INSECTÐSYMBIONT INTERACTIONS Distribution and Ecology of occidentalis (Thysanoptera: ) Bacterial Symbionts

1 LISA J. CHANBUSARAKUM AND DIANE E. ULLMAN

Department of Entomology, University of California-Davis, Davis, CA 95616

Environ. Entomol. 38(4): 1069Ð1077 (2009) Downloaded from https://academic.oup.com/ee/article/38/4/1069/493304 by guest on 27 September 2021 ABSTRACT Bacterial populations in Frankliniella occidentalis (Pergande) (Thysanoptera: Thripi- dae) collected in diverse California environments consisted of two bacterial symbionts: BFo-1 and BFo-2 (B ϭ bacteria, Fo ϭ Frankliniella occidentalis, numbers reßect different types). Dual infections of BFo-1 and BFo-2 were found in 50% of the , 18% had neither bacterium, and 24 and 8% were infected solely with BFo-1 and BFo-2, respectively. No other bacteria consistently infected F. occi- dentalis. Dual infections occurred more often in male thrips and in thrips of both sexes from southern mountain and valley sites. As average collection year or month minimum temperature decreased, infections of BFo-1, alone or in dual infections, increased signiÞcantly. As yearly precipitation increased, infection with BFo-1 alone also increased. F. occidentalis color morphology did not affect bacterial infection. BFo-1 created weak bioÞlms at 25 and 32ЊC; BFo-2 made strong bioÞlms at 25ЊC and no bioÞlms at 32ЊC. When the bacteria were grown in culture together, weak bioÞlms formed at both temperatures studied, although there was no way to determine what each bacterium contributed to the bioÞlm. BFo-1 and BFo-2 grew at similar rates at 25 and 30ЊC. Our data show BFo-1 and BFo-2 occur in natural populations of F. occidentalis and support the hypothesis BFo have a symbiotic relationship with F. occidentalis. Regional differences in bacterial prevalence suggest bacterial infec- tion is associated with environmental conditions, and altitude, temperature, and precipitation may be important factors.

KEY WORDS Frankliniella occidentalis, bacterial symbionts, prevalence, distribution

The importance of bacterial gut symbionts can clearly An investigation of a Hawaiian Islands F. occidentalis be seen among many groups of . Bacteria re- laboratory colony, a German laboratory colony, and a siding in termite guts Þx nitrogen and recycle nitrog- sample of thrips collected in 1965 from Davis, CA, enous waste produced during termite metabolism showed two consistent types of bacteria in each of the (Ohkuma 2003, Gomathi et al. 2005, Doolittle et al. F. occidentalis populations, named BFo-1 and BFo-2 2008); the house cricket Acheta domesticus has gut (Bacteria from F. occidentalis, types 1 and 2) (Chan- bacteria that help the cricket process polysaccharides busarakum and Ullman 2008), suggesting a widespread from plants (Kaufman and Klug 1991). The ßy Cyclo- and an at least 40-yr-old association between the bac- rrhapha actually digests some of its gut bacteria teria and the . These bacteria could survive in- (Lemos and Terra 1991). However, numerous insectÐ dependently of the host and were transmitted among bacteria interactions are not well understood, even for F. occidentalis by defecation and subsequent feeding those bacteria that can be examined independently by unrelated thrips. Although these Þndings provided from their insect host (Harada et al. 1997, Watanabe strong evidence for an association between F. occi- and Sato 1998). One such relationship involves the dentalis and BFo-1 and BFo-2, and although BFo-1 Western ßower thrips, Frankliniella occidentalis (Per- seemed to share ancestry with environmental bacte- gande) (Thysanoptera: Thripidae), and their gut bac- ria, nothing was known about prevalence of the bac- teria (de Vries et al. 2001a, b, 2004; Chanbusarakum teria in contemporary wild F. occidentalis populations. and Ullman 2008). Understanding how the bacteria InsectÐmicrobe relationships in the optimized and interact with the thrips could lead to innovative strat- standardized settings in laboratories are not always egies for management, a goal that is important because reßected in the natural environment, where preda- F. occidentalis are serious and largely unmanageable tors, changing food sources, and ßuctuating weather pests of numerous food, Þber, and ornamental crops could affect the insect host as well as the microbe it (Boonham et al. 2002). harbors. This has been seen in Tobacco thrips, Fran- kliniella fusca, where not all bacteria species found in laboratory thrips were found in Þeld thrips and titer 1 Corresponding author, e-mail: [email protected]. levels differed between Þeld and laboratory collec-

0046-225X/09/1069Ð1077$04.00/0 ᭧ 2009 Entomological Society of America 1070 ENVIRONMENTAL ENTOMOLOGY Vol. 38, no. 4 Downloaded from https://academic.oup.com/ee/article/38/4/1069/493304 by guest on 27 September 2021

Fig. 1. Thrips collection locations and relative infection patterns. Numbers in shapes refer to samples analyzed by real-time PCR (Table 1); letters indicate samples used when searching for new symbionts by cloning analysis (Table 3). Numbers in parenthesis refer to sample size at each site. Star, location of Sacramento; oval, location of Lake Tahoe. tions (Wells et al. 2002). In fact, microbes associated whether other bacteria species regularly associate with other organisms often behave differently as their with F. occidentalis. To get an idea of how much BFo environment changes (Brown and Barker 1999). phenotypes might vary in changing environments, we Phenotypic characteristics of bacteria, such as bio- explored temperature affects on bioÞlm and growth of Þlm formation and growth, often are affected by tem- the two bacteria types using in vitro studies. peratures. BioÞlms have been known to help bacteria with adhesion, distribution of nutrients, resistance to Materials and Methods antibiotics, and resistance to desiccation (Branda et al. 2005, Romanova et al. 2006, Schaudinn et al. 2007). Specimen Collection. Naturally occurring thrips BioÞlms can also be comprised of many bacterial spe- populations were collected AprilÐMay and June 2004 cies that each contribute something unique to the and March 2007 in sites across California (Fig. 1). GPS overall bioÞlm (Sutherland 2001b, Sauer et al. 2007). coordinates were noted for each collection site. A Rapid growth can lead to dominating numbers that out “collection site” is deÞned as one or more plants of the compete other bacteria (Harder and Veldkamp 1971, same species within a 1-m radius. Plants were beaten Thomas and Wimpenny 1993). Variations in growth over a white tray to shake thrips loose. Thrips were and bioÞlm formation can inßuence how well bacteria placed into 100% ethanol with a soft paintbrush and colonize an area. Environmental effects on the host as stored at Ϫ80ЊC. Adult thrips from these collections well as the bacteria therefore might determine the were examined under a dissecting microscope to ver- prevalence and distribution of a symbiotic association. ify they were F. occidentalis (OÕDonnell et al. 2000). Given the variability of microbeÐhost relationships Host Characteristics and Environmental Data. En- in laboratory settings compared with the Þeld, and the vironmental data from collection sites were deter- potential importance of these bacteria to F. occiden- mined from nearby weather stations (Western Re- talis Þtness, we conducted a study of F. occidentalis gional Climate Center 2008) and Google Earth sampled from diverse habitats and environments (McClendon 2007). Sites also were sorted into general across California. We determined the prevalence of California regions: coastal, southern valley (associated BFo in natural thrips populations and explored how with the San Joaquin Valley), northern valley (relat- environmental factors may be linked to both bacterial ing to the Sacramento Valley), and northern and infection and host characteristics. We also studied southern mountains divided by the position of Lake August 2009 CHANBUSARAKUM AND ULLMAN:ENVIRONMENT, F. occidentalis, AND GUT BACTERIA 1071

