The gram-negative sensing receptor PGRP-LC contributes to grooming induction in Drosophila

Aya Yanagawa; Claudine Neyen; Bruno Lemaitre; Frédéric Marion-Poll

doi:10.1371/journal.pone.0185370

Abstract

Behavioral resistance protects insects from microbial infection. However, signals inducing insect hygiene behavior are still relatively unexplored. Our previous study demonstrated that olfactory signals from microbes enhance insect hygiene behavior, and gustatory signals even induce the behavior. In this paper, we postulated a cross-talk between behavioral resistance and innate immunity. To examine this hypothesis, we employed a previously validated behavioral test to examine the function of taste signals in inducing a grooming reflex in decapitated flies. Microbes, which activate different pattern recognition systems upstream of immune pathways, were applied to see if there was any correlation between microbial perception and grooming reflex. To narrow down candidate elicitors, the grooming induction tests were conducted with highly purified bacterial components. Lastly, the role of DAP-type peptidoglycan in grooming induction was confirmed. Our results demonstrate that cleaning behavior can be triggered through recognition of DAP-type PGN by its receptor PGRP-LC.

Citation: Yanagawa A, Neyen C, Lemaitre B, Marion-Poll F (2017) The gram-negative sensing receptor PGRP-LC contributes to grooming induction in Drosophila. PLoS ONE 12(11): e0185370. https://doi.org/10.1371/journal.pone.0185370

Editor: Efthimios M. C. Skoulakis, Biomedical Sciences Research Center Alexander Fleming, GREECE

Received: March 27, 2017; Accepted: September 12, 2017; Published: November 9, 2017

Copyright: © 2017 Yanagawa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files. Movie 1. Grooming test with standard LPS is available from Figshare, Accession link: https://figshare.com/s/5e9729a1d2a82cc2d43, DOI: 10.6084/m9.figshare.5211898.

Funding: This research is supported by Itochu research foundation and the Research Institute of Sustainable Humanosphere, Kyoto University (grant no. 2016-5-1-5), http://www.rish.kyoto-u.ac.jp/mission_e/mission5/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Insect defenses against pathogens have been mostly studied from an immunological point of view, unravelling the biochemical components as well as the signaling pathways involved in triggering these responses [1]. However, little data is available concerning the role of behavior in these innate defenses. Although hygienic activities like grooming (have been shown recently to be) are remarkably developed in social insects [2–4], little is known about grooming reflexes in Drosophila. Drosophila clean themselves vigorously (i.e. grooming) when touched with bacterial extracts, or with bitter chemicals (like quinine), but not with sugar or water [5]. Our objective was to study self-grooming in a solitary insect amenable to genetic studies. We used Drosophila melanogaster to establish whether grooming behavior relies on the immune system, and if so, which chemical signals from microbes (bacteria, fungi) might stimulate self-grooming.

In Drosophila, antimicrobial defense mechanisms include maintenance of physical barriers (epithelia), secretion of humoral mediators (antimicrobial peptides, reactive oxygen species), activation of proteolytic cascades leading to melanisation and cellular functions including phagocytosis and encapsulation [1]. The regulation of the antimicrobial peptide gene expression during systemic infection has been studied in great detail. Production of antimicrobial peptides by the fat body, an analogue of the mammalian liver, is orchestrated through two signaling modules: the Toll and Imd pathways are activated in response to microbial infection and lead to activation of NF-κB-like factors. Two types of pattern recognition systems assure microbial sensing upstream of the Toll and Imd pathways. Peptidoglycan recognition proteins (PGRPs), which can be soluble or membrane-bound, activate both Toll and Imd, and soluble Glucan-binding proteins (GNBPs), which recognize yeast glucans and activate exclusively Toll pathways. All PGRPs recognize bacterial peptidoglycan (PGN) but with specificity towards the nature of peptidoglycan: Lysine-type PGN from Gram-positive bacteria engages the soluble form PGRP-SA in hemolymph and leads to Toll activation, while the Imd pathway, active against Gram-negative bacteria, is triggered by the binding of DAP-type PGN to PGRP-LC in cooperation with PGRP-LE [6–8]. The Imd pathway mainly responds to Gram-negative bacterial infection and controls antibacterial peptide genes via the activation of the Rel protein Relish. PGRP-LB is an amidase specific of DAP-type PGN and its expression is controlled by the Imd pathway [9, 10]. PGRP-LB negatively regulates the Imd pathway by scavenging extracellular peptidoglycan. PGRP-LC is a transmembrane receptor with three alternative splice isoforms (PGRP-LCa, -LCx and -LCy) that have somewhat different ligand specificities. Antimicrobial peptides are also expressed in many epithelia in contact with the external world such as the gut, genital tracts and trachea. Tissue-specific expression of surface and intracellular receptors ensures a correct level of immune activation in tissues exposed to the environment versus tissues in contact with the sterile interior milieu [11, 12].

Here we postulated that microbial factors, notably peptidoglycan from Gram-negative bacteria, plays an important role in inducing hygiene behavior in Drosophila. Grooming behavior in Drosophila may contribute to diminishing the exposure of flies to pathogens. Previous studies have shown that a stereotyped grooming reflex can be triggered in decapitated flies by tactile [13, 14] or chemical stimulation [5, 15, 16]. As a consequence, use of decapitated flies enable simple but reliable assessments of grooming activity upon stimulation of sensilla [17, 18]. We have carefully modified the method to remove the influence from mechanical stimulus together with water influence and confirmed it with several approaches using UAS-GAL4 experiments and optogenetic experiments[5], then applied this classical method to assess the reflex triggered by chemical stimuli associated with pathogens. We scored the occurrence of grooming responses following a contact with a solution of water mixed with different solutions. First, microbes, which activate different pattern recognition systems, were applied to decapitated flies to assess the function of bacterial components in the induction of grooming. Then the grooming induction tests were conducted with highly purified bacterial components to pinpoint specific elicitors. These tests support an important role of DAP-type PGN in eliciting grooming. Lastly we showed that two pattern-recognition receptor capable of recognizing DAP-type PGN working upstream of the Imd pathway are required for grooming. In contrast, PGRP-LB and intracellular components of the Imd pathway, Relish, and the Imd adaptor [19] were not involved in the grooming reaction (Fig 1A). In this paper, we successfully demonstrate that DAP-type PGN from Gram-negative bacteria triggers the cleaning behavior when it is recognized by its receptor PGRP-LC.

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Fig 1.

(A) Grooming induction test. Circles on body parts indicate location of gustatory receptors. Tested locations of gustatory receptor are indicated by red circle (front legs, hind legs and wing). (B)–(E) show the results. Grooming behavior induced by (B) heat-killed E. coli on w¹¹¹⁸, (C) A. aceti on CS flies, (D) L. bulgaricus on CS flies and (E) B. bassiana on CS flies. n = 40 (n = 20 from each sex). G-/+ means Gram-negative/positive bacteria. Significant increase from the control response (water) is indicated by asterisks: * indicates p <0.05, **indicates p < 0.01 and *** indicates p < 0.001 (Dunnett’s test). Data represent mean ± standard error (SE). (F) shows results of Chi-square test on grooming induction by various microbes in wild type flies. Microbes, which induced significant concentration-dependent behavioral increase were in white zone, and no-significance in gray zone. White zone in a table shows significant difference (p < 0.05), and grey zone indicates no-significance (p > 0.05).

https://doi.org/10.1371/journal.pone.0185370.g001

Results

Contact with microbes induces grooming

To determine whether microbial compounds elicit grooming behavior in Drosophila, grooming activity in decapitated flies was examined after applying different microbial species. Gram-negative bacteria, gram-positive bacteria, and pathogenic fungi were tested.

