, and similar function; that is, cross-feeding lactate with the G.R.G. Clover. 2009. 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