Tahoe (Fig. 1). Five sites from each region were ran- chine. Standard ampliÞcation conditions were used: domly selected for analysis. F. occidentalis were sorted 50ЊC for 2 min, 95ЊC for 10 min, 40 cycles of 95ЊC for into light, bicolor, and dark color morphologies using 15 s, and 60ЊC for 60 s. Fluorescent signals were col- Њ established methods (OÕDonnell 2007).Whenever lected during the 60 C annealing temperature. A Ct possible, up to two thrips from light, bicolor, and dark below 30 cycles was considered a negative result. color morphology were chosen from a given site. Ad- Investigation of Other Potential Symbionts. Natural ditional sites were examined to bring the total number F. occidentalis populations were also surveyed for ad- of thrips used per region to Ϸ25. In total, 120 adult ditional bacterial symbionts not present in laboratory- thrips from 31 unique sites were analyzed (Fig. 1; reared thrips colonies (Fig. 1; Table 3). DNA from one Table 1). or groups of 10 F. occidentalis adults from a single Real-Time Polymerase Chain Reaction for Detec- collection site were directly extracted using the Qia- tion of Infection Patterns. Individual F. occidentalis gen DNeasy kit. The bacterial 16S rDNA was ampliÞed

were sent to the Lucy Whittier Molecular and Diag- and cloned following previously described methods Downloaded from https://academic.oup.com/ee/article/38/4/1069/493304 by guest on 27 September 2021 nostic Facility (University of California-Davis, Davis, (Chanbusarakum and Ullman 2008). PCR products CA) for DNA extraction using the ABI PRISM 6100 were puriÞed using the Qiagen QIAquick PCR puri- Nucleic Acid PrepStation (Applied Biosystems, Foster Þcation kit before being sent to the Division of Bio- City, CA). Subsequent eluate (100 ␮l) was concen- logical Sciences DNA Sequencing Facility (College of trated by DNA precipitation and resuspended in 20 ␮l Biological Sciences, UC-Davis, Davis, CA) for se- gamma-irradiated diethyl pyrocarbonate-treated dis- quencing with the primers 27f, 514r, 530f, and 860r to tilled water. obtain approximately the Þrst 1,000 bp of the 16S Both the Lucy Whittier Molecular and Diagnostic rDNA (Table 2). The resulting 16S rDNA sequences Facility and our laboratory attempted to detect thrips were assembled using Invitrogen Vector NTI Contig bacteria via real-time polymerase chain reaction Express Software (Invitrogen, Carlsbad, CA) and (PCR) from extracted and precipitated DNA; how- manually checked. The 16S rDNA sequences were ever, no signal was ever detected. Therefore, to in- BLASTn searched in GenBank (June 2008) to identify crease concentrations of bacterial 16S rDNA to a de- closest matching known sequences. In total, 53 clones tectable level and to minimize ampliÞcation of from 32 thrips in Þve California sites were analyzed. undesired product, we performed a nested PCR on Analysis of Infection Patterns Relative to Host and total DNA extracted from thrips. In the Þrst round of Environmental Conditions. We assessed the correla- PCR, Advantage2 Polymerase buffer (Clontech, tion between infection patterns and host color mor- Mountain View, CA), 1 U Advantage2 polymerase, 0.2 phology, host sex, collection region, average maximum mM dNTPs, 0.4 ␮M 27f and 1525r primers (Table 2), and minimum temperatures for the collection month 5 ␮l concentrated sample, and distilled water up to 25 and year, altitude, precipitation for the year and ␮l were run in a PTC 200 Peltier Thermal Cycler month, collection date, and host plant type with a (Bio-Rad, San Francisco, CA). The program was 95ЊC series of Fisher exact test, simple logistic regression, or for 1 min, 30 cycles of 95ЊC30s,62ЊC40s,70ЊC 1 min multinomial logistic regression where appropriate, us- 30 s, and 70ЊC for 5 min before sitting at 4ЊC until use. ing SAS (SAS Institute 2004). Statistical signiÞcance Nested PCR was performed on the resulting product. was declared at the 5% level. Where marginal signif- The reaction for the nested PCR consisted of the icance was observed, P-values of 0.06Ð0.09 are shown. same concentrations of polymerase, buffer, dNTPs, The analysis was performed in two main steps: ana- and primers as stated above. The 27f and 1525r primers lyzing each of the BFo infections independently of the were replaced with internal 16S rDNA primers 53f and presence or absence of the other BFo (i.e., BFo-1 514r (Table 2). Only 1 ␮l of round 1 PCR product was regardless of being alone or in a dual infection with used in the reaction, and additional distilled water was BFo-2), and analyzing the infection patterns of the used to bring the total reaction volume to 25 ␮l. De- two bacteria together so that possible infection pat- sired product yielded a 500-bp band on ethidium bro- terns were ϩϩ, ϩϪ, Ϫϩ, ϪϪ. Because of sampling mideÐstained gels. The Qiagen Gel Extraction Kit limitations, only main effect models were assessed. (Qiagen, Valencia, CA) was used to extract 50 ␮lof Influence of Temperature on BFo Growth and Bio- nested PCR. DNA was eluted with 40 ␮l EB buffer. film. BioÞlm formation was measured using a micro- Two unique forward and reverse primers and two titer plate following established protocols (Merritt et unique probes speciÞc to BFo-1 and BFo-2 were de- al. 2005). Brießy, BFo-1 and BFo-2 were grown to signed from the available 16S rDNA sequences in stationary phase in LB broth. Each bacterial culture GenBank (accession numbers AF024607ÐAF024613 was diluted 1:100, and 100 ␮l was placed into 8 wells and EU029105ÐEU029106). The FAM-labeled probes of a 96-well microtiter plate. When using both bacteria and primers were designed and tested by the Lucy in the same culture, 50 ␮l of each diluted culture was Whittier Facility (Table 2). placed into one well. Plates were covered and incu- For real-time PCR, 400 nM of each primer, 80 nM bated at 25 and 32ЊC for 24 h. Plates were washed, and probe, 1ϫ PCR master mix (Applied Biosystems) and wells received 0.1% crystal violet to stain bioÞlms. distilled water were combined with Ϸ100 ng puriÞed After excess stain was rinsed away, 100% ethanol was 16S DNA for a total volume of 25 ␮l per well. Samples added to the wells to redissolve remaining crystal were run in triplicate in 96-well plates and ampliÞed violet. The intensity of stain was read at 595 nm. in a 7300 Applied Biosystems Real-Time PCR Ma- SigniÞcant bioÞlm formation was assessed using three- 02E 1072

