A potential tradeoff between feeding rate and aversive learning determines intoxication in a Caenorhabditis elegans host-pathogen system

Journal Pre-proof A potential tradeoff between feeding rate and aversive learning determines intoxication in a Caenorhabditis elegans host-pathogen system Pallavi Velagapudi, Rachel Ghoubrial, Ratnavi Shah, Helana Ghali, Meghan Haas, Krunal S. Patel, Ashleigh Riddell, Christopher A. Blanar, Robert P. Smith PII:

S1286-4579(20)30024-1

DOI:

https://doi.org/10.1016/j.micinf.2020.01.002

Reference:

MICINF 4684

To appear in:

Microbes and Infection

Received Date: 3 June 2019 Revised Date:

15 January 2020

Accepted Date: 16 January 2020

Please cite this article as: P. Velagapudi, R. Ghoubrial, R. Shah, H. Ghali, M. Haas, K.S. Patel, A. Riddell, C.A. Blanar, R.P. Smith, A potential tradeoff between feeding rate and aversive learning determines intoxication in a Caenorhabditis elegans host-pathogen system, Microbes and Infection, https://doi.org/10.1016/j.micinf.2020.01.002. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Masson SAS on behalf of Institut Pasteur.

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A potential tradeoff between feeding rate and aversive learning determines intoxication in a

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Caenorhabditis elegans host-pathogen system.

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Pallavi Velagapudi*, Rachel Ghoubrial*, Ratnavi Shah, Helana Ghali, Meghan Haas, Krunal S. Patel,

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Ashleigh Riddell, Christopher A. Blanar and Robert P. Smith

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Department of Biological Sciences, Halmos College of Natural Sciences and Oceanography, Nova Southeastern University, Fort Lauderdale FL, 33314

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Correspondence should be addressed to Robert P Smith. E-mail: [email protected] Tel: 954 262

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7979

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*

these authors contributed equally

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Abstract

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Despite being the first line of defense against infection, little is known about how host-

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pathogen interactions determine avoidance. Caenorhabditis elegans can become infected by

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chemoattractant-producing bacteria through ingestion. The worms can learn to associate these

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chemoattractants with harm through aversive learning. As a result, the worms will avoid the

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pathogen. Evolutionary constraints have likely shaped the attraction, intoxication and learning

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dynamics between bacteria and C. elegans, but these have not been explored. Using bacteria

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engineered to express an acylhomoserine lactone chemoattractant and a nematicidal protein, we

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explored how manipulating the amount of attractant produced by the bacteria affects learning and

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intoxication in mixed stage populations of C. elegans. We found that increasing the production rate of

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the chemoattractant increased the feeding rate in C. elegans, but decreased the time required for C.

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elegans to learn to avoid the chemoattractant. Learning generally coincided with a decreased feeding

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rate. We also observed that the percentage of intoxicated worms was maximized at intermediate

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production rates of the attractant. We propose that interactions between attractant driven feeding

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rate and aversive learning are likely responsible for this trend. Our results increase our understanding

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of behavioral avoidance in C. elegans and have implications in understanding host-pathogen dynamics

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that shape avoidance.

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Keywords: evolutionary tradeoff; acylhomoserine lactone; avoidance; crystal toxin protein; feeding

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rate; learning deficient;

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1. Introduction

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The interactions between a host and a pathogen have been described as an evolutionary

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arms race [1]. The pathogen develops strategies to infect the host. The host counteracts by

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developing its own strategies to prevent and defend against infection. The strategies that the host

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can develop to defend against infection are diverse but can be grouped into avoidance [2], tolerance

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[3] and resistance [4]. Avoidance has been observed in diverse organisms and can involve strategies

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to avoid infected conspecifics [5], vectors [6] and environmental conditions [7] that promote disease

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transmission, and strategies to modify habitat to decrease the chance of infection [8]. While the

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immunological and biochemical interactions that govern infection processes have been explored,

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less attention has been paid to studying behaviors that are used for avoidance [2]. Understanding

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avoidance within the context of host-pathogen interactions is important, as such strategies serve as

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the first line of defense against infection [9] and are more cost-effective relative to mounting an

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immunological response [2].

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Some pathogens emit (e.g., [10]), or alter their host to emit (e.g., [11]), chemoattractants that

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serve to promote infection and/or transmission. Pathogens that emit chemoattractants infect a wide

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range of hosts including nematodes [12], insects [10], plants [13], and humans [14]. Towards

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avoiding these pathogens, potential hosts have developed avoidance strategies that can rely on the

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ability to detect and avoid the chemoattractant, which serves to reduce infection (e.g., [5]). These

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interactions are observed in the nematode Caenorhabditis elegans, which has served as a model system

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for host-pathogen interactions [15, 16]. C. elegans can become infected by several species of bacteria,

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fungi, and viruses [15, 17]. One mechanism that bacteria use to gain entry into C. elegans is through

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ingestion [15]. The primary food source for C. elegans in both natural [18] and laboratory settings [19]

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is bacteria. The worms use gradients of bacteria-produced chemicals, including amino acids [20], and

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autoinducers involved in quorum sensing [21], to locate food sources. Some bacterial pathogens of

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C. elegans produce chemicals that initially attract the worms [12, 22, 23]. However, upon

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consumption of these pathogens, C. elegans can learn to associate the attractive chemical with harm

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and will alter its behavior by reducing its feeding rate [24], entering into the dauer stage [25],

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becoming repulsed by the chemical [26] and/or leaving areas where the pathogenic bacteria are

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growing [22]. Ultimately, these strategies allow C. elegans to avoid the pathogen.