Grooming induction by E. coli, a representative Gram-negative bacterium, was already shown in Canton S (CS) flies [5] (Figure a in S1 Fig). Since it cannot be excluded that the grooming behavior is highly dependent on the genetic background of the flies, we tried to reproduce E. coli–induced grooming in another wild-type strain, w¹¹¹⁸. E. coli suspension successfully induced the behavior in w¹¹¹⁸ in a dose dependent manner (Fig 1B). In addition, we employed another common Gram-negative bacterium, Acetobacter aceti, to confirm that Gram-negative bacterial components induced the cleaning behavior in flies. A. aceti, exhibited a concentration dependent behavioral induction upon contact with hind legs (p = 0.02, x² = 11.51, Chi-square test, Fig 1F) and wing margins (p < 0.01, x² = 28.34, Chi-square test, Fig 1F), but not with front legs (p = 0.90, x² = 1.05, Chi-square test, Fig 1F) (Fig 1C). There was no difference in behavioral induction between females and males both in CS and W¹¹¹⁸ flies to Gram-negative bacteria (front legs: p > 0.10, hind legs: p > 0.05, wing margins: p > 0.10, Chi-square test, Fig 1F), so we pooled male and female responses in all further experiments.

On the other hand, no grooming induction was observed upon contact with Gram-positive bacteria, Lactobacillus bulgaricus and Mycoplasma fermentans (Fig 1D, Figure b in S1 Fig, respectively) and fungi, Beauveria bassiana (Fig 1E) (p > 0.1 for all body parts regardless of concentration and sex parameters).

These results indicate that bacterial components from Gram-negative bacteria contribute to inducing grooming behavior in decapitated flies.

Highly purified microbial components induce grooming

To determine which molecules from Gram-negative bacteria are detected by Drosophila, grooming activity in decapitated flies was examined using a standard solution of LPS from SIGMA containing also bacterial peptidoglycan and lipopeptide (referred to as Sigma ‘LPS’)[20] and highly purified microbial elicitors, including Lipopolysaccharide (LPS), two preparations of peptidoglycans and glucan.

Although we had previously seen a clear grooming induction by a standard solution of Sigma ‘LPS’ from E. coli (L2630, Sigma) (Fig 2A) [5], this behavioral induction disappeared with a highly purified solution of LPS from E. coli (tlrl-eblps, InvivoGen) (p > 0.1 for all body parts regardless of concentration) (Fig 2B). In contrast, peptidoglycan from E. coli (gift from Dominique Mengin-Lecreulx) induced the cleaning behavior significantly (p < 0.01, x² = 15.95 at concentration parameter, p < 0.01, x² = 0.39 at sex parameter, Chi-square test) (Fig 2C). Grooming induction was also observed after stimulation with tracheal cytotoxin (TCT, gift from Dominique Mengin-Lecreulx) (p = 0.02, x² = 12.22 at concentration parameter, p < 0.01, x² = 8.43 at sex parameter, Chi-square test) (Fig 2D). TCT is a soluble fragment of peptidoglycan composed of a monomer with an anhydro bound released from the cell wall of proliferating Gram-negative bacteria [21]. It has been shown to be the minimal peptidoglycan unit capable of activating an immune response [20, 22]. However, peptidoglycan from the DAP-type Gram-positive bacterium, Bacillus subtilis (tlrl-pgnb3, InvivoGen) induced grooming less conclusively (Figure c in S1 Fig: p > 0.1 for all body parts at concentration parameter) and the soluble ß-glucan laminarin (tlrl-lam, InvivoGen) did not induce the behavior (Figure d in S1 Fig: p > 0.1 for all body parts at concentration parameter). Results of Chi-square test are listed in Fig 2E. Of note, a low behavioral induction was observed in the comparison between control and peptidoglycan of B. subtilis when it was deposited on hind legs, but only at the highest concentration (Figure c in S1 Fig, Dunnett’s test). The results of Chi-square test are listed in Fig 2E and S1 Table.

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Fig 2.

Grooming behavior induced by microbial components in wild-type flies, (a) standard LPS from E. coli on w¹¹¹⁸ flies (b) pure LPS from E.coli on CS flies, (C) peptidoglycan from E. coli, (D) TCT on CS flies. High grooming response was induced by Standard LPS from E. coli on CS flies. The data was available in Yanagawa et al. [5] A significant increase in response from that of the control (water) is indicated by asterisks: * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 (Dunnett’s test). (E) shows results of Chi-square test on grooming induction by highly purified microbial components in wild type flies. Compounds, which induced significant concentration-dependent behavioral increase were in white zone, and no-significance in gray zone. White zone in a table shows significant difference (p < 0.05), and grey zone indicates no-significance (p > 0.05).

https://doi.org/10.1371/journal.pone.0185370.g002

Taken together, these results suggest that peptidoglycan from Gram-negative bacteria plays a role in inducing the grooming reflex. In all following experiments, we kept using the standard LPS from Sigma because it is a cheap and convenient source of peptidoglycan.

Grooming induction is lost in mutants of pattern-recognition receptors but not of signaling components of the Imd pathway

Since Drosophila senses Gram-negative bacterial peptidoglycan via two PGRPs, membrane-bound PGRP-LC and intracellular PGRP-LE [7, 8, 11, 12], we used mutants of these receptors (w;;PGRP-LC^E12 and y, w, PGRP-LE¹¹²) to test their role in the activation of grooming by DAP-type PGN. Additionally, we tested a mutant of other Imd pathway components, namely the negative regulator PGRP-LB (w;;PGRP-LB^Δ), a secreted amidase which binds to and degrades extracellular peptidoglycan (Fig 3A)[23, 24]. The results of Chi-square test are listed in Fig 3D and S2 Table.

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Fig 3.

(A) Schematic indicating how the Imd pathway is activated by Gram-negative bacteria. Grooming behavior induced by LPS contact on wings in (B) black square: w;;PGRP-LC^E12 with Cantonized 2nd chromosome, green square: y,w,PGRP-LE¹¹² and grey circle: w;;PGRP-LBΔ (C) black square: w;;PGRP-LC^E12 and orange square: w;P[acman]-PGRP-LC;PGRPLC^E12 rescue flies. Standard LPS was used as a stimulus. A significant increase in response from that of the control (water) is indicated by asterisks: * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 (Dunnett’s test); n = 40 (n = 20 for each sex). Data represents mean +/- SE, analyzed as in Fig 1. (D) shows results of Chi-square test on grooming induction. Mutants, which showed significant concentration-dependent behavioral increase were in white zone, and no-significance in gray zone. White zone in a table shows significant difference (p < 0.05), and grey zone indicates no-significance (p > 0.05).

https://doi.org/10.1371/journal.pone.0185370.g003

Grooming induction was significantly suppressed in all mutants lacking PGRPs (either PGRP-LCs or PGRP-LE), indicating an involvement of these pattern-recognition receptors in the activation of grooming upon DAP-type PGN (Fig 3B, S2 Fig). In this study, we confirm PGRP-LC function in grooming induction both in 4-day old flies and 10 day-old flies (Fig 4A and Figure a in S3 Fig) since, in previous results, age dependent increase was observed in grooming induction [5]. To control for potential background effects, we first used several strains deleted for PGRP-LC in different wild-type backgrounds (w¹¹¹⁸, Can^S) and obtained similar results. The response was rescued by the reinsertion of the PGRP-LC locus into the PGRP-LC^E12 mutant background (full genotype: w;P[acman]-PGRP-LC;PGRP-LC^E12) (Fig 3C). There was no significant difference in the response in mutants w;;PGRP-LC^E12 and y, w, PGRP-LE¹¹² (p = 0.41, x² = 0.68 at strain parameter, Chi-square test), while w;;PGRP-LB^Δ showed drastic behavioral increase. Grooming was still observed in PGRP-LB deficient flies at normal levels, suggesting that PGRP-LB is not involved in this process. Moreover w;P[acman]-PGRP-LC;PGRPLCE12 rescue clearly recovered the response in comparison with w;;PGRP-LC mutants (p < 0.01, x² = 162.43 at strain parameter, Chi-square test)(Fig 3C). We also checked whether grooming induction by gustatory stimulus (quinine, bitter taste) was dependent on the same receptor, PGRP-LC. However, it seemed to be independent (Fig 4B, Figure b in S3 Fig). The results of Chi-square test are listed in Fig 4C and S3 Table.