Table 1. Collected F. occidentalis location, host plant, environmental data, and color morphology sorted by infection pattern

mo, yr Location (city/jnct, Mo max, min. yr max, No. Host plant Region Collect date Dual infection BFo-1 BFo-2 None precipitation Lat, long, alt. (m) county) min temperature (ЊC) (m) 1 Redding, Shasta Scotchbroom NM 15 June 2004 LF LM 33.85, 17.50, 24.58, 10.47 0.02, 1.01 40.45, Ϫ122.19, 419 2 Tulelake, Siskiyou Medicago NM 14 June 2004 DF LF(2) DF 25.89, 6.76, 17.46, 0.73 0.00, 0.59 41.58, Ϫ121.29, 1,219 3 Susanville, Lassen Wyethia ovatia NM 14 June 2004 LF DF(2) LF BF BF 26.461, 7.53, 17.66, 1.71 0.01, 1.11 40.25, Ϫ120.41, 1,554 4 Hallelujah Junction, Lassen Melilotus indica NM 14 June 2004 LF(2) BF DF DF 27.50, 9.57, 18.79, 3.39 0.01, 0.80 39.46, Ϫ120.02, 1,542 5 Weed, Siskiyou Shasta Daisy NM 15 June 2004 LF DF LF DF 28.94, 7.89, 20.78, 3.67 0.00, 1.84 41.24, Ϫ122.22, 1,067 Ϫ

6 Grass Valley, Nevada Rosa NM 14 June 2004 LF(2) LF 27.71, 12.21, 20.28, 6.57 0.00, 1.06 39.14, 121.02, 735 NVIRONMENTAL 7 Jct hwy 88&89, Alpine Ceanothus SM 7 June 2004 LF LM DF DF 24.62, 7.59, 16.94, 2.13 0.02, 2.85 38.46, Ϫ119.49, 2,134 8 Markleeville, Alpine Ranunculus SM 7 June 2004 LF(2) DF(2) BF 24.29, 3.56, 17.21, Ϫ1.64 0.01, 2.92 38.41, Ϫ119.46, 1,829 9 Bridgeport, Mono Wyethia ovatia SM 6 June 2004 LF(2) BF DF BF DF 24.13, 2.17, 11.91, Ϫ4.73 0.01, 2.34 38.15, Ϫ119.13, 1,981 10 Kyburz, El Dorado Ceanothus SM 7 June 2004 LF LF BF DF 17.69, 4.86, 11.48, 0.18 0.10, 11.68 38.46, Ϫ120.17, 1,768 11 Placerville, El Dorado Melilotus indica SM 7 June 2004 LF LM BF DF DF 30.04, 12.83, 20.49, 7.12 0.00, 0.82 38.43, Ϫ120.47, 579 12 Tulare, Tulare Nerium oleander SV 5 June 2004 LF LM 32.32, 16.78, 24.04, 11.22 0.00, 0.21 36.11, Ϫ119.20, 16 Ϫ

13 BakersÞeld, Kern Gladiolas SV 5 June 2004 LF(2) 32.68, 17.76, 25.36, 12.26 0.00, 0.14 35.22, 119.01, 16 E

14 Turlock, Stanislaus Hemerocallis SV 19 Mar. 2007 LM(2) DF(2) 22.38, 8.91, 21.82, 9.14 0.48, 0.15 37.30, Ϫ120.51, 31 NTOMOLOGY 15 Turlock, Stanislaus Triticum aestivum SV 19 Mar. 2007 LM BF DF BF DF 22.38, 8.91, 21.82, 9.14 0.48, 0.15 37.30, 120.51, 31 16 Modesto, Stanislaus Rosa SV 19 Mar. 2007 LM BF DF BF DF 23.60, 8.67, 25.24, 10.54 0.01, 0.19 37.38, Ϫ120.59, 27 17 Ripon, San Joaquin Malus sp. SV 19 Mar. 2007 BF(2) DF 23.55, 7.46, 25.12, 9.32 0.00, 0.21 37.44, Ϫ121.07, 6 18 Stockton, San Joaquin Brassica alba SV 19 Mar. 2007 LM BF(2) LF DF 23.55, 7.46, 25.12, 9.32 0.00, 0.21 37.56, Ϫ121.17, 5 19 Berkeley, Almeda Prunos triloba C 11 Mar. 2007 LF DF BM LF DF BF 19.69, 7.24, 20.12, 8.43 0.00, 0.44 37.53, Ϫ122.16, 52 20 Petaluma, Sonoma Hemerocallis C 11 June 2004 LF LF 25.72, 10.65, 21.73, 9.36 0.08, 0.68 38.14, Ϫ122.37, 7 21 Davis, Yolo Dietes NV 31 May 2004 BM LF LM BF LF 28.14, 10.85, 23.24, 8.96 0.00, 0.48 38.31, Ϫ121.45, 15.85 22 Davis, Yolo Hypericum patulum NV 31 May 2004 LF LM 28.14, 10.85, 23.24, 8.96 0.00, 0.48 38.31, Ϫ121.45, 15.85 23 Davis, Yolo Syringa NV 8 July 2004 LM LF 33.46, 13.83, 23.24, 8.96 0.00, 0.48 38.31, Ϫ121.45, 15.85 24 Davis, Yolo Agapanthus NV 8 July 2004 LF 33.46, 13.83, 23.24, 8.96 0.00, 0.48 38.31, Ϫ121.45, 15.85 25 Cloverdale, Sonoma Coleonema pulchron C 11 June 2004 LF LF 31.26, 13.00, 24.43, 9.33 0.00, 1.01 38.48, Ϫ123.00, 112 26 Arbuckle, Calusa Lupinos NV 8 Mar. 2007 DF(2) LF(2) BF 18.78, 5.17, 24.19, 8.61 0.05, 0.42 39.01, Ϫ122.03, 37 27 Woodland, Yolo Brassica alba NV 8 Mar. 2007 DF DF(2) 22.63, 8.79, 24.62, 10.16 0.00, 0.30 39.01, Ϫ122.03, 37 28 Weott, Humboldt Chrysanthem C 12 June 2004 LF LM BF DF BF DF 20.72, 12.04, 17.81, 8.75 0.00, 1.01 40.19, Ϫ123.55, 87 29 Corning, Tehama Ranunculus NV 16 June 2004 DF LF LM DF 32.11, 15.82, 24.12, 10.06 0.00, 0.58 39.95, Ϫ122.20, 90 30 Loleta, Humboldt Lupinos C 12 June 2004 DF BF(2) DF 17.93, 11.02, 16.04, 8.51 0.00, 0.97 40.38, Ϫ124.13, 12 31 Eureka, Humboldt Raphanus C 12 June 2004 BF DF BF DF 17.93, 11.02, 16.04, 8.51 0.00, 0.97 40.47, Ϫ124.09, 2

