Pantoea) Agglomerans and Klebsiella Pneumoniae Through All Life Stages of the Mediterranean Fruit Fly (Diptera: Tephritidae

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 by ßuorescence using a ßuorescent stubs (Ted Pella, Inc.) with carbon conductive adhe- microscope (Leica, Heerbrugg, Switzerland). Trans- sive (Ted Pella, Inc.). The edges of the coverslips were formation was determined to be stable through tacked down to the stub with either colloidal silver numerous subcultures. liquid or Electrodag 502 (Ted Pella, Inc.). Samples Experiment 1. Six hundred (100 ßies ϫ 3 repli- were sputter-coated (Hummer VII Sputter Coating cates ϫ 2 groups) newly eclosed adults were provi- System, Anatech, Ltd., SpringÞeld, VA) with 30 nm of sioned with the following diets for 2 d: sucrose/yeast gold-palladium. A Philips XL-40 scanning electron mi- hydrolysate enzymatic (3:1, vol:vol), or the same su- croscope (FEI/Philips, Eidenhoven, The Nether- crose/yeast hydrolysate enzymatic diet containing lands) at an accelerating voltage of 12 kV was used to ßuorescently marked E. agglomerans and K. pneu- view these samples. moniae (e.g., probiotic mixture). The ßies had a con- Experiment 2. Sixty adult females fed ad libitum on tinuous water supply. The probiotic mixture was either probiotic-amended standard diet or standard diet, added to the dry diet and included 750 ␮l of individual and water for 2 d. Females were transferred to clean preparations of bacteria grown to mid-log phase, cages containing males, standard diet and water. Fertil- January 2009 LAUZON ET AL.: VERTICAL TRANSFER OF SYMBIONTS IN C. capitata 87

Fig. 1. CLSM image of ßuorescent bacteria, EGFP K. pneumoniae and DsRed E. agglomerans, assembled as a bioÞlm in a Mediterranean fruit ßy esophageal bulb. Blue regions (arrows) indicate colocalization of the bacterial species. MagniÞcation, 25ϫ. (Online Þgure in color.) ized eggs were collected, seeded onto larval diet, and faces, within the alimentary canal and on the egg pupae were collected. Pupae were held until eclosion. surface of adult C. capitata after ingestion (Figs. 1Ð3). Some adults were killed and examined for the presence The bioÞlm Þlled the insect lumen (Fig. 2). The bio- of the ßuorescent probiotics. Others were kept, and the Þlms within esophageal bulbs, and crops were not as process was repeated for two subsequent generations. dense and seemed architecturally less complex than Adult ßies were transferred to previously unused cages bioÞlms within the intestines of the ßies. The distri- and given fresh standard adult diet and water routinely. bution of E. agglomerans and K. pneumoniae seemed Experiment 3: Irradiation of Pupae Containing E. random throughout most areas of the bioÞlms. How- agglomerans and K. pneumoniae. One hundred Medi- ever, using software for the CLSM that determines terranean fruit ßy eggs were artiÞcially seeded with E. overlap of emission signals, we found that E. agglom- agglomerans and K. pneumoniae and reared to the pupal erans and K. pneumoniae colocalized at openings stage. Fifty microliters of a mixture of the bacteria was within bioÞlms (Fig. 4). applied to eggs on Þlter paper. Specimens from all life E. agglomerans and K. pneumoniae were found in stages were sampled to conÞrm the presence of the ovaries and assembled as bioÞlms on the surface of ßuorescently marked bacteria by using CLSM. Pupae developing eggs, developed eggs, or both (see below; were placed into petri dishes and sealed within Zip-Loc Fig. 10a and b). The two bacterial species colocalized, bags (S.C. Johnson & Son, Inc., Racine, WI) for 30 min. and the degree of colocalization was greater than that Pupae were irradiated at 145 Gy following the standard observed with digestive bioÞlms (data not shown). protocol for Mediterranean fruit ßy pupae used in sterile BioÞlms on egg surfaces were less dense than those insect release programs. Equal numbers of irradiated and observed in intestines, but they were thicker than nonirradiated pupae were placed within separate insect those observed in esophageal bulbs and crops. E. ag- cages that contained standard adult diet and water until glomerans and K. pneumoniae were not found in male they emerged as adult ßies. Adults were allowed to feed reproductive organs. Neither ßuorescently marked for2donstandard diet and water and then were sac- bacteria were found in adults that fed on standard diet. riÞced by freezing. The ßies were dissected and exam- Fate of E. agglomerans and K. pneumoniae in Con- ined using CLSM described above. CLSM will conÞrm secutive Generations of C. capitata. E. agglomerans the presence, location, and arrangement of transformed and K. pneumoniae were detected in all life stages of endosymbionts. Mediterranean fruit ßy through two generations. Upon visual inspection, adult C. capitata that acquired E. agglomerans and K. pneumoniae through feeding Results had the greatest amount of each bacterial species Fate of E. agglomerans and K. pneumoniae in C. within their alimentary canal organs. Two consecutive capitata. E. agglomerans and K. pneumoniae estab- generations of C. capitata both had ßuorescent E. lished as bioÞlms, or aggregates adhered to tissue sur- agglomerans and K. pneumoniae in each life stage 88 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 102, no. 1