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Fig 4.

(A) Grooming behavior induced in 10-day old PGRP-LC^E12 mutants in orange circle with orange line: CS background and white circle with blue line: w¹¹¹⁸ background. (B) Grooming behavior induced by quinine in PGRP-LC^E12 mutants of orange circle with orange line: CS background and white circle with blue line: w¹¹¹⁸ background flies. A significant increase in response from that of the control (water) is indicated by asterisks: * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 (Dunnett’s test). (C) shows the results of Chi-square test in PGRP-LC mutants to Quinine and in 10-day old PGRP-LC mutants. Mutants, which showed significant concentration-dependent behavioral increase were in white zone, and no-significance in gray zone. White zone in a table shows significant difference (p < 0.05), and grey zone indicates no-significance (p > 0.05).

https://doi.org/10.1371/journal.pone.0185370.g004

Next, we performed ubiquitous mis-expression of PGRP-LCx using the Gal4-UAS system and RNAi/overexpression constructs. Flies expressing RNAi against PGRP-LC did not show the response (Fig 5A, Figure a in S4 Fig), in agreement with what was observed in PGRP-LC mutants. When PGRP-LCx was overexpressed, flies became highly sensitive and induced behavior at 1000 times lower peptidoglycan concentration (Fig 5B, Figure b in S4 Fig). Then the grooming reflex had diminished. This pattern of output decrease appears sometimes also in other insect behaviors. It is considered as the neural adjustment cause by too strong stimulus, which caused bursts of electrophysiological signals in the neural circuit or by the systematic inhibition to regulate a reaction. The results of Chi-square test are listed in Fig 5C and S4 Table.

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Fig 5.

(A) Grooming behavior induced in offspring of act-Gal4 x PGRP-LC^RNAi flies black circle with black line: actGal4x -PGRP-LC^RNAi and white circle with red line control (siblings). Standard LPS was used as a stimulus. (B) Grooming behavior induced by LPS contact on wing and front/hind legs in PGRP-LCx overexpressing flies by Gal4-UAS system. Concentration dependent 40 repetitions were conducted by two concentration range: 10⁻⁵–10⁻² mg/ml range of standard LPS and 10⁻¹–10 mg/ml range of standard LPS. Open circles illustrated the grooming induction in control flies (siblings). Standard LPS was used as a stimulus. A significant increase in response from that of the control (water) is indicated by asterisks: * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 (Dunnett’s test). n = 40 (n = 20 for each sex). Data represents mean +/- SE, analysed as in Fig 1. (C) shows the results of Chi-square test. Mutants, which showed significant concentration-dependent behavioral increase were in white zone, and no-significance in gray zone. White zone in a table shows significant difference, and grey zone indicates no-significance.

https://doi.org/10.1371/journal.pone.0185370.g005

Taken together, these observations suggest that sensing of peptidoglycan from Gram-negative bacteria by PGRPs induces cleaning behavior. All results suggested a role of PGRP-LC in the induction of grooming behavior.

Discussions

We examined the reflex of hygiene behavior of flies to microbial contact. Bacterial cell wall components were applied to decapitated flies to see the behavioral reflex to soluble bacterial compounds⁵. Drosophila senses Gram-negative bacteria via PGRP-LC and PGRP-LE and relays sensing of extracellular or intracellular DAP-type PGN to the Imd pathway [7, 25]. This study demonstrates that initial sensing of Gram-negative bacteria at epithelial surfaces may serve to resist microbial infection by inducing grooming behavior.

It is often said that insects combine behavioral resistance and immune activity [4, 26]. Since behavior likely has the lowest cost to prevent pathogenic infection for insects, they might use behavior as the first protection together with the cuticle barrier, and spend less resource on immune responses in the gut or the haemolymph, which might be a good trade-off system to save bio-energy [26]. We have reported that contacting wing marginal sensilla with E. coli and standard LPS solution induced grooming activity [5], which suggested that hygiene behavior in Drosophila can be triggered by a detection of microbial surface compounds by similar mechanisms/receptors to those involved in triggering immune responses. Using highly purified bacterial cell wall components, we could show that grooming can be induced upon deposition of peptidoglycan extracts derived from gram-negative bacteria. Peptidoglycan is recognized by PGRPs, which activate the Imd pathway [27, 28]. In this study, using the same methods that we applied to see the role of gustatory perception in grooming induction reflex in decapitated flies, we showed that the innate immune system also serves in behavioral resistance at surface level in D. melanogaster. The results show that classical components involved in bacterial recognition can be re-used to induce behavioral response.

Insect perception of microbes is still ambiguous. As for relations between the immune and the sensory systems, several transcriptomic studies noted that microbial infections are associated with changes of expression of olfactory-related genes like odorant binding proteins [29, 30]. Previously, we demonstrated that grooming in response to microbes relies in part on the activation of taste neurons because poxn70 did not show any cleaning behavior by touching with bacterial extracts [5]. Poxn mutation in poxn flies induces a failure in the development of external chemoreceptors [31], and therefore, they are tasteless. Interestingly, we now show that grooming induction by microbes seems to depend on pattern-recognition receptors involved in immunity, since PGRP-LC and PGRP-LE mutants stopped responding to peptidoglycan. Whether PGRP-LC is expressed in taste neurons is presently unknown. To learn PGRP expression and interactions in the hemolymph of sensory sensilla, further studies are needed. It suggests that the surface immune-components serve in behavioral resistance. This is probably to prevent the loss of bioenergy because behavioral resistance works most effectively over the first contact with microbes. The interaction among signals and their cascades can much more complicated than we have thought. Even many other signals and its related genes can be involved in this behavior [16, 32] cooperating with local neurons [18]. Grooming induction by Sigma LPS was dramatic, while that by pure PGN was rather modest. In addition, sex dependent difference appeared more in behavioral responses induced by the compounds including those from gram-positive bacteria. It indicated that not only DAP-type PGNs but also the other bacterial surface compounds like lipopeptide would have some impacts on this behavioral reflex [5, 15, 32]. Moreover, although whether PGRP-LC is expressed in taste neurons is unknown, it is highly possible that standard LPS contains some substances perceived as aversive stimulus by Drosophila since the ion channel, which relates with aversive gustation, is known to have some interaction with standard LPS [33].

The role of grooming behavior seems diverse and many factors involved in this behavior are still unknown [32]. Grooming possibly helps removing dust particles [34] and also cleaning external chemosensory receptors [35]. Although self-grooming activities can be induced by a number of situations involving aversive stimuli, mechanical [36], chemicals [37] or complex behaviors like feeding and oviposition [38, 39], its importance for the survival of the species has rarely been considered. The hypotheses of grooming function to protect themselves against microbial infection were expressed independently in recent papers but not within a common unifying frame. In 2016, Soldano et al. [33] demonstrated that the role of ion channel dTRPA1 in the behavior after the chemical contact with Lipopolysaccharides (LPS), and in our knowledge, it is the first report of detailed mechanism on the link between behavioral immunity and insect perception. Vice versa, bacteria is known to use insect perception, and control its behavior to aid their survivals [40]. There is still a long way to go to recognize behavior as an integral part of the strategies used by insects to cope with pathogens [2, 3]. The behavioral process of grooming is highly complex and further study will open highly interesting aspects on the strategy for survival in insects which can bring new technique in the way of organic agriculture using biocontrol agents, insect mass production and sanitations in medical field.