Region: C, coast; NV, north valley; SV, south valley; NM, north mountains; SM, south mountains.

Infection pattern: L, light; B, bicolor; D, dark. F, female; M, male. Numbers in parentheses indicate no. in sample. 4 no. 38, Vol. Downloaded from https://academic.oup.com/ee/article/38/4/1069/493304 by guest on 27 September 2021 September 27 on guest by https://academic.oup.com/ee/article/38/4/1069/493304 from Downloaded August 2009 CHANBUSARAKUM AND ULLMAN:ENVIRONMENT, F. occidentalis, AND GUT BACTERIA 1073

Table 2. Primers used for PCR and real-time PCR the same plant were varied (Fig. 1), with two or more infection patterns present in 22 of the 30 sites. This Ј Ј Primer name Sequence (5 to 3 ) prevalence differed noticeably from Hawaiian Islands 27fa AGAGTTTGATCMTGGCTCAG (M ϭ A ϩ laboratory colony infection rates, where cloning C wobble) 53fa CACATGCAAGTCGAACGG showed 30% were dually infected, 60% had exclusively 514ra ATTACCGCGGCKGCTGGCAC (K ϭ G ϩ BFo-1, 10% had BFo-2 only, and no thrips were with- T wobble) out bacteria (Chanbusarakum and Ullman 2008). The 530fa GTGCCAGCMGCCGCGG (M ϭ A ϩ C wobble) Fisher exact test showed a signiÞcant association ex- 860ra GGCGGTCGACTTAACGCGTTAGC isted between the presence of BFo-1 and BFo-2 (P ϭ 1525ra AAGGAGGTGWTCCARCC (R ϭ G ϩ A wobble) 0.001); speciÞcally, more thrips possessed both bac- BFo-1 45f GACCAAAGTGGGGGACCTTC teria compared with being uninfected or having only BFo-1 125r GTGAGCCGTTACCCCACCTA one of the bacterial types. BFo-1 probe 212p CCTCACACCATCGGATGTGCCCA Bfo-2 169f AATACCGCATAATGTCGCAAGAC Analysis of Infection Patterns Relative to Host and Downloaded from https://academic.oup.com/ee/article/38/4/1069/493304 by guest on 27 September 2021 BFo-2 275r GCCTAGGTAAGCCATTACCTTACCTAC Environmental Conditions. SigniÞcantly more thrips BFo-2 probe 212p CCTCGCACCATCAGATGAACCCAGA were infected with BFo-1, regardless of simultaneous a Primers based on the alignment published by Lane (1991). Real- presence or absence of BFo-2, in the southern valley time PCR primers denoted by references to BFo 16S rDNA. Numbers and mountain regions (F ϭ 60, P ϭ 0.05; Fig. 1). refer to approximate 16S-bp position. Infection with BFo-1, regardless of the presence or absence of BFo-2, was also signiÞcantly associated way analysis of variance (ANOVA) with terms for with the collection month average minimum temper- BFo-1, BFo-2, temperature, and interactions. ModelÞt ature and the collection year average minimum tem- ϭ Ϫ Ϯ ␹2 ϭ ϭ was assessed using graphical analysis of visuals and the perature (P 0.04, 0.067 0.033, 4.17 and P Ϯ ␹2 ϭ Shapiro-Wilks test for normality. Post hoc tests were 0.05, 0.115 0.059, 3.87, respectively). As min- performed using Tukey-Kramer adjustments for mul- imum monthly and yearly temperatures increased by Њ tiple comparisons. 1 C, the likelihood of a thrips being infected with To examine how temperature might inßuence each BFo-1 decreased by 6 and 10%, respectively. Altitude bacteria type, we performed a growth curve analysis was marginally signiÞcant relative to BFo-1 infection ϭ of cultured BFo when raised at 25 and 30ЊC. BFo-1 and (P 0.08), with the likelihood of seeing BFo-1 in- BFo-2 from previously studied cultures (Chanbusara- creasing by 0.1% with every meter increase in eleva- kum and Ullman 2008) were grown on LB agar plates tion. Other environmental characteristics and host (Sigma, St. Louis, MO). A single colony forming unit traits were not signiÞcantly linked to overall BFo-1 (cfu) was transferred to LB broth and allowed to grow infection success. overnight at the experiment temperature. The bacte- In contrast, BFo-2, regardless of the presence of rial culture was diluted 10Ϫ5 and allowed to grow for BFo-1, was not signiÞcantly associated with environ- 24 h. Periodically, we took a sample of the growing mental characteristics analyzed. Only the host char- culture and plated dilutions of the bacteria onto LB acteristic of sex seemed to share a link with BFo-2 agar plates. The plates were incubated at the same infection: males tended to be infected (81%) rather temperature as the cultures and cfus were counted the than not (P ϭ 0.06). next morning. Total colonies in the sample at a given Thrips located in the southern valley and mountain time were calculated. regions were signiÞcantly more likely to have both bacteria (ϩϩ) than either or no bacteria (P ϭ 0.03; Fig. 1). An association between dual infection and Results environmental measurements was not observed, how- Prevalence and Distribution of BFo-1 and BFo-2 in ever. The prevalence of dual infections was also not Natural F. occidentalis Populations. Infection patterns signiÞcantly different between the two southern re- of natural California F. occidentalis populations were gions. Males tended to have dual infections more than not signiÞcantly related to collection time or thrips other infections (P ϭ 0.06). host plant. One half of the thrips studied possessed When comparing infection with only BFo-1 (ϩϪ) both bacteria, 8 and 24% had only BFo-1 or BFo-2, to other infection patterns (ϩϩ, Ϫϩ, ϪϪ), the respectively, and 18% of thrips surveyed had neither amount of precipitation for the collection year was bacterium. Infection patterns of individual thrips from marginally signiÞcant (P ϭ 0.06, Ϫ0.018 Ϯ 0.096, ␹2 ϭ