Fig. 2. CLSM image of ßuorescent bacteria, EGFP K. pneumoniae and DsRed E. agglomerans, assembled as a bioÞlm in the midgut of a Mediterranean fruit ßy. MagniÞcation, 63ϫ. (Online Þgure in color.)

(Figs. 5Ð7). We detected only a few bacterial cells in have been satisÞed, such as responses of planktonic each stage, with the exception of adults which had bacteria to each other, the environment, and a surface moderate numbers of each bacteria established within (Loo et al. 2000). Thus, the establishment of E. agglo- their gut. merans and K. pneumoniae as a bioÞlm in the C. capi- Scanning Electron Microscopic Examination of tata gut suggests that these bacteria naturally and Eggs and Intestines. Intestinal bioÞlms were massive routinely assemble there. Enterobacter and Klebsiella and seemed to occlude the lumen (Fig. 8). The ridged spp. are bacterial species most consistently isolated surface of the apical end of dissected C. capitata eggs from several tephritid species, and their assembly as a supported the attachment of bacteria (Fig. 9). Bacte- bioÞlm may explain why. ria were observed within the conÞnes of the micropyle BioÞlms are metabolically active structures (Cos- (Fig. 10a). A similar pattern of establishment is shown terton et al. 1995) that often operate as a coordinated using CLSM (Fig. 10b). unit, i.e., a digestive organ, and they are complex and Radiation Effects on the Spread of E. agglomerans diverse in their metabolic abilities. Lauzon et al. and K. pneumoniae. E. agglomerans and K. pneumoniae (2000) suggested that E. agglomerans and K. pneu- were detected in adult C. capitata intestines that were moniae jointly participate in the catabolism of nitro- irradiated previously as pupae (Fig. 11). No apprecia- gen in the gut of R. pomonella. Behar et al. (2005) ble variation between nonirradiated and irradiated demonstrated nitrogen Þxation in the gut of C. capitata ßies was seen. in which Enterobacteriaceae were the dominant microbiota. The colocalization of E. agglomerans and K. pneumoniae at areas where food from the gut lumen Discussion would enter the bioÞlm supports this hypothesis. Bio- We found that E. agglomerans and K. pneumoniae Þlms within C. capitata may serve functions beyond establish rapidly after ingestion and form a bioÞlm in that of nitrogen cycling (Lauzon et al. 2000), although the gut of female and male C. capitata. The bioÞlm was we can only speculate about these functions now. extensive and contained many open pores, channels, Lauzon et al. (2003) found that E. agglomerans de- and arteries that ranged in length and width. E. ag- grades and detoxiÞes phloridzin, a toxic and potent glomerans and K. pneumoniae colocalized near the antifeeding compound typical of Malus spp. found in entry sites of the bioÞlm. The rapid establishment and plant leachate. The intestinal bioÞlm likely catabolizes coordinated assembly of microorganisms as bioÞlms a variety of compounds that enter the insect gut, such (Costerton et al. 1987, 1995; Davies et al. 1998; OÕToole as plant antifeeding compounds, before they reach and Kolter 1998) come only after prerequisite criteria epithelial tissue. The thickness of the intestinal bioÞlm January 2009 LAUZON ET AL.: VERTICAL TRANSFER OF SYMBIONTS IN C. capitata 89

Fig. 3. CLSM image of ßuorescent bacteria, EGFP K. pneumoniae and DsRed E. agglomerans, assembled as a bioÞlm on an egg surface. MagniÞcation, 63ϫ. (Online Þgure in color.) may physically protect epithelial tissue from toxins, agglomerans and K. pneumoniae to the diet of mass- toxicants, or irritants that C. capitata might ingest. reared male C. capitata increases their mating efÞ- The bioÞlm Þlled the gut lumen forming both nar- ciency (Niyazi et al. 2004). Lauzon and Potter (2008) row and large passages for food and water to meander also found that the gut of C. capitata damaged by or ßow through. The thickness and architecture of the irradiation at the standard 145 Gy for sterilization of bioÞlm may regulate the transit time of food and water males released in control programs improved when passing through the gut (Gjersing et al. 2005). This these irradiated adults consumed a diet that contained would allow for maximized digestion and absorption E. agglomerans and K. pneumoniae. of nutrients, efÞcient removal of materials, or both. It The question remains, however, of how the Þtness of is possible that the bioÞlm acts both as a nonspeciÞc E. agglomerans and K. pneumoniae increases. Both bac- digestive organ and a protective barrier for C. capitata terial species can, and do, exist in a free-living state in and other tephritids. nature (Brennan 1992). It is possible that these bacteria We found that E. agglomerans and K. pneumoniae survive and reproduce more readily in the gut of C. migrate per os to the ovaries and assemble in a bioÞlm capitata than in water, soil, or plant environments. at the apical end of developing and developed eggs. Tephritids consume a variety of microorganisms Thus, E. agglomerans and K. pneumoniae are vertically while feeding on natural food substrates (Lauzon et al. transmitted in C. capitata. To our knowledge, this is the Þrst report of maternal transmission of symbionts in 1998) Adult C. capitata and other tephritids are known tephritids. The passage of these two bacteria from to acquire E. agglomerans and K. pneumoniae through female to egg strongly suggests that these bacteria are natural food sources that have a high nitrogen content, important in the life history of C. capitata. i.e., bird feces and insect frass on leaf surfaces (un- It is generally accepted if symbionts are spread published data). We found that E. agglomerans and K. maternally through a host population, then two hostÐ pneumoniae ingested by adult C. capitata are passed symbiont relationships likely occur, mutualism or par- vertically through all life stages and along successive asitism (Dedeine et al. 2003). Mutualism includes a generations. The numbers of ßuorescently marked reciprocal increase in Þtness for the host and the bacteria varied among life stages and tissues. The adult symbiont and is the likely association that exists be- intestine, the egg surface in the female reproductive tween E. agglomerans, K. pneumoniae, and C. capitata. tract, and the egg surface postoviposition seem to Several observations support the hypothesis that the support the greatest numbers of bacteria, respectively. presence of E. agglomerans and K. pneumoniae in- Very few bacteria were detected in larval and pupal crease the Þtness of C. capitata. The addition of E. samples and were not detected at all in the testes. 90 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 102, no. 1

Fig. 4. CLSM image of colocalization of EGFP K. pneumoniae and DsRed E. agglomerans, shown as blue regions. Notice localization dominates near openings within the bioÞlm. MagniÞcation, 63ϫ. (Online Þgure in color.)

Mixed patterns of symbiont transmission behavior of symbionts associated with tephritids may be one in insects are not uncommon (Agnew et al. 2003). A explanation for the difÞculty in deÞning the symbiotic mixed pattern of vertical and horizontal transmission associations of tephritids. This mixed pattern of sym-

Fig. 5. CLSM image of a second generation larval gut containing EGFP K. pneumoniae and DsRed E. agglomerans cells indicated by arrows. MagniÞcation, 63ϫ. (Online Þgure in color.) January 2009 LAUZON ET AL.: VERTICAL TRANSFER OF SYMBIONTS IN C. capitata 91

Fig. 6. CLSM image of internal structures within a second generation Mediterranean fruit ßy pupa showing the presence of ßuorescent bacteria, EGFP K. pneumoniae and DsRed E. agglomerans. (Online Þgure in color.) biont transmission behavior may indicate a compro- among life stages may be related to stage-speciÞc dif- mise between vertical transmission efÞciency and cost ferences in nutritional requirements. Adults require to the host. The variation in symbiont abundance high levels of protein (i.e., nitrogenous compounds),

Fig. 7. CLSM image of a second generation adult Mediterranean fruit ßy intestine containing ßuorescent bacteria EGFP K. pneumoniae and DsRed E. agglomerans. (Online Þgure in color.) 92 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 102, no. 1

Fig. 8. SEM image of a Mediterranean fruit ßy intestine showing numerous bacteria Þlling the intestinal lumen. MagniÞcation, 1,500ϫ. for completion of sexual maturation and egg produc- the low abundance of these bacteria in the larval stages tion. Adults not only enhance symbiont horizontal may reßect a compromise between vertical transmis- transmission efÞciency by attraction to, and feeding sion efÞciency and cost to the fruit ßy host. The insect on, these bacteria in nature (MacCollom et al. 1992, does not maintain a high level of symbionts during the 1994; Prokopy et al. 1993, Lauzon et al. 1998) but also larval stage that requires relatively low levels of ni- ingest the carrier substrates with a high nitrogen con- trogenous compounds and when it is exposed to a tent. That is, the ßies are simultaneously ingesting high more homogeneous environment within the fruit. In levels of nitrogenous compounds and the symbionts addition, the highly active larval gut from proliÞc feed- that facilitate nitrogen catabolism. The observed role ing may physically diminish the ability of the symbi- of E. agglomerans in detoxiÞcation (Lauzon et al. 2003) onts to establish. Turbulence and ßushing are known suggests that larger numbers of this species would be factors that can interfere with bioÞlm formation beneÞcial to the host during the adult stage when the (Sutherland 2001). This may be another factor asso- insect is exposed to a diverse environment. Similarly, ciated with the larval stage that diminishes symbiont

Fig. 9. SEM image of a Mediterranean fruit ßy egg with bacteria attached primarily on the apical end and adhere primarily near surface ridges. MagniÞcation, 3,000ϫ. January 2009 LAUZON ET AL.: VERTICAL TRANSFER OF SYMBIONTS IN C. capitata 93

Fig. 10. (a) SEM image of bacteria near and within micropyle of a Mediterranean fruit ßy egg. MagniÞcation, 12,000ϫ. (b) CLSM image of bacteria, EGFP K. pneumoniae and DsRed E. agglomerans, on the surface of a Mediterranean fruit ßy egg showing a similar pattern of placement as seen in Fig. 12a. MagniÞcation, 63ϫ. (Online Þgure in color.) transmission efÞciency. Although few bacteria are tional demands of ßies as they age and mature repro- present in the pupal stage, bacteria are likely carried ductively. into the adult gut in the meconium. Symbionts trans- The presence of E. agglomerans and K. pneumoniae mitted in this way have an opportunity to attach and on the egg surface pre- and postoviposition suggests proliferate in the relatively calm gut of newly eclosed another role for the symbionts in larval development. adults. Nutrients, such as precursor materials of the The bacteria were observed within the micropyle, peritrophic matrix, would support bacterial growth. A suggesting that this may be an entry point into the egg more steady ßow of nutrients would come later in the for maternal transmission. Bacteria on the surface of gut with feeding behavior changes that meet nutri- the egg may also inoculate the larval host at oviposi-

Fig. 11. CLSM image of ßuorescent bacteria EGFP K. pneumoniae and DsRed E. agglomerans within the gut of an adult sterile male Mediterranean fruit ßy. MagniÞcation, 25ϫ. (Online Þgure in color.) 94 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 102, no. 1 tion, providing a second means of transmission to the Drew, R.A.I., and A. C. Lloyd. 1987. Relationship of fruit immature insect through feeding. Furthermore, these ßies (Diptera: Tephritidae) and their bacteria to host bacteria may play an important role in the breakdown plants. Ann. Entomol. Soc. Am. 80: 629Ð636. of the host fruit and subsequent development of the Drew, R.A.I., and H. A. Faye. 1988. Elucidation of the roles larvae. of ammonia and bacteria in the attraction of Dacus tryoni Mixed patterns of symbiont transmission behavior (Froggatt) (Queensland fruit ßy) to proteinaceous sus- in insects are not uncommon (Agnew et al. 2003) and pensions. J. Plant Prot. Trop. 5: 127Ð130. Drew, R.A.I., A. C. Courtice, and D. S. Teakle. 1983. Bac- likely reßect a hostÕs adaptation to various ecological teria as a natural source of food for adult fruit ßies niches. Multiple acquisitions and replacements of (Diptera: Tephritidae). Oecologia (Berl.) 60: 279Ð284. symbionts in C. capitata suggest that these bacteria Epsky, N. D., R. R. Heath, B. D. Dueben, C. R. Lauzon, A. T. serve important roles in overcoming biological con- Proveaux, and G. B. MacCollom. 1998. Attraction of straints encountered by these insects in nature. 3-methyl-1-butanol and ammonia identiÞed from Enter- Knowledge and understanding of symbiont inheri- obacter agglomerans to Anastrepha suspensa. J. Chem. tance should lead to a more complete understanding Ecol. 24: 1867Ð1880. of C. capitata dispersal and biology. Franz, G., P. Kerremans, P. Rendon, and J. Hendrichs. 1996. Development and application of genetic sexing systems for Mediterranean fruit ßy based on temperature sensi- Acknowledgments tive lethal, pp. 185Ð191. In B. A. McPheron and G. J. Steck [eds.], Fruit ßy pests, a world assessment of their biology We thank Kingsley Fisher, Stuart Stein, and Jon Nishimoto and management. St. Lucie Press, Delray Beach, FL. of the USDA APHIS for lending technical support, and Drs. Gjersing, E. L., S. L. Codd, J. D. Seymour, and P. S. Stewart. Denise Schichnes and Steve Ruzin of the Biological Imaging 2005. Magnetic resonance microscopy analysis of advec- Facility at The University of California, Berkeley, for assis- tive transport in a bioÞlm reactor. Biotech. Bioengineer. tance and use of the facilities. We also thank Neil Wright and 89: 822Ð834. Song-So Min of the California Department of Food and Hendrichs, J., C. R. Lauzon, S. S. Cooley, and R. J. Prokopy. Agriculture for supplying pupae and David Robacker and 1993. 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