Materials and methods

Fly stocks

D. melanogaster was maintained on a standard cornmeal agar food at 20°C and at 80% humidity. CantonS (CS) and white w¹¹¹⁸ flies were used as wild-type controls. As for immunity mutants, w;;PGRP-LC^E12 (in w¹¹¹⁸ background or with Cantonized 2^nd chromosome) y, w, PGRP-LE¹¹² and w;;PGRP-LB^△ were used. PGRP-LC^E12, PGRP-LB^Δand PGRP-LE¹¹² lines have been described previously [6, 7, 9, 41–43]. 4 day old flies were used.

The Gal4-UAS system [44] was used to mis-express PGRP-LC. The y, w;act5-Gal4 driver line (DGRC# 107727) was crossed to w;;UAS-PGRP-LCx or to w;;UAS- PGRP-LC^RNAi. In Gal4-UAStests, balanced siblings were employed as controls.

Microbial preparations

We tested the Gram-negative bacteria Escherichia coli and Acetobacter aceti, the Gram-positive bacteria Lactobacillus bulgaricus, Listeria monocytogenes and Mycoplasma fermentans, and the entomopathogenic fungus Beauveria bassiana.

The E. coli strain of TOP 10 was grown in liquid LB medium at 37°C. For heat-killed preparations, E. coli was washed with distilled water and heated at 95°C for 5 minutes. A. aceti ATCC 53264 was grown in liquid SH medium at 28°C. A. aceti was washed by distilled water and heated at 95°C for 5 minutes. 7.9 x 10⁹ /ml bacterial suspension was diluted 10⁰, 10², 10⁴ and 10⁶ fold. L. bulgaricus was grown in liquid MRS medium at 40°C, then it was washed by distilled water and heated at 95°C for 5 minutes.

Heat-killed Listeria monocytogenes and Mycoplasma fermentans were purchased from InvivoGen, (InvivoGen, Lot # HKLM-35-03 and HKMF-32-01, respectively) and adjusted to 1.0 x 10⁹ / ml. The suspensions were diluted 10⁰, 10², 10⁴ and 10⁶ fold. B. bassiana was grown in liquid LB medium for fungi. B. bassiana was washed by distilled water and heated at 95°C for 5 minutes. All microbial suspensions were adjusted to highest concentration, and then was diluted 10⁰, 10², 10⁴ and 10⁶ fold. They have a pH of about 7.0.

Chemicals

Standard LPS from E. coli was purchased from Sigma (L2630)[5], and highly purified LPS from E. coli was purchased from InvivoGen (tlrl-eblps). Peptidoglycan from the Gram-positive bacterium Bacillus subtilis and the soluble ß-glucan laminarin were purchased from InvivoGen (tlrl-pgnb3, tlrl-lam, respectively). Peptidoglycan from E. coli and TCT were gifts from Dominique Mengin-Lecreulx (University of Orsay, Orsay, France). All chemical suspensions have a pH of about 7.0.

Grooming induction and scoring

Briefly, 10 flies were beheaded by a single cut made at the neck with micro-scissors. Micro-scissors were washed and wiped by 70% ethanol before and after use. Beheaded flies were placed in an upright position on a clean paper sheet and allowed to recover. To stimulate them, the wings, front legs, or hind legs were gently touched with a sharpened toothpick that was previously soaked in a test solution. To avoid contamination, the paper sheet was changed between each test, and a new toothpick was sharpened before each test. Grooming behavior after touching by toothpick was observed, and its intensity scored as 0, 1, 2, or 3. A score of 0 indicated no grooming, and a score of 1 or greater indicated that grooming occurred. Because grooming duration has been shown varied widely in previous studies, the strength of induction was scored as follows: 1, grooming that stopped within 10 seconds; 2, grooming that lasted more than 10 seconds and less than 20 seconds; and 3, grooming that lasted over 20 seconds. Four-day-old flies were used in testing. Each substance was tested on 20 females and 20 males (n = 40).

Statistical analysis

To examine concentration-dependent increases in grooming behavior in headless flies with respect to sex, chemical, and fly strain, Chi-square test (JMP 10.0 software, SAS) was applied. Additionally, Dunnett’s test (JMP 10.0 software, SAS) was conducted to examine behavioral induction at each concentration.

Supporting information

S1 Fig. Grooming behavior induced by heat-killed microbes and its related chemicals in CS flies, (a) E.coli (b) M. fermentans (c) peptidoglycan from B. subtilis (d) ß-glucan from algae (laminarin) on CS flies were used for all tests, which employed w¹¹¹⁸.

A significant increase in response from that of the control (water) is indicated by asterisks: * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 (Dunnett’s test). Please note that the data of CS flies to E. coli is provided as a reference. This data belongs to the previous publication in Front. Behav. Neurosci. [5].

https://doi.org/10.1371/journal.pone.0185370.s001

(TIF)

S2 Fig. Grooming behavior induced by LPS contact on front legs and hind legs in black square: w;;PGRP-LC^E12 with Cantonized 2nd chromosome, green square: y, w, PGRP-LE¹¹², grey circle: w;;PGRP-LB^Δ, black square: w;;PGRP-LC^E12 and orange square: w;P[acman]-PGRP-LC;PGRP-LC^E12 rescue flies.

Standard LPS was used as a stimulus. n = 40 (n = 20 for each sex). Data represents mean +/- SE, analyzed as in Fig 2. A significant increase in response from that of the control (water) is indicated by asterisks: * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 (Dunnett’s test).

https://doi.org/10.1371/journal.pone.0185370.s002

(TIF)

S3 Fig.

(a) Grooming behavior induced in 10-day old PGRP-LC^E12 mutants in orange circle with orange line: CS background and white circle with blue line: w¹¹¹⁸ background. (b) Grooming behavior induced by quinine in PGRP-LC^E12 mutants of orange circle with orange line: CS background and white circle with blue line: w¹¹¹⁸ background flies. Standard LPS was used as a stimulus. n = 40 (n = 20 for each sex). Data represents mean +/- SE, analysed as in Fig 2. A significant increase in response from that of the control (water) is indicated by asterisks: * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 (Dunnett’s test).

https://doi.org/10.1371/journal.pone.0185370.s003

(TIF)

S4 Fig.

(a) Grooming behavior induced in offspring of act-Gal4 x PGRP-LC^RNAi flies black circle with black line: actGal4x-PGRP-LC^RNAi and white circle with red line control (siblings). (b) Grooming behavior induced by LPS contact on front/hind legs in PGRP-LCx overexpressing flies by Gal4-UAS system. Concentration dependent 40 repetitions were conducted by two concentration range: 10⁻⁵–10⁻² mg/ml range of standard LPS and 10⁻¹–10 mg/ml range of standard LPS. (c) and (d) illustrated the grooming induction in control flies (siblings). Standard LPS was used as a stimulus. n = 40 (n = 20 for each sex). Data represents mean +/- SE, analysed as in Fig 2. A significant increase in response from that of the control (water) is indicated by asterisks: * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 (Dunnett’s test).

https://doi.org/10.1371/journal.pone.0185370.s004

(TIF)

S1 Table. Results of Chi-square test on grooming induction by G+ microbe and highly purified microbial compounds in wild type flies.

Microbes, which induced significant concentration-dependent behavioral increase were in white zone, and no-significance in gray zone. White zone in a table shows significant difference (p < 0.05), and grey zone indicates no-significance (p > 0.05).

https://doi.org/10.1371/journal.pone.0185370.s005

(TIF)

S2 Table. Results of Chi-square test on grooming induction in IMD pathway related mutants.

Mutants, which showed significant concentration-dependent behavioral increase were in white zone, and no-significance in gray zone. White zone in a table shows significant difference (p < 0.05), and grey zone indicates no-significance (p > 0.05).

https://doi.org/10.1371/journal.pone.0185370.s006

(TIF)

S3 Table. Results of Chi square test on grooming induction in PGRP-LC mutants to quinine and in 10-day old PGRP-LC mutants.

