Pantoea) Agglomerans and Klebsiella Pneumoniae Through All Life Stages of the Mediterranean Fruit Fly (Diptera: Tephritidae
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ARTHROPOD BIOLOGY Establishment and Vertical Passage of Enterobacter (Pantoea) agglomerans and Klebsiella pneumoniae through All Life Stages of the Mediterranean Fruit Fly (Diptera: Tephritidae) 1,2 3 1 3 C. R. LAUZON, S. D. MCCOMBS, S. E. POTTER, AND N. C. PEABODY Ann. Entomol. Soc. Am. 102(1): 85Ð95 (2009) ABSTRACT We investigated the fate of ingested Enterobacter (Pantoea) agglomerans and Klebsiella pneumoniae within adult Mediterranean fruit ßy, Ceratitis capitata (Wiedemann) (Diptera: Tephriti- dae), in a mass rearing facility. This examination revealed the establishment of both bacterial strains as bioÞlms within the adult intestines, on the apical end of developing and developed eggs, and throughout all subsequent life stages. The bacteria were detected in adults through two generations. Irradiation treatment for the sterile insect technique did not disrupt the vertical transmission of E. (P.) agglomerans or K. pneumoniae. This is the Þrst demonstration of maternal spread of Enterobacter/ Pantoea spp. and Klebsiella spp. through populations of C. capitata. A mixed pattern of vertical and horizontal transmission of symbionts associated with tephritids may be one explanation for the difÞculty in deÞning the symbiotic associations of tephritids. KEY WORDS symbiosis, reproduction, sterile insect technique The importance of bacteria in the life history of certain Tephritids consume a variety of microorganisms in pest Tephritidae has been in question since the 1930s their natural food (Drew et al. 1983, Drew and Lloyd when Allen et al. (1934) described the association 1987, Prokopy et al. 1993). Of these, species in two between Phytomonas (Pseudomonas) melopthora and bacterial genera Enterobacter/Pantoea and Klebsiella Rhagoletis pomonella Walsh, the apple maggot. Since consistently inhabit the tephritid gut (Lauzon et al. that time, the main focus of work into the nature of 1998). Lauzon et al. (2000) suggested that these two tephritidÐbacteria interactions has been the attraction bacteria participate in nitrogen cycling within the of certain pest Tephritidae to bacteria (MacCollom et tephritid gut and serve as important contributors to al. 1992, 1994; Lauzon et al. 1998, 2000) or to odors tephritid survival in nature. ArtiÞcial rearing of fruit produced by bacteria in culture (Bateman and Morton ßies, such as the Mediterranean fruit ßy, Ceratitis capi- 1981, Drew and Faye 1988, Martinez et al. 1994, tata (Wiedemann) (Diptera: Tephritidae), for control Robacker and Flath 1995, Robacker and Bartelt 1997, programs precludes the exposure to, and establish- Robacker et al. 1998, Epsky et al. 1998, Robacker and ment of, these bacterial genera from natural sources. Lauzon 2002). This work sought to establish lures for C. capitata are cultured in large-scale production fa- detection of these important agricultural pests rather cilities around the world and typically in these situa- than to determine the intimacy and meaning of any tions normal gut microbiota are absent (C.R.L., un- symbiotic relationship that exists in nature. published data). If speciÞc bacteria in the gut of C. The mechanisms by which tephritids acquire and capitata confer a Þtness advantage, then their absence maintain their symbionts have not been fully deÞned. in mass production may result in decreased Þtness of Although ingestion of bacteria is one way that tephrit- sterile males released in control programs. Indeed, ids acquire their normal gut bacteria, it has not been reports exist that describe poor performance of sterile established that this is the sole mechanism. Moreover, male ßies in the Þeld (Shelly and Whittier 1996). the type of symbiotic relationships tephritids possess Understanding the relationship between bacteria and with bacteria, i.e., facultative or obligatory, has never C. capitata in nature may allow for improvements to been conclusively determined. mass production protocols (e.g., probiotic diets) that would increase the effectiveness of sterile males. This work is part of an investigation to determine Mention or use of a particular commercial product does not con- stitute endorsement by the USDA. whether artiÞcial introduction of symbionts (i.e., pro- 1 Department of Biological Sciences, California State University, biotic diets) will appreciably improve the Þtness of East Bay, Hayward, CA 94542. mass-reared sterile males. Three experiments are de- 2 Corresponding author, e-mail: [email protected]. scribed in this article and begin to address the question 3 USDA APHIS PPQ Center for Plant Health Science and Tech- nology, Fruit Fly Genetics and Management Laboratory, Waimanalo, of symbiont establishment and spread. The Þrst