Table 3. F. occidentalis examined in search for novel thrips symbionts

Site (city, county) Collected N Plant species GPS A Hallelujah Junction, Lassen 14 June 2004 10 Melilotus indica 39.8, Ϫ120.0 B BakersÞeld, Kern 5 June 2004 1 Chrysanthemum 35.3, Ϫ119.3 C Santa Monica, Los Angeles 24 April 2004 1 Chapparel pea 34.0, Ϫ120.3 D Petaluma, Sonoma 11 June 2004 10 Agapanthus 38.2, Ϫ121.6 E Davis, Yolo 29 June 2004 10 Rosa 38.5, Ϫ122.8

Letters indicate locations on collection map (Fig. 1). 1074 ENVIRONMENTAL ENTOMOLOGY Vol. 38, no. 4

Table 4. Bacteria found from clones of samples

Total BFo-1 BFo-2 Non-BFo clones Phyla sequences Site (locale, county) Non-BFo accession numbers clones clones clones matched Hallelujah Junction, Lassen 9 0 2 511 Cyanobacteriaa Bacteriodetes EU863588 EU863589 EU863590 ␤-Proteobacteria BakersÞeld, Kern 10 3 0 7 ␥-Proteobacteria EU863591 Santa Monica, Los Angeles 6 1 0 23 ␥-Proteobacteria Þrmicutes EU863592 EU863593 Petaluma, CA 16 0 15 1 ␤-Proteobacteria EU863594 Davis, Yolo 12 10 0 11 ␣-Proteobacteria Cyanobacteriaa EU863586 EU863587

Phyla determined by examination of the top three matches from Blastn search. Genbank accession numbers for new sequences in last column. a Identical sequences. Downloaded from https://academic.oup.com/ee/article/38/4/1069/493304 by guest on 27 September 2021 3.57), with the odds of BFo-1 alone infecting thrips control (Fig. 2). Interestingly, we saw a shift between increasing by 0.02% with every millimeter increase in the bioÞlm levels of BFo-1 and BFo-2 as temperature precipitation. Infection of thrips with BFo-2 alone increased: BFo-1 at 25ЊC had a weak bioÞlm not sta- (Ϫϩ) approached a signiÞcant association with col- tistically greater than the sterile control, but created lection month average minimum temperature (P ϭ a signiÞcant amount of bioÞlm at 32ЊC. BFo-2 had the 0.09): as minimum average temperature increased by highest bioÞlm readings at 25ЊC, but no detectable 1ЊC, the odds of seeing BFo-2 only infections increased bioÞlm at 32ЊC. by 16%. A lack of infection with either BFo was not At each temperature studied, BFo-1 and BFo-2 signiÞcantly linked to host or environmental condi- growth rates were not signiÞcantly different from one tions. another (doubling time Ϸ45 min), although cfus from Investigation of Other Potential Symbionts. Efforts overnight cultures of BFo-1 were signiÞcantly higher to Þnd other F. occidentalis symbionts showed no new than BFo-2 (1.277 Ϯ 0.649, df ϭ 89, P ϭ 0.007). Growth species of closely associated bacteria from the thrips rates of both bacteria were not surprisingly signiÞ- populations studied. Studies of bacterial 16S rDNA cantly lower at 25ЊC compared with 30ЊC(Ϫ0.107 Ϯ cloned from thrips often matched BFo-1 or BFo-2, 0.054, df ϭ 89, P ϭ 0.04). although both bacteria were not detected in clones from the same sample (Table 4). Other bacteria de- Discussion tected in DNA extractions included environmental bacteria belonging to a variety of Phyla (Table 4). Our data showed that BFo-1 and BFo-2 infect con- Only two sites had identical bacterial clones; all other temporary F. occidentalis populations in diverse hab- clones varied by Ͼ90% 16S rDNA. itats and environments in California. The prevalence Influence of Temperature on BFo Growth and Bio- of these speciÞc bacteria with wild F. occidentalis re- film. Temperature and each bacteria type were sig- gardless of plant host, collection time, environmental niÞcantly associated with bioÞlm production (F ϭ characteristics, or host attributes conÞrms laboratory- 16.18; df ϭ 2; P ϭ 0.0001). At both temperatures an- based indications that this relationship is stable and alyzed, when both bacteria were present in the cul- widespread, Þtting the traditional deÞnition of a sym- ture, bioÞlm levels signiÞcantly surpassed the sterile biosis (Sapp 2004) and supporting the contention that

Fig. 2. OD readings of crystal violet-stained bioÞlms at 25 and 32ЊC. *Readings signiÞcantly higher than respective controls (P ϭ 0.05). Deviation bars are shown. August 2009 CHANBUSARAKUM AND ULLMAN:ENVIRONMENT, F. occidentalis, AND GUT BACTERIA 1075