Mutants, which showed significant concentration-dependent behavioral increase were in white zone, and no-significance in gray zone. White zone in a table shows significant difference (p < 0.05), and grey zone indicates no-significance (p > 0.05).

https://doi.org/10.1371/journal.pone.0185370.s007

(TIF)

S4 Table. Results of Chi square test on grooming induction in PGRP-LC related mutants.

Mutants, which showed significant concentration-dependent behavioral increase were in white zone, and no-significance in gray zone. White zone in a table shows significant difference (p < 0.05), and grey zone indicates no-significance (p > 0.05).

https://doi.org/10.1371/journal.pone.0185370.s008

(TIF)

Acknowledgments

E. coli strain of TOP 10 was kindly supplied by Dr. E. Jacquin-Joly (INRA, France) and A.aceti was kindly supplied by Dr. T. Imai (Kyoto University, Japan). PGN was from Dr. D. Mengin-Lecreulx (University of Orsay, Orsay, France). This research is supported by Itochu research foundation and the Research Institute of Sustainable Humanosphere, Kyoto University (grant no. 2016-5-1-5).

References

1. Lemaitre B, Hoffmann J. The host defense of Drosophila melanogaster. Annu. Rev. Immunol. 2007; 25: 697–743. pmid:17201680
- View Article
- PubMed/NCBI
- Google Scholar
2. Roy HE, Steinkraus DC, Eilenberg J, Hajek AE, Pell JK. Bizarre interactions and endgames: Entomopathogenic Fungi and Their Arthropod Hosts. Annu. Rev. Entomol. 2006; 51: 331–357. pmid:16332215
- View Article
- PubMed/NCBI
- Google Scholar
3. Fernando E, Vega H, Kaya K. Insect pathology. 2nd ed. Academic Press; 2011.
4. DeGrandi-Hoffman G, Chen Y. Nutrition, immunity and viral infections in honey bees. Current Opinion in Insect Science 2015; 10: 170–176.
- View Article
- Google Scholar
5. Yanagawa A, Guigue A, Marion-Poll F. Hygienic grooming is induced by contact chemicals in Drosophila melanogaster. Front. Behav. Neurosci. 2014; 8: article 254. pmid:25100963
- View Article
- PubMed/NCBI
- Google Scholar
6. Choe KM, Werner T, Stöven S, Hultmark D, Anderson KV. Requirement for a peptidoglycan recognition protein (PGRP) in Relish activation and antibacterial immune responses in Drosophila. Science 2002; 296: 359–362. pmid:11872802
- View Article
- PubMed/NCBI
- Google Scholar
7. Takehana A, Yano T, Mita S, Kotani A, Oshima Y, Kurata S. Peptidoglycan recognition protein (PGRP)-LE and PGRP-LC act synergistically in Drosophila immunity. EMBO J. 2004; 23: 4690–4700. pmid:15538387
- View Article
- PubMed/NCBI
- Google Scholar
8. Kaneko T, Yano T, Aggarwal K, Lim JH, Ueda K, Oshima Y, et al. PGRP-LC and PGRP-LE have essential yet distinct functions in the Drosophila immune response to monomeric DAP-type peptidoglycan. Nat. Immunol. 2006; 7: 715–723. pmid:16767093
- View Article
- PubMed/NCBI
- Google Scholar
9. Zaidman-Rémy A, Hervé M, Poidevin M, Pili-Floury S, Kim M-S, Blanot D, et al. The Drosophila amidase PGRP-LB modulates the immune response to bacterial infection. Immunity 2006; 24: 463–473. pmid:16618604
- View Article
- PubMed/NCBI
- Google Scholar
10. Girardin SE, Philpott DJ. PGRP-LB minds the fort. Immunity 2006; 24: 363–366. pmid:16618594
- View Article
- PubMed/NCBI
- Google Scholar
11. Neyen C, Poidevin M, Roussel A, Lemaitre B. Tissue- and ligand-specific sensing of Gram-negative infection in Drosophila by PGRP-LC isoforms and PGRP-LE. The Journal of Immunology 2012; 189: 1886–1897. pmid:22772451
- View Article
- PubMed/NCBI
- Google Scholar
12. Bosco-Drayon V, Poidevin M, Boneca IG, Narbonne-Reveau K, Royet J, Charroux B. Peptidoglycan sensing by the receptor PGRP-LE in the Drosophila gut induces immune responses to infectious bacteria and tolerance to microbiota. Cell Host Microbe. 2012; 12: 153–65 (2012). pmid:22901536
- View Article
- PubMed/NCBI
- Google Scholar
13. Burg MG, Hanna L, Kim Y-T, Wus C-F. Development and maintenance of a simple reflex circuit in small-patch mosaics of Drosophila: effects of altered neuronal function and developmental arrest. J. Neurobiol. 1993; 24: 803–823. pmid:8331340
- View Article
- PubMed/NCBI
- Google Scholar
14. Cofras G, Dudai Y. Habituation and dishabituation of a cleaning reflex in normal and mutant Drosophila. The Journal of Neuroscience 1989; 9: 56–62. pmid:2913213
- View Article
- PubMed/NCBI
- Google Scholar
15. Ashton K, Wagoner AP, Carrillo R, Gibson G. Quantitative trait loci for the monoamine-related traits heart rate and headless behavior in Drosophila melanogaster. Genetics 2001; 157: 283–294. pmid:11139509
- View Article
- PubMed/NCBI
- Google Scholar
16. Raad H, Ferveur JF, Ledger N, Capovilla M, Robichon A. Functional Gustatory Role of Chemoreceptors in Drosophila Wings. Cell Report 2016; 15: 1442–1454.
- View Article
- Google Scholar
17. Vandervorst P, Ghysen A. Genetic control of sensory connections in Drosophila. Nature 1980; 286: 65–67. pmid:7393324
- View Article
- PubMed/NCBI
- Google Scholar
18. Seeds AM, Ravbar P, Chung P, Hampel S, Midgley FM Jr, Mensh BD, et al. A suppression hierarchy among competing motor programs drives sequential grooming in Drosophila. eLife 2014; 3: e02951: pmid:25139955
- View Article
- PubMed/NCBI
- Google Scholar
19. Buchon N, Broderick NA, Lemaitre B. Gut homeostasis in a microbial world: insights from Drosophila melanogaster. Nature Reviews Microbiology 2014; 11: 615–626.
- View Article
- Google Scholar
20. Kaneko T, Goldman WE, Mellroth P, Steiner H, Fukase K, Kusumoto S, et al. Monomeric and polymeric gram-negative peptidoglycan but not purified LPS stimulate the Drosophila IMD pathway. Immunity 2004; 20: 637–649. pmid:15142531
- View Article
- PubMed/NCBI
- Google Scholar
21. Mattoo S, Cherry JD. Molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies. Clinical Microbiology Reviews 2005; 18: 326–382. pmid:15831828
- View Article
- PubMed/NCBI
- Google Scholar
22. Stenbak CR, Ryu JH, Leulier F, Pili-Floury S, Parquet C, Herve M, et al. Peptidoglycan molecular requirements allowing detection by the Drosophila immune deficiency pathway. J. Immunol. 2004; 173: 7339–7348. pmid:15585858
- View Article
- PubMed/NCBI
- Google Scholar
23. Guan R, Mariuzza RA. Peptidoglycan recognition proteins of the innate immune system. Trends in Microbiology 2007; 15: 127–134. pmid:17275309
- View Article
- PubMed/NCBI
- Google Scholar
24. Ferrandon D, Imler J-L, Hetru C, Hoffmann JA. The Drosophila systemic immune response: sensing and signalling during bacterial and fungal infections. Nat. Rev. Immunol. 2007; 7: 862–874. pmid:17948019
- View Article
- PubMed/NCBI
- Google Scholar
25. Kurata S. Extracellular and intracellular pathogen recognition by Drosophila PGRP-LE and PGRP-LC. International Immunology 2010; 22: 143–148. pmid:20089584
- View Article
- PubMed/NCBI
- Google Scholar
26. Hughes WOH, Eilenberg J, Boomsma JJ. Trade-offs in group living: transmission and disease resistance in leaf-cutting ants. Proc. R. Soc. Lond. B 2002; 269: 1811–1819.
- View Article
- Google Scholar
27. Gendrin M, Welchman DP, Poidevin M, Herve M, Lemaitre B. Long-Range Activation of Systemic Immunity through Peptidoglycan Diffusion in Drosophila. PlosPathogens 2009; 5: e1000694.
- View Article
- Google Scholar
28. Gottar M, Gobert V, Matskevich AA, Reichhart JM, Wang CS, Buft TM, et al. Dual detection of fungal infections in Drosophila via recognition of glucans and sensing of virulence factors. Cell 2006; 127: 1425–1437. pmid:17190605
- View Article
- PubMed/NCBI
- Google Scholar
29. Bartholomay LC, Cho WL, Rocheleau TA, Boyle JP, Beck ET, Fuchs JF, et al. Description of the transcriptomes of immune response-activated Hemocytes from the mosquito vectors Aedes aegypti and Armigeres subalbatus. Infect. Immun. 2004; 72: 4114–4126. pmid:15213157
- View Article
- PubMed/NCBI
- Google Scholar
30. Aguilar R, Jedlicka AE, Mintz M, Mahairaki V, Scott AL, Dimopoulos G. Global gene expression analysis of Anopheles gambiae responses to microbial challenge. Insect Biochem. Mol. Biol. 2005; 35: 709–719. pmid:15894188
- View Article
- PubMed/NCBI
- Google Scholar
31. Nottebohm E, Usui A, Therianos S, Kimura K, Dambly-Chaudière C, Ghysen A. The gene poxn controls different steps of the formation of chemosensory organs in Drosophila. Neuron 1994; 12: 25–34. pmid:8292359
- View Article
- PubMed/NCBI
- Google Scholar
32. Zhukovskaya M, Yanagawa A, Forschler BT. Grooming behavior as a mechanism of insect disease defense. Insects 2013; 4: 609–630. pmid:26462526
- View Article
- PubMed/NCBI
- Google Scholar
33. Soldano A, Alpizar YA, Boonen B, Franco L, López-Requena A, Liu G, et al. Gustatory-mediated avoidance of bacterial lipopolysaccharides via TRPA1 activation in Drosophila. eLife 2016; 5: e13133. pmid:27296646
- View Article
- PubMed/NCBI
- Google Scholar
34. Phillis RW, Bramlage AT, Wotus C, Whittaker A, Gramates LS, Seppala D, et al. Isolation of mutations affecting neural circuitry required for grooming behavior in Drosophila melanogaster. Genetics 1993; 133: 581–592. pmid:8454205
- View Article
- PubMed/NCBI
- Google Scholar
35. Böröczky K, Wada-Katsumata A, Batchelor D, Zhukovskaya M, Schal C. Insects groom their antennae to enhance olfactory acuity. PNAS 2013; 110: 3615–3620. pmid:23382193
- View Article
- PubMed/NCBI
- Google Scholar
36. Page KL, Matheson T. Wing hair sensilla underlying aimed hindleg scratching of the locust. J. Exp. Biol. 2004; 207: 2691–2703. pmid:15201302
- View Article
- PubMed/NCBI
- Google Scholar
37. Newland PL. Avoidance reflexes mediated by contact chemoreceptors on the legs of locusts. J. Comp. Physiol. A 1998; 183: 313–324.
- View Article
- Google Scholar
38. Rieger D, Fraunholz C, Popp J, Bichler D, Dittmann R, Helfrich-Forster C. The fruit fly Drosophila melanogaster favors dim light and times its activity peaks to early dawn and late dusk. J. Biol. Rhythms 2007; 22: 387–399. pmid:17876060
- View Article
- PubMed/NCBI
- Google Scholar
39. Yang C-h, Belawat P, Hafen E, Jan LY, Jan Y-N. Drosophila egg-laying site selection as a system to study simple decision-making processes. Science 2008; 319: 1679–1683. pmid:18356529
- View Article
- PubMed/NCBI
- Google Scholar
40. Kapsetaki SE, Tzelepis I, Avgousti K, Livadaras I, Garantonakis N, Varikou K, Apidianakis Y. The bacterial metabolite 2-aminoacetophenone promotes association of pathogenic bacteria with flies. Nature Communications 2014; 5: article 4401
- View Article
- Google Scholar
41. Georgel P, Naitza S, Kappler C, Ferrandon D, Zachary D, Swimmer C, et al. Drosophila immune deficiency (IMD) is a death domain protein that activates antibacterial defense and can promote apoptosis. Dev. Cell 2001; 1: 503–514. pmid:11703941
- View Article
- PubMed/NCBI
- Google Scholar
42. Gottar M, Gobert V, Michel T, Belvin M, Duyk G, Hoffmann JA, et al. The Drosophila immune response against Gram-negative bacteria is mediated by a peptidoglycan recognition protein. Nature 2002; 416: 640–644. pmid:11912488
- View Article
- PubMed/NCBI
- Google Scholar
43. Paredes JC, Welchman DP, Poidevin M, Lemaitre B. Negative regulation by amidase PGRPs shapes the Drosophila antibacterial response and protects the fly from innocuous infection. Immunity 2011; 35: 770–779. pmid:22118526
- View Article
- PubMed/NCBI
- Google Scholar
44. Brand AH, Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 1993; 118: 401–415. pmid:8223268
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Lemaitre B, Hoffmann J. The host defense of Drosophila melanogaster. Annu. Rev. Immunol. 2007; 25: 697–743. pmid:17201680
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Roy HE, Steinkraus DC, Eilenberg J, Hajek AE, Pell JK. Bizarre interactions and endgames: Entomopathogenic Fungi and Their Arthropod Hosts. Annu. Rev. Entomol. 2006; 51: 331–357. pmid:16332215
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Fernando E, Vega H, Kaya K. Insect pathology. 2nd ed. Academic Press; 2011.

