The Fly Factor Phenomenon Is Mediated by Interkingdom Signaling Between Bacterial Symbionts and Their Blow Fly Hosts
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Insect Science (2018) 00, 1–10, DOI 10.1111/1744-7917.12632 ORIGINAL ARTICLE The fly factor phenomenon is mediated by interkingdom signaling between bacterial symbionts and their blow fly hosts Yonathan Uriel , Regine Gries, Lorna Tu, Cassandra Carroll, Huimin Zhai, Margo Moore and Gerhard Gries Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada Abstract We tested the recent hypothesis that the “fly factor” phenomenon (food cur- rently or previously fed on by flies attracts more flies than the same type of food kept inaccessible to flies) is mediated by bacterial symbionts deposited with feces or regur- gitated by feeding flies. We allowed laboratory-reared black blow flies, Phormia regina (Meigen), to feed and defecate on bacterial Luria–Bertani medium solidified with agar, and isolated seven morphologically distinct bacterial colonies. We identified these us- ing matrix-assisted laser desorption/ionization mass spectrometry and sequencing of the 16S rRNA gene. In two-choice laboratory experiments, traps baited with cultures of Pro- teus mirabilis Hauser, Morganella morganii subsp. sibonii Jensen, or Serratia marcescens Bizio, captured significantly more flies than corresponding control jars baited with tryptic soy agar only. A mixture of seven bacterial strains as a trap bait was more attractive to flies than a single bacterial isolate (M. m. sibonii). In a field experiment, traps baited with agar cultures of P.mirabilis and M. m. sibonii in combination captured significantly more flies than traps baited with either bacterial isolate alone or the agar control. As evident by gas chromatography-mass spectrometry, the odor profiles of bacterial isolates differ, which may explain the additive effect of bacteria to the attractiveness of bacterial trap baits. As “generalist bacteria,” P. mirabilis and M. m. sibonii growing on animal protein (beef liver) or plant protein (tofu) are similarly effective in attracting flies. Bacteria-derived airborne semiochemicals appear to mediate foraging by flies and to inform their feeding and oviposition decisions. Key words blow flies; enteric bacteria; fly factor; interkingdom communication; micro- bial symbionts; semiochemical attractants Introduction feeding activity by flies promotes the recruitment of other flies to a food source (Danchin & Wagner, 1997; Lihoreau The “fly factor” refers to observations that food currently & Rivault, 2011; Holl & Gries, 2018). or previously fed on by flies attracts more flies than the The fly factor has been observed in diverse dipteran same type of food kept inaccessible to flies (Barnhart taxa, including black blow flies (Phormia regina Meigen) & Chadwick, 1953). Several studies have reported that (Dethier, 1955), house flies (Musca domestica Linnaeus) (Barnhart & Chadwick, 1953; Acree et al., 1959; Holl & Gries, 2018), face flies (Musca autumnalis De Geer) Correspondence: Yonathan Uriel, Department of Biological (Teskey, 1969), and green bottle flies (Lucilia sericata Sciences, Simon Fraser University, 8888 University Drive, Burn- Meigen) (Brodie et al., 2015). The fly factor is also ef- aby, British Columbia V5A 1S6, Canada. Tel: +1 778 782 5939; fective across taxa; for example, feeding black blow flies fax: +1 778 782 3496; email: [email protected] attract foraging house flies, and vice versa (Dethier, 1955), 1 C 2018 Institute of Zoology, Chinese Academy of Sciences 2 Y.Urieletal. and feeding green bottle flies attract foraging black blow Working with the black blow fly, Phormia regina,asa flies, and vice versa (Brodie et al., 2015). model species we tested the hypotheses that (i) specific, Previously, in investigating the mechanisms underly- fly-deposited bacteria are attractive to flies; (ii) multiple ing the fly factor we showed that neither temperature strains of fly-deposited bacteria are more attractive to flies nor relative humidity differed between feeding and non- than single-strain bacteria; and (iii) fly-attractive bacteria feeding flies. In contrast, feeding flies produced signif- are attractive to flies irrespective of the substrate the bac- icantly more CO2 than nonfeeding flies (Holl & Gries, teria grow on. 2018), suggesting that elevated levels of CO2 may sig- nify fly feeding activity and thus the presence of a food source or oviposition site. However, experimental test- Materials and methods ing of CO2 as a foraging cue for flies did not reveal any behavior-modifying effect (Holl & Gries, 2018), confirm- Rearing of experimental flies ing that house flies do not respond to CO2 (Richards, 1922; Wieting & Hoskins, 1939). Attraction of house flies to fed- We maintained blow fly colonies in the Simon Fraser on food and to house fly feces and regurgitate prompted University (SFU) insectary (Burnaby, BC, Canada), ° Holl and Gries (2018) to hypothesize that house flies re- at 25 C, 65% relative humidity, and a photoperiod of spond to minute amounts of semiochemicals (message- L14 : D10. We reared fly