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For naïve worms, avoiding pathogens through learning appears not to be instantaneous. C.

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elegans must consume the pathogen over several hours to drive aversive learning [26]. The time

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required to drive aversive learning appears to vary among pathogens [17, 26]. Similarly, death of C.

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elegans due to infection also varies among pathogens [12, 23]. This variability may be accounted for

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by differences in the structure and diffusion rates of the odor and/or mechanisms of action during

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pathogenesis. It is likely that pathogens of C. elegans face an evolutionary conundrum in the

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attraction process. Pathogens that produce large amounts of attractant are likely to be consumed

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often in the environment but are more likely to drive aversive learning in C. elegans at a more rapid

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pace. Evidence of this stems from studies demonstrating that aversive learning and avoidance

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develop rapidly in C. elegans when the strength of a negative stimulus increases [27]. Pathogens that

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reduce the amount of attractant too much risk reducing their infectivity by lowering the likelihood

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that C. elegans consumes them. Indeed, reducing the amount of a chemoattractant has been shown to

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decrease attraction of C. elegans [28]. While this tradeoff is plausible, it remains unstudied, likely

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owing to the inability to manipulate attractant production in natural pathogens of C. elegans. Many

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interactions between C. elegans and its pathogens, including aversive learning, are thought to be

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evolutionarily conserved across diverse species [19]. Thus, the study of this potential evolutionary

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tradeoff might have implications in additional host-pathogen systems.

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In this manuscript, we used an engineered strain of Escherichia coli to examine how changing

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the production rate of a chemoattractant affects aversive learning and intoxication in mixed stage

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populations of C. elegans. Unlike their natural counterparts, using engineered bacteria allowed us to

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manipulate the parameters of attraction to examine how changing attractant production rate affects

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aversive learning. Previous studies have used engineered bacteria to determine evolutionary tradeoffs

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that are nearly impossible to examine using naturally occurring counterparts [29]. To address our

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question, we used mixed-stage populations of C. elegans, which have been used in the past to simulate

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naturally occurring nematode populations (e.g., [30]). We chose to use mixed-stage worms as the

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evolutionary constraints that drove the interactions between learning. Chemoattraction and toxicity

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likely arose under these conditions.

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2. Materials and methods

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2.1 Strains and growth conditions

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All experiments were performed in modified M9 medium [1X M9 salts (48 mM Na2HPO4,

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22 mM KH2PO4, 862 mM NaCl, 19 mM NH4Cl) 0.4% glucose, 0.1% casamino acids (Teknova,

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Hollister, CA), 0.5% thiamine (Calbiochem, San Diego, CA), 2.5 mM MgSO4, 0.1 mM CaCl2, 100

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mM 3-(N-morpholino)propanesulfonic acid (MOPS, Amresco, Solon, OH)] buffered to pH 7.0 with

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5 M KOH with 1% agar (Alfa Aesar, Ward Hill, MA). Single colonies were inoculated overnight into

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3 mL of Luria-Bertani (LB) medium (MP Biomedicals, Solon OH). Culture media contained 50

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µg/mL kanamycin and 100 µg/mL ampicillin for plasmid maintenance. Expression from the Plac

115

promoter was induced using 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, Promega,

116

Madison, WI). Expression from the Ptet promoter was induced using anhydrotetracycline (atc, Acros

117

Organics, Geel, Belgium) at various concentrations.

118

We used a previously engineered strain of E. coli [31] that expresses a crystal toxin protein

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(Cry5B), and a protein that produces an acylhomoserine lactone (AHL) chemoattractant [31] (LuxI,

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Fig. 1a). An IPTG inducible Plac promoter controls expression of cry5B. An anhydrotetracycline (atc)

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inducible Ptet promoter controls expression of luxI. The Cry5B protein causes intoxication in

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nematodes by damaging the worm’s intestine, which results in arrested movement, vacuolated

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intestines, and/or loss of internal structures [32].

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C. elegans strains N2 (wildtype, Carolina Biological, Burlington, NC), lrn-1, mod-1 and eat-1

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(Caenorhabditis Genetics Center, University of Minnesota) were maintained on nematode growth agar

126

(Carolina Biological) containing a lawn of E. coli strain MG1655 (F- lambda- ilvG- rfb-50 rph-1, a

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derivative of E. coli K-12 [33, 34]). Cultures were grown on 100 mm x 15 mm agar plates at room

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temperature and sub-cultured by rinsing the plate with sterile dH2O and transferring 10 µL of

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worms to a new plate [35]. We used mixed developmental stage worms grown on nematode growth

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agar for approximately 7 days to examine how a population of C. elegans consisting of individuals in

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multiple stages of development would behave. The average distribution of life stages (across three

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independent plates) in these cultures was L1 = 50.6% ± 3.06, L2 = 27.74% ± 4.25, L3 = 16.25% ±

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6.56, and L4 = 5.41% ± 3.52. C. elegans cultures that were grown independently across multiple days

134

were used as biological replicates. The average number of worms used in each assay is located in the

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figure legend and was rounded to the nearest whole number.

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2.2 Feeding rate

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We grew either luxI-expressing and luxI/cry5B-expressing bacteria overnight on M9 agar

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plates containing kanamycin (luxI) or kanamycin and ampicillin (luxI/cry5B) and various

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concentrations of atc. When examining the luxI/cry5B-expressing strain, the medium did, or did not

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as a control, contain 1 mM IPTG. 10 µL of M9 medium containing C. elegans was placed in the

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center