[ref4] 4. DeGrandi-Hoffman G, Chen Y. Nutrition, immunity and viral infections in honey bees. Current Opinion in Insect Science 2015; 10: 170–176.
View Article
Google Scholar

[11] View Article

[12] Google Scholar

[ref5] 5. Yanagawa A, Guigue A, Marion-Poll F. Hygienic grooming is induced by contact chemicals in Drosophila melanogaster. Front. Behav. Neurosci. 2014; 8: article 254. pmid:25100963
View Article
PubMed/NCBI
Google Scholar

[14] View Article

[15] PubMed/NCBI

[16] Google Scholar

[ref6] 6. Choe KM, Werner T, Stöven S, Hultmark D, Anderson KV. Requirement for a peptidoglycan recognition protein (PGRP) in Relish activation and antibacterial immune responses in Drosophila. Science 2002; 296: 359–362. pmid:11872802
View Article
PubMed/NCBI
Google Scholar

[18] View Article

[19] PubMed/NCBI

[20] Google Scholar

[ref7] 7. Takehana A, Yano T, Mita S, Kotani A, Oshima Y, Kurata S. Peptidoglycan recognition protein (PGRP)-LE and PGRP-LC act synergistically in Drosophila immunity. EMBO J. 2004; 23: 4690–4700. pmid:15538387
View Article
PubMed/NCBI
Google Scholar