F. occidentalis have a symbiotic relationship with The presence or absence of certain aphid secondary BFo-1 and BFo-2 (Chanbusarakum and Ullman 2008). symbionts has been linked to host plants, parasitoid de- Interestingly, BFo-1 and BFo-2 interact with one an- fense, and pathogen defense (Oliver et al. 2005, 2008; other, and their association with the thrips host is inßu- Scarborough et al. 2005. Our data suggest certain abiotic enced by region and, in some cases, environment. The factors readily affect symbionts colonization patterns; statistically signiÞcant association between BFo-1 and further study is needed to understand how biotic factors BFo-2 infection and the prevalence of dual infection might affect such a relationship. suggest this may be a relationship in the early stages of Mobility of F. occidentalis adults may have also con- bacterial mutualism. Notably, the ability of each bacte- founded the data. Bacterial infection is known to occur rium to survive without the other indicates the beneÞts during the larval stages (de Vries et al. 2001b), which are of association may be minor or also exact a cost, as is often wingless, whereas this study focused on adult thrips who seen with organisms that can thrive both independently are winged and migratory (Pickett et al. 1988). More

of and associated with one another (Frank 1995, Poulsen deÞnitive relationships between infection and environ- Downloaded from https://academic.oup.com/ee/article/38/4/1069/493304 by guest on 27 September 2021 et al. 2003, Oliver et al. 2006). The association observed ment may have emerged if it were possible to study between certain symbiont infections and monthly col- infection in naturally occurring larval populations, which lection temperature has been seen before in other in- would have allowed us to analyze relationships before sectÐbacteria symbioses. A number of pea aphid sec- migration to new environments could occur. This is not ondary symbionts have conveyed heat tolerance to their currently possible because nondestructive species iden- host, increasing the hostÕs rate of survival and fecundity tiÞcation methods are only possible with adult insects. (Chen et al. 2000, Russell and Moran 2006). Recently, Thus, our analysis of adults provided the best contem- mutations in the aphid primary symbiont Buchnera have porary idea of the relationships among F. occidentalis, been linked to temperature tolerance in the host (Dun- their bacteria, and the environment. bar et al. 2007). Many bacteria are affected by temper- It is unclear why a marginally signiÞcant link existed ature variations, and the resulting biological changes in between certain bacterial infections (ϩϩ and Ϫϩ) and microorganisms could inßuence how well the microbes thrips sex. This is a very unusual relationship that has not might colonize their host. Similarly, the indicated impact to our knowledge been reported elsewhere. In most of altitude on organisms has also been seen in other study cases, where a sex bias has been reported relative to a systems. Examinations of insects and mammals have symbiosis, the population has been female-biased or all shown that altitude gradients can inßuence biological female (Werren 1997, Bourtzis and OÕNeill 1998, Hurst processes such as hatch rate and organ size (Trapani and and Jiggins 2000, Arakaki et al. 2001). Although we did Campbell 1959; Fielding et al. 1999; Hammond et al. 1999, not design our study to look at sex ratios of each collec- 2001). It would not be surprising to see similar effects in tion site, the presence of males and the ability of F. thrips and their microorganisms at different altitudes. A occidentalis to lose bacterial symbionts on reaching more in-depth investigation into this system could shed adulthood (de Vries et al. 2001b) leads us to believe more light on this potentially complicated relationship. thrips bacteria to not engage in sex manipulation. Males Given the signiÞcant relationship between dual infec- may simply be more susceptible to continual BFo-1 and tion and the southern valley and southern mountain BFo-2 colonization compared with their female coun- regions, we were surprised that there were not similarly terparts. strong relationships between dual infection and the en- BFo-1 and BFo-2 seem to be recurring facultative vironmental characteristics of each region. A lack of clear bacterial symbionts within F. occidentalis populations. link between dual infection and environmental charac- Although BFo-1 and BFo-2 were not detected in the teristics may be caused by limited sample size. When same batch of cloned thrips, we found it interesting BFo-1 only infections (ϩϪ) were analyzed in conjunc- that one of the types was detected in each cloned tion with dual infections (ϩϩ), a signiÞcant relationship sample. We did not see such prevalence with any emerged for BFo-1 infections, not only with southern other bacteria analyzed from our samples. This sug- valley and southern mountain regions, but also with av- gests BFo-1 and BFo-2 are the main bacteria associated erage minimum monthly and yearly temperatures and with natural thrips populations. with altitude. These associations were not detected if We did not Þnd a deÞnitive explanation for why dual each infection pattern was examined independently. infection was most prevalent or why certain infection Thus, a larger sample size may be needed to determine patterns were favored in certain environments. Among such intricate associations. The data we collected do the possibilities we considered were bacterial bioÞlm suggest that bacterial infections that involve BFo-1 alone production and bacterial growth in different conditions and/or in dual infection may be favored in environments (i.e., temperature). Although our in vitro data cannot be at increasing altitude, with cooler temperatures and transferred directly to in vivo behavior, BFo-1 and BFo-2 higher precipitation. Further studies may show other produced bioÞlms differentially. Theoretically, the associations and may clarify marginally signiÞcant links, stronger bioÞlm in dual cultures might help maintain the such as that between BFo-2 only infection and collection potential adhesive, defensive, communicative, and nu- month minimum temperature. trient-sharing beneÞts of a bioÞlm (Costerton et al. 1987; Food quality and predation may also affect the thripsÐ James et al. 1995; OÕToole et al. 2000; Lewis 2001; Suth- microbe relationship in ways not yet understood. Sur- erland 2001a, b; Jefferson 2004; Romanova et al. 2006; veys of psyllids found parasitism was associated with the Sauer et al. 2007) without requiring both bacteria to presence of facultative symbionts (Hansen et al. 2007). make their own bioÞlm at all times and in all conditions. 1076 ENVIRONMENTAL ENTOMOLOGY Vol. 38, no. 4

