INSECTÐSYMBIONT INTERACTIONS Distribution and Ecology of Frankliniella occidentalis (Thysanoptera: Thripidae) 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 thrips, 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 insects. 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 insect. 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,