[22] View Article

[23] PubMed/NCBI

[24] Google Scholar

[ref8] 8. Kaneko T, Yano T, Aggarwal K, Lim JH, Ueda K, Oshima Y, et al. PGRP-LC and PGRP-LE have essential yet distinct functions in the Drosophila immune response to monomeric DAP-type peptidoglycan. Nat. Immunol. 2006; 7: 715–723. pmid:16767093
View Article
PubMed/NCBI
Google Scholar

[26] View Article

[27] PubMed/NCBI

[28] Google Scholar

[ref9] 9. Zaidman-Rémy A, Hervé M, Poidevin M, Pili-Floury S, Kim M-S, Blanot D, et al. The Drosophila amidase PGRP-LB modulates the immune response to bacterial infection. Immunity 2006; 24: 463–473. pmid:16618604
View Article
PubMed/NCBI
Google Scholar

[30] View Article

[31] PubMed/NCBI

[32] Google Scholar

[ref10] 10. Girardin SE, Philpott DJ. PGRP-LB minds the fort. Immunity 2006; 24: 363–366. pmid:16618594
View Article
PubMed/NCBI
Google Scholar

[34] View Article

[35] PubMed/NCBI

[36] Google Scholar

[ref11] 11. Neyen C, Poidevin M, Roussel A, Lemaitre B. Tissue- and ligand-specific sensing of Gram-negative infection in Drosophila by PGRP-LC isoforms and PGRP-LE. The Journal of Immunology 2012; 189: 1886–1897. pmid:22772451
View Article
PubMed/NCBI
Google Scholar

[38] View Article

[39] PubMed/NCBI

[40] Google Scholar

[ref12] 12. Bosco-Drayon V, Poidevin M, Boneca IG, Narbonne-Reveau K, Royet J, Charroux B. Peptidoglycan sensing by the receptor PGRP-LE in the Drosophila gut induces immune responses to infectious bacteria and tolerance to microbiota. Cell Host Microbe. 2012; 12: 153–65 (2012). pmid:22901536
View Article
PubMed/NCBI
Google Scholar

[42] View Article

[43] PubMed/NCBI

[44] Google Scholar

[ref13] 13. Burg MG, Hanna L, Kim Y-T, Wus C-F. Development and maintenance of a simple reflex circuit in small-patch mosaics of Drosophila: effects of altered neuronal function and developmental arrest. J. Neurobiol. 1993; 24: 803–823. pmid:8331340
View Article
PubMed/NCBI
Google Scholar

[46] View Article

[47] PubMed/NCBI

[48] Google Scholar

[ref14] 14. Cofras G, Dudai Y. Habituation and dishabituation of a cleaning reflex in normal and mutant Drosophila. The Journal of Neuroscience 1989; 9: 56–62. pmid:2913213
View Article
PubMed/NCBI
Google Scholar

[50] View Article

[51] PubMed/NCBI

[52] Google Scholar

[ref15] 15. Ashton K, Wagoner AP, Carrillo R, Gibson G. Quantitative trait loci for the monoamine-related traits heart rate and headless behavior in Drosophila melanogaster. Genetics 2001; 157: 283–294. pmid:11139509
View Article
PubMed/NCBI
Google Scholar

[54] View Article

[55] PubMed/NCBI

[56] Google Scholar

[ref16] 16. Raad H, Ferveur JF, Ledger N, Capovilla M, Robichon A. Functional Gustatory Role of Chemoreceptors in Drosophila Wings. Cell Report 2016; 15: 1442–1454.
View Article
Google Scholar

[58] View Article

[59] Google Scholar

[ref17] 17. Vandervorst P, Ghysen A. Genetic control of sensory connections in Drosophila. Nature 1980; 286: 65–67. pmid:7393324
View Article
PubMed/NCBI
Google Scholar

[61] View Article

[62] PubMed/NCBI

[63] Google Scholar

[ref18] 18. Seeds AM, Ravbar P, Chung P, Hampel S, Midgley FM Jr, Mensh BD, et al. A suppression hierarchy among competing motor programs drives sequential grooming in Drosophila. eLife 2014; 3: e02951: pmid:25139955
View Article
PubMed/NCBI
Google Scholar

[65] View Article

[66] PubMed/NCBI

[67] Google Scholar

[ref19] 19. Buchon N, Broderick NA, Lemaitre B. Gut homeostasis in a microbial world: insights from Drosophila melanogaster. Nature Reviews Microbiology 2014; 11: 615–626.
View Article
Google Scholar

[69] View Article

[70] Google Scholar

[ref20] 20. Kaneko T, Goldman WE, Mellroth P, Steiner H, Fukase K, Kusumoto S, et al. Monomeric and polymeric gram-negative peptidoglycan but not purified LPS stimulate the Drosophila IMD pathway. Immunity 2004; 20: 637–649. pmid:15142531
View Article
PubMed/NCBI
Google Scholar

[72] View Article

[73] PubMed/NCBI

[74] Google Scholar

[ref21] 21. Mattoo S, Cherry JD. Molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies. Clinical Microbiology Reviews 2005; 18: 326–382. pmid:15831828
View Article
PubMed/NCBI
Google Scholar

[76] View Article

[77] PubMed/NCBI

[78] Google Scholar

[ref22] 22. Stenbak CR, Ryu JH, Leulier F, Pili-Floury S, Parquet C, Herve M, et al. Peptidoglycan molecular requirements allowing detection by the Drosophila immune deficiency pathway. J. Immunol. 2004; 173: 7339–7348. pmid:15585858
View Article
PubMed/NCBI
Google Scholar

[80] View Article

[81] PubMed/NCBI

[82] Google Scholar

[ref23] 23. Guan R, Mariuzza RA. Peptidoglycan recognition proteins of the innate immune system. Trends in Microbiology 2007; 15: 127–134. pmid:17275309
View Article
PubMed/NCBI
Google Scholar

[84] View Article

[85] PubMed/NCBI

[86] Google Scholar

[ref24] 24. Ferrandon D, Imler J-L, Hetru C, Hoffmann JA. The Drosophila systemic immune response: sensing and signalling during bacterial and fungal infections. Nat. Rev. Immunol. 2007; 7: 862–874. pmid:17948019
View Article
PubMed/NCBI
Google Scholar

[88] View Article

[89] PubMed/NCBI

[90] Google Scholar

[ref25] 25. Kurata S. Extracellular and intracellular pathogen recognition by Drosophila PGRP-LE and PGRP-LC. International Immunology 2010; 22: 143–148. pmid:20089584
View Article
PubMed/NCBI
Google Scholar

[92] View Article

[93] PubMed/NCBI

[94] Google Scholar

[ref26] 26. Hughes WOH, Eilenberg J, Boomsma JJ. Trade-offs in group living: transmission and disease resistance in leaf-cutting ants. Proc. R. Soc. Lond. B 2002; 269: 1811–1819.
View Article
Google Scholar

[96] View Article

[97] Google Scholar

[ref27] 27. Gendrin M, Welchman DP, Poidevin M, Herve M, Lemaitre B. Long-Range Activation of Systemic Immunity through Peptidoglycan Diffusion in Drosophila. PlosPathogens 2009; 5: e1000694.
View Article
Google Scholar

[99] View Article

[100] Google Scholar

[ref28] 28. Gottar M, Gobert V, Matskevich AA, Reichhart JM, Wang CS, Buft TM, et al. Dual detection of fungal infections in Drosophila via recognition of glucans and sensing of virulence factors. Cell 2006; 127: 1425–1437. pmid:17190605
View Article
PubMed/NCBI
Google Scholar

[102] View Article

[103] PubMed/NCBI

[104] Google Scholar

[ref29] 29. Bartholomay LC, Cho WL, Rocheleau TA, Boyle JP, Beck ET, Fuchs JF, et al. Description of the transcriptomes of immune response-activated Hemocytes from the mosquito vectors Aedes aegypti and Armigeres subalbatus. Infect. Immun. 2004; 72: 4114–4126. pmid:15213157
View Article
PubMed/NCBI
Google Scholar