ex- HI 96795. periment was conducted to monitor the fate of two 86 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 102, no. 1 endosymbionts that were ingested by C. capitata for cleansed of medium, and resuspended in sterile water the purposes of describing endosymbiont establish- to an optical density of 0.40 at 550 nm. Quality control ment and retention in the adult C. capitata gut. Our was conducted on aliquots of the bacterial diets sam- Þndings also indicated that the endosymbionts mi- pled before and after use for the presence of the grated to the ovaries and established on the apical end probiotics following standard methods (Brennan of eggs. These Þndings led to the second experiment 1992) and using API 20E biochemical identiÞcation where we monitored the fate of the endosymbionts strips (bioMerieux Vitek, Inc., Hazelwood, MO). through successive life stages of C. capitata starting After 2 d of feeding ad libitum, 30 individual male with females that ingested the endosymbionts, and and female Mediterranean fruit ßy from each group two successive generations of C. capitata thereafter. were dissected, and their alimentary canal and repro- The third experiment was designed to determine ductive organs were removed (Lauzon et al. 1998) and whether radiation exposure at 145 Gy used in the prepared for examination using confocal scanning la- sterile insect technique would disrupt the vertical ser microscopy (CLSM). transmission of the endosymbionts. CLSM. Alimentary canal and reproductive organs were placed onto clean, glass slides. Intestines were often teased apart using sterile needles (Yale hypo- Materials and Methods dermic needles, BD Biosciences., Franklin Lakes, NJ). Insect Rearing. All experiments were conducted at Approximately 20 l of antifade reagent (Citißuor, the USDA APHIS Hawaii Fruit Fly Production Facility Ltd., City University, London, United Kingdom) were (HFFPF) in Waimanalo, HI. The standard Maui-93 added to each sample. A coverslip was gently applied, strain of C. capitata (McInnis et al. 1996) was used in and the edges were sealed to the slide using Þngernail experiments 1 and 2. Maui-93 had been in production polish. The samples were optically sectioned using a at HFFPF for Ϸ9 yr. The Vienna VII temperature- 510 confocal laser scanning microscope (Zeiss Optical sensitive lethal genetic sexing strain (Franz et al. 1996) Systems, Inc., Thornwood, NY) located at The Bio- used in experiment 3 was obtained from the California logical Imaging Facility at The University of California Department of Food and Agriculture Medßy Rearing in Berkeley, CA. Facility in Waimanalo, HI. Scanning Electron Microscopy (SEM). Intestines Transformation of Endosymbionts. Strains of P (E.) from 15 males and 15 females from each group were agglomerans herein referred to as E. agglomerans, and removed and placed individually on Thermanox plas- K. pneumoniae of tephritid origin were previously tic sterile coverslips (Nunc Brand Products, Nalge transformed to express the ßuorescent protein en- Nunc International, Naperville, IL) coated with poly- hanced green ßuorescent protein (EGFP) (Peloquin L-lysine (Ted Pella, Inc., Redding, CA). Once the et al. 2000), or they were transformed (Sambrook et samples were afÞxed to coverslips, they were im- al. 2000) to express DsRed as follows: bacterial cells mersed into 2.5% glutaraldehyde (Ted Pella, Inc.) in were grown to late-stationary phase in Luria Bertani 0.05 M cacodylate buffer, pH 7.2, for1hat4ЊC. Eggs (LB) broth (Difco, Detroit, MI) and centrifuged at were removed and placed into microfuge tubes con- 10,000 ϫ g for 15 min at 18ЊC. The bacterial pellet was taining the same Þxative in buffer and held for1hat resuspended in 250 l of ice-cold calcium chloride 4ЊC. All samples were rinsed twice for 10 min each (aq) in microfuge tubes and kept on ice. Ten micro- using 0.05 M cacodylate buffer, pH 7.2, for1hatroom liters of DsRed plasmid DNA (Invitrogen, Carlsbad, temperature, followed by two 10-min rinses using the CA.) was added to the bacteria:calcium chloride so- cacodylate buffer, and two additional 10-min rinses lution, the solution was mixed well, and returned to ice using distilled water, pH 7.0. Samples were postÞxed for 15 min. Bacterial cells were then heat-shocked in using osmium tetroxide and dehydrated in a graded a42ЊC water bath for 90 s under gentle agitation. Cells ethanol series: 50, 70, 95 (two times), 100% (three were placed in ice for 1 min and received 250 lofLB times) for 10 min each. Samples were infused with broth. After 10 min, cells were plated onto LB agar and CO2 and processed accordingly in a critical point incubated overnight at 24ЊC. Transformed bacterial dryer (Polaron Instruments, Inc., HatÞeld, PA). The cells displayed a pink-red colonial phenotype and samples were then individually mounted on aluminum were conÞrmed