Further studies may want to focus on the relevance of Brown, M. R., and J. Barker. 1999. Unexplored reservoirs of BFo-1 and BFo-2 bioÞlm formation to their occurrence pathogenic bacteria: protozoa and bioÞlms. Trends Mi- and survival in thrips living at different temperatures. crobiol. 7: 46Ð50. Previous observations of BFo-1 and BFo-2 in culture Chanbusarakum, L., and D. Ullman. 2008. Characterization and plates showed that BFo-1 was more abundant than of bacterial symbionts in Frankliniella occidentalis (Per- BFo-2 at room temperature (Chanbusarakum and Ul- gande), western ßower thrips. J. Invertebrate Pathol. 99: lman 2008). This analysis conÞrmed that BFo-1 is more 318Ð325. Chen, D.-Q., C. B. Montllor, and A. H. Purcell. 2000. Fitness abundant within overnight cultures; however, the two effects of two facultative endosymbiotic bacteria on the bacterial types actually have similar growth rates. We pea aphid, Acyrthosiphon pisum, and the blue alfalfa believe this observation was caused by the delayed aphid, A. kondoi. Entomol. Exp. Appl. 95: 315Ð323. growth by BFo-2 when moved from an agar plate to Costerton, J. W., K. J. Cheng, G. G. Geesey, T. I. Ladd, J. C. nutrient broth. Because we know the bacteria grow at Nickel, M. Dasgupta, and T. J. Marrie. 1987. Bacterial equal rates when reared at the same temperature, it is bioÞlms in nature and disease. Annu. Rev. Microbiol. 41: Downloaded from https://academic.oup.com/ee/article/38/4/1069/493304 by guest on 27 September 2021 likely BFo-1 adapts more quickly to the liquid broth 435Ð464. and would therefore have a head start multiplying de Vries, E. J., J.A.J. Breeuwer, G. Jacobs, and C. Mollema. compared with BFo-2. This supports previous Þndings 2001a. The association of western ßower thrips, Fran- of metabolic differences between the two types of kliniella occidentalis, with a near Erwinia species gut bac- bacteria (Chanbusarakum and Ullman 2008) and pro- teria: transient or permanent? J. Invertebrate Pathol. 77: vides more support for BFo-1 and BFo-2 to be con- 120Ð128. de Vries, E. J., G. Jacobs, and J.A.J. Breeuwer. 2001b. sidered two unique species. Growth and transmission of gut bacteria in the western Thrips infected with BFo-1 and BFo-2 occurred on ßower thrips, Frankliniella occidentalis. J. Invertebrate a wide diversity of plant hosts and in virtually every Pathol. 77: 129Ð137. environment and habitat in which we sampled. Par- de Vries, E. J., G. Jacobs, M. W. Sabelis, S.B.J. Menken, and ticular bacterial infection patterns were inßuenced by J.A.J. Breeuwer. 2004. Diet-dependent effects of gut environmental characteristics, suggesting that alti- bacteria on their insect host: the symbiosis of Erwinia sp. tude, temperature, and precipitation may exert an and western ßower thrips. Proc. R. Soc. Lond. Ser. B. Biol. inßuence on the insectÐmicrobe relationship. Our Sci. 271: 2171Ð2178. Þndings relative to bacterial growth and bioÞlm for- Doolittle, M., A. Raina, A. Lax, and R. Boopathy. 2008. Pres- mation at different temperatures raise the hypothesis ence of nitrogen Þxing Klebsiella pneumoniae in the gut that infection pattern variation in particular environ- of the Formosan subterranean termite (Coptotermes for- ments may occur because of effects on these biological mosanus). Bioresource Technol. 99: 3297Ð3300. Dunbar, H. E., A.C.C. Wilson, N. R. Ferguson, and N. A. processes. These data provided increasing support for Moran. 2007. Aphid thermal tolerance is governed by a considering BFo-1 and BFo-2 as two unique species point mutation in bacterial symbionts. PLoS Biol. 5: e96. and began unraveling the potential interactions oc- Fielding, C. A., J. B. Whittaker, J.E.L. Butterfield, and J. C. curring between them. Further analyses of this system Coulson. 1999. Predicting responses to climate change: should focus on the driving forces behind this wide- the effect of altitude and latitude on the phenology of the spread relationship and seek to clarify how these bac- Spittlebug Neophilaenus lineatus. Funct. Ecol. 13: 65Ð73. teria impact their thrips host. Frank, S. A. 1995. The origin of synergistic symbiosis. J. Theoret. Biol. 176: 403Ð410. Gomathi, V., A. Ramalakshmi, and K. Ramasamy. 2005. Iso- lation of nitrogen Þxing bacteria from fungus termites. Acknowledgments Entomol. Res. 35: 75Ð78. We thank J. Braun for statistical assistance and C. Hammond, K. A., J. Szewczak, and E. Krol. 2001. Effects of OÕDonnell and C. Zietlow for plant identiÞcation assistance. altitude and temperature on organ phenotypic plasticity along an altitudinal gradient. J. Exp. Biol. 204: 1991Ð2000. Hammond, K. A., J. Roth, D. N. Janes, and M. R. Dohm. 1999. Morphological and physiological responses to altitude in References Cited deer mice Peromyscus maniculatus. Physiol. Biochem. Arakaki, N., T. Miyoshi, and H. Noda. 2001. Wolbachia-me- Zool. 72: 613Ð622. diated parthenogenesis in the predatory thrips Fanklin- Hansen, A. K., G. Jeong, T. D. Paine, and R. Stouthamer. tothrips vespiformis (Thysanoptera: Insecta). Proc. R. 2007. Frequency of secondary symbiont infection in an Soc. Lond. Ser. B. Biol. Sci. 268: 1011Ð1016. invasive psyllid relates to parasitism pressure on a geo- Boonham, N., P. Smith, K. Walsh, J. Tame, J. Morris, N. graphic scale in California. Appl. Environ. Microbiol. 73: Spence, J. Bennison, and I. Barker. 2002. The detection 7531Ð7535. of Tomato spotted wilt virus (TSWV) in individual thrips Harada, H., H. Oyaizu, Y. Kosako, and H. Ishikawa. 1997. using real time ßuorescent RT-PCR (TaqMan) J. Virol. Erwinia aphidicola, a new species isolated from pea aphid, Methods 101: 37Ð48. Acyrthosiphon pisum. J. Gen. Appl. Microbiol. 43: 349Ð Bourtzis, K., and S. O’Neill. 1998. Wolbachia infections and 354. reproductionÑWolbachia can cause cytoplas- Harder, W., and H. Veldkamp. 1971. Competition of marine mic incompatibility, parthenogenesis, and feminization in psychrophilic bacteria at low temperatures. Antonie van many . Bioscience 48: 287Ð293. Leeuwenhoek 37: 51Ð63. Branda, S. S., A. Vik, L. Friedman, and R. Kolter. 2005. Hurst, G.D.D., and F. M. Jiggins. 2000. Male-killing bacteria BioÞlms: the matrix revisited. Trends Microbiol. 13: in insects: mechanisms, incidence, and implications. 20Ð26. Emerg. Infect. Dis. 6: 329Ð336. August 2009 CHANBUSARAKUM AND ULLMAN:ENVIRONMENT, F. occidentalis, AND GUT BACTERIA 1077

James, G. A., L. Beaudette, and J. W. Costerton. 1995. In- Poulsen, M., A.N.M. Bot, C. R. Currie, M. G. Nielsen, and J. J. terspecies bacterial interactions in bioÞlms J. Industrial Boomsma. 2003. Within-colony transmission and the Microbiol. Biotechnol. 15: 257Ð262. cost of a mutualistic bacterium in the leaf-cutting ant Jefferson, K. K. 2004. What drives bacteria to produce a Acromyrmex octospinosus. Funct. Ecol. 17: 260Ð269. bioÞlm? FEMS Microbiol. Lett. 236: 163Ð173. Romanova, Y. M., T. A. Smirnova, A. L. Andreev, T. S. Il’ina, Kaufman, M. G., and M. K. Klug. 1991. The contribution of L. V. Didenko, and A. L. Gintsburg. 2006. Formation of hindgut bacteria to dietary carbohydrate utilization by bioÞlms as an example of the social behavior of bacteria. crickets (Orthoptera: Gryllidae). Comp. Biochem. Microbiology 75: 481Ð485. Physiol. A. 98A: 117Ð123. Russell, J. A., and N. A. Moran. 2006. Costs and beneÞts of Lane, D. J. 1991. 16s/23s rRNA sequencing, pp. 115Ð176. In symbiont infection in aphids: variation among symbionts E. Stackebrandt and M. Goodfellow (eds.), Nucleic acid and across temperatures. Proc. Biol. Sci. 273: 603Ð610. techniques in bacterial systematics. Wiley, Chichester, Sapp, J. 2004. The dynamics of symbiosis: an historical over- United Kingdom. view. Can. J. Bot. 82: 1046Ð1056. Lemos, F. J., and W. R. Terra. 1991. Digestion of bacteria SAS Institute. 2004. SAS/STAT 9.1 userÕs guide. SAS Insti- Downloaded from https://academic.oup.com/ee/article/38/4/1069/493304 by guest on 27 September 2021 and the role of midgut lysozyme in some insect larvae. tute, Cary, NC. Comp. Biochem. Physiol. B. 100: 265Ð268. Sauer, K., A. H. Rickard, and D. G. Davies. 2007. BioÞlms Lewis, K. 2001. Riddle of bioÞlm resistance. Antimicrob. and biocomplexity. Microbe 2: 347Ð353. Agents Chemother. 45: 999Ð1007. Scarborough, C. L., J. Ferrari, and H. C. Godfray. 2005. McClendon, B. 2007. Google Earth computer program, ver- Aphid protected from pathogen by endosymbiont. Sci- sion 3.0. Google, Inc. Mountain View, CA. ence 310: 1781. Merritt, J. H., D. E. Kadouri, and G. A. O’Toole. 2005. Grow- Schaudinn, C., P. Stoodley, A. Kainovic, T. O’Keeffe, B. Cos- ing and analyzing static bioÞlms. Wiley, Hoboken, NJ. terton, D. Robinson, M. Baum, G. Ehrlich, and P. Web- O’Donnell, C. 2007. Color morphology of the western ster. 2007. Bacterial bioÞlms, other structures seen as ßower thrips of California and virus-vector relationships mainstream concepts. Microbe 2: 231Ð237. in the : Thyasnoptera. Entomology, Univer- Sutherland, I. 2001a. BioÞlm exopolysaccharides: a strong sity of California-Davis, Davis, CA. and sticky framework. Microbiology 147: 3Ð9. O’Donnell, C., L. Mound, and M. P. Parrella. 2000. The Sutherland, I. 2001b. The bioÞlm matrixÑan immobilized biology and identiÞcation of selected North American but dynamic microbial environment. Trends Microbiol. 9: thysanoptera associated with ornamental plants. Ento- 222Ð227. mology, University of California-Davis, Davis, CA. Thomas, L. V., and J. W. Wimpenny. 1993. Method for in- O’Toole, G., H. B. Kaplan, and R. Kolter. 2000. BioÞlm for- vestigation of competition between bacteria as a function mation as microbial development. Annu. Rev. Microbiol. of three environmental factors varied simultaneously. 54: 49Ð79. Appl. Environ. Microbiol. 59: 1991Ð1997. Ohkuma, M. 2003. Termite symbiotic systems: efÞcient bio- Trapani, I. L., and D. H. Campbell. 1959. Passive antibody recycling of lignocellulose. Appl. Microbiol. Biotechnol. decay in rabbits under cold or altitude stress. J. Appl. 61: 1Ð9. Physiol. 14: 424Ð426. Oliver, K. M., N. A. Moran, and M. S. Hunter. 2005. Varia- Watanabe, K., and M. Sato. 1998. Plasmid-mediated gene tion in resistance to parasitism in aphids is due to sym- transfer between insect-resident bacteria, Enterobacter bionts not host genotype. Proc. Natl. Acad. Sci. U.S.A. 102: cloacae, and plant-epiphytic bacteria, Erwinia herbicola, 12795Ð12800. in guts of silkworm larvae. Curr. Microbiol. 37: 352Ð355. Oliver, K. M., N. A. Moran, and M. S. Hunter. 2006. Costs Wells, M. L., R. D. Gitaitis, and F. H. Sanders. 2002. Asso- and beneÞts of a superinfection of facultative symbionts ciation of tobacco thrips, Frankliniella fusca (Thysan- in aphids. Proc. R. Soc. Lond. Ser. B. Biol. Sci. 273: 1273Ð optera: Thripidae) with two species of bacteria of the 1280. genus Pantoea. Ann. Entomol. Soc. Am. 95: 719Ð723. Oliver, K. M., J. Campos, N. A. Moran, and M. S. Hunter. Werren, J. H. 1997. Biology of Wolbachia. Annu. Rev. En- 2008. Population dynamics of defensive symbionts in tomol. 42: 587Ð609. aphids. Proc. Biol. Sci. 275: 293Ð299. Western Regional Climate Center, D.R.I. 2008. Current Pickett, C. H., L. T. Wilson, and D. Gonzalez. 1988. Popu- and historical climatological data from western regional lation dynamics and within-plant distribution of the west- climate center weather observation stations. Western Re- ern ßower thrips (Thysanoptera: Thripidae), an early- gional Climate Center, Reno, NV. season predator of spider mites infesting cotton. Environ. Entomol. 17: 551Ð559. Received 15 October 2008; accepted 24 April 2009.