[106] View Article

[107] PubMed/NCBI

[108] Google Scholar

[ref30] 30. Aguilar R, Jedlicka AE, Mintz M, Mahairaki V, Scott AL, Dimopoulos G. Global gene expression analysis of Anopheles gambiae responses to microbial challenge. Insect Biochem. Mol. Biol. 2005; 35: 709–719. pmid:15894188
View Article
PubMed/NCBI
Google Scholar

[110] View Article

[111] PubMed/NCBI

[112] Google Scholar

[ref31] 31. Nottebohm E, Usui A, Therianos S, Kimura K, Dambly-Chaudière C, Ghysen A. The gene poxn controls different steps of the formation of chemosensory organs in Drosophila. Neuron 1994; 12: 25–34. pmid:8292359
View Article
PubMed/NCBI
Google Scholar

[114] View Article

[115] PubMed/NCBI

[116] Google Scholar

[ref32] 32. Zhukovskaya M, Yanagawa A, Forschler BT. Grooming behavior as a mechanism of insect disease defense. Insects 2013; 4: 609–630. pmid:26462526
View Article
PubMed/NCBI
Google Scholar

[118] View Article

[119] PubMed/NCBI

[120] Google Scholar

[ref33] 33. Soldano A, Alpizar YA, Boonen B, Franco L, López-Requena A, Liu G, et al. Gustatory-mediated avoidance of bacterial lipopolysaccharides via TRPA1 activation in Drosophila. eLife 2016; 5: e13133. pmid:27296646
View Article
PubMed/NCBI
Google Scholar

[122] View Article

[123] PubMed/NCBI

[124] Google Scholar

[ref34] 34. Phillis RW, Bramlage AT, Wotus C, Whittaker A, Gramates LS, Seppala D, et al. Isolation of mutations affecting neural circuitry required for grooming behavior in Drosophila melanogaster. Genetics 1993; 133: 581–592. pmid:8454205
View Article
PubMed/NCBI
Google Scholar

[126] View Article

[127] PubMed/NCBI

[128] Google Scholar

[ref35] 35. Böröczky K, Wada-Katsumata A, Batchelor D, Zhukovskaya M, Schal C. Insects groom their antennae to enhance olfactory acuity. PNAS 2013; 110: 3615–3620. pmid:23382193
View Article
PubMed/NCBI
Google Scholar

[130] View Article

[131] PubMed/NCBI

[132] Google Scholar

[ref36] 36. Page KL, Matheson T. Wing hair sensilla underlying aimed hindleg scratching of the locust. J. Exp. Biol. 2004; 207: 2691–2703. pmid:15201302
View Article
PubMed/NCBI
Google Scholar

[134] View Article

[135] PubMed/NCBI

[136] Google Scholar

[ref37] 37. Newland PL. Avoidance reflexes mediated by contact chemoreceptors on the legs of locusts. J. Comp. Physiol. A 1998; 183: 313–324.
View Article
Google Scholar

[138] View Article

[139] Google Scholar

[ref38] 38. Rieger D, Fraunholz C, Popp J, Bichler D, Dittmann R, Helfrich-Forster C. The fruit fly Drosophila melanogaster favors dim light and times its activity peaks to early dawn and late dusk. J. Biol. Rhythms 2007; 22: 387–399. pmid:17876060
View Article
PubMed/NCBI
Google Scholar

[141] View Article

[142] PubMed/NCBI

[143] Google Scholar

[ref39] 39. Yang C-h, Belawat P, Hafen E, Jan LY, Jan Y-N. Drosophila egg-laying site selection as a system to study simple decision-making processes. Science 2008; 319: 1679–1683. pmid:18356529
View Article
PubMed/NCBI
Google Scholar

[145] View Article

[146] PubMed/NCBI

[147] Google Scholar

[ref40] 40. Kapsetaki SE, Tzelepis I, Avgousti K, Livadaras I, Garantonakis N, Varikou K, Apidianakis Y. The bacterial metabolite 2-aminoacetophenone promotes association of pathogenic bacteria with flies. Nature Communications 2014; 5: article 4401
View Article
Google Scholar

[149] View Article

[150] Google Scholar

[ref41] 41. Georgel P, Naitza S, Kappler C, Ferrandon D, Zachary D, Swimmer C, et al. Drosophila immune deficiency (IMD) is a death domain protein that activates antibacterial defense and can promote apoptosis. Dev. Cell 2001; 1: 503–514. pmid:11703941
View Article
PubMed/NCBI
Google Scholar

[152] View Article

[153] PubMed/NCBI

[154] Google Scholar

[ref42] 42. Gottar M, Gobert V, Michel T, Belvin M, Duyk G, Hoffmann JA, et al. The Drosophila immune response against Gram-negative bacteria is mediated by a peptidoglycan recognition protein. Nature 2002; 416: 640–644. pmid:11912488
View Article
PubMed/NCBI
Google Scholar

[156] View Article

[157] PubMed/NCBI

[158] Google Scholar

[ref43] 43. Paredes JC, Welchman DP, Poidevin M, Lemaitre B. Negative regulation by amidase PGRPs shapes the Drosophila antibacterial response and protects the fly from innocuous infection. Immunity 2011; 35: 770–779. pmid:22118526
View Article
PubMed/NCBI
Google Scholar

[160] View Article

[161] PubMed/NCBI

[162] Google Scholar

[ref44] 44. Brand AH, Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 1993; 118: 401–415. pmid:8223268
View Article
PubMed/NCBI
Google Scholar

[164] View Article

[165] PubMed/NCBI

[166] Google Scholar

Figures

Abstract

Introduction

Results

Contact with microbes induces grooming

Highly purified microbial components induce grooming

Grooming induction is lost in mutants of pattern-recognition receptors but not of signaling components of the Imd pathway

Discussions

Materials and methods

Fly stocks

Microbial preparations

Chemicals

Grooming induction and scoring

Statistical analysis

Supporting information

S1 Fig. Grooming behavior induced by heat-killed microbes and its related chemicals in CS flies, (a) E.coli (b) M. fermentans (c) peptidoglycan from B. subtilis (d) ß-glucan from algae (laminarin) on CS flies were used for all tests, which employed w1118.

S2 Fig. Grooming behavior induced by LPS contact on front legs and hind legs in black square: w;;PGRP-LCE12 with Cantonized 2nd chromosome, green square: y, w, PGRP-LE112, grey circle: w;;PGRP-LBΔ, black square: w;;PGRP-LCE12 and orange square: w;P[acman]-PGRP-LC;PGRP-LCE12 rescue flies.

S3 Fig.

S4 Fig.

S1 Table. Results of Chi-square test on grooming induction by G+ microbe and highly purified microbial compounds in wild type flies.

S2 Table. Results of Chi-square test on grooming induction in IMD pathway related mutants.

S3 Table. Results of Chi square test on grooming induction in PGRP-LC mutants to quinine and in 10-day old PGRP-LC mutants.

S4 Table. Results of Chi square test on grooming induction in PGRP-LC related mutants.

Acknowledgments

References

S1 Fig. Grooming behavior induced by heat-killed microbes and its related chemicals in CS flies, (a) E.coli (b) M. fermentans (c) peptidoglycan from B. subtilis (d) ß-glucan from algae (laminarin) on CS flies were used for all tests, which employed w¹¹¹⁸.

S2 Fig. Grooming behavior induced by LPS contact on front legs and hind legs in black square: w;;PGRP-LC^E12 with Cantonized 2nd chromosome, green square: y, w, PGRP-LE¹¹², grey circle: w;;PGRP-LB^Δ, black square: w;;PGRP-LC^E12 and orange square: w;P[acman]-PGRP-LC;PGRP-LC^E12 rescue flies.