maggots on bovine liver bearing chemicals) produced by microbes in these sub- (Supreme Meat Supplies Ltd., Burnaby, BC, Canada) and strates. Their hypothesis was inspired by findings that provisioned adult flies with sugar, and, 3 d post-eclosion, feeding and defecating flies can inoculate food sources with milk powder ad libitum as a protein source. with symbiotic microbes (Hendrichs et al., 1992) present in their digestive tract (Gupta et al., 2012) and salivary Isolating and culturing of bacteria associated with flies glands (Singh et al., 2015). One of these microbes is the bacterium Proteus mirabilis Hauser (Gupta et al., 2012) To isolate bacteria associated with P.regina, we released that produces semiochemicals such as indole and dimethyl 50–60 flies into a metal mesh cage (61 cm3; BioquipR , trisulfide (Ma et al., 2012; Tomberlin et al., 2012), which Compton, CA, USA), which contained a water wick and are well-documented fly attractants (Mulla et al., 1977; Luria–Burtani agar (LBA) in an open Petri dish (85 mm × Cosse´ & Baker, 1996; Brodie et al., 2014, 2016). 15 mm). After flies had fed and defecated on the agar dish Proteus mirabilis colonizes animal feces and car- for1hat25°C, we removed and incubated it aerobically at rion that attract foraging flies (Chaudhury et al., 2010; 25 °C for 24 h. Thereafter, we restreaked morphologically Tomberlin et al., 2017). Interestingly, some fly-attractive distinct colonies onto new LBA plates, until we obtained odorants produced by P. mirabilis also serve as signaling pure colonies, and incubated them at 25 °C for 24 h. Plates molecules in its quorum sensing (QS) behavior (Sturgill were stored at 4 °C. We were able to isolate seven bacterial & Rather, 2004; Ma et al., 2012; Tomberlin et al., 2012), a colonies. We made storage cultures of each isolate and phenomenon whereby secondary metabolites secreted by stored them in 20% glycerol medium at –80 °C. the bacteria accumulate to a critical threshold concentra- tion which then triggers transcription of swarming genes in bacterial conspecifics (Verstraeten et al., 2008). As QS Identification of bacteria is linked to bacterial proliferation (Kearns, 2010), the rel- ative abundance of QS-associated odorants may signal the We identified five of the seven bacterial colonies to nutritional quality of a food source to foraging flies. the species level by mass spectrometry using matrix- Prior studies have not yet demonstrated a direct link be- assisted laser desorption/ionization (MALDI-TOF MS; tween the feeding activity of blow flies, their deposition of Bruker Corp., Billerica, MA, USA) (Ibarra Jimenez et al., microbial symbionts, and the attractiveness of these sym- 2018). We used a Bruker bacterial test standard (Bruker bionts to foraging flies. Furthermore, if microbes were to Corp., Millica, MA, USA) for calibration in accordance emit signature odorants, multiple strains of microbes in with manufacturer instructions. For each strain, we pro- combination (with a combined and more complex semio- cessed two preparations, and analyzed all spectra using chemical blend) might be more readily detectable and Biotyper software (Bruker Corp., Billerica, MA, USA). thus be more attractive to flies than single-strain bacteria. This Biotyper software calculates an arbitrary score for Finally, as generalist detritivores, we would expect blow each sample between 0 and 3 by comparing sample mass flies to respond to the odorants from generalist bacteria spectra to reference mass spectra; we accepted species growing on diverse detritus. assignments at scores of >2.0 in accordance with the C 2018 Institute of Zoology, Chinese Academy of Sciences, 00, 1–10 The fly factor is microbe mediated 3 manufacturer’s recommended protocol. We identified the we incubated all slices of beef liver and tofu for 24 h at remaining two colonies to the genus level by amplify- 25 °C. ing and sequencing the 16S rRNA gene, and by com- paring the resulting partial sequences to the database of the National Center for Biotechnology Information Gen- Specific experiments Bank, using the online BLASTn tool (Bethesda, MD; http://www.ncbi.nlm.nih.gov/BLAST.cgi). Hypothesis 1: Fly-deposited bacteria are attractive To prepare bacteria for sequencing, we cultured them in to conspecific flies Experiments 1–7 followed the general LB broth at 25 °C for 18–20 h. We extracted chromosomal laboratory two-choice experimental design (see above). In DNA using a standard phenol/chloroform extraction pro- each experimental replicate, we baited one of the paired tocol (Sambrook et al., 1989), and amplified 1.5 kb of the 450 mL amber glass jars with a 1/4 section cut from 16S rRNA gene by polymerase chain reaction using the a Petri dish (85 mm diameter) containing sterile TSA Eubacteria-specific primers 16F27 (5ʹ-CCA GAG TTT (control stimulus) and the other with a 1/4 section of GAT CMT GGC TCA G-3ʹ) and 16R1525XP (5ʹ-TTC TSA previously inoculated with a 24 h culture of one TGC AGT CTA GAA GGA GGT GWT CCA GCC-3ʹ), of the bacterial isolates, that is, Exiguobacterium spp. A following the PCR protocol from Pidiyar et al. (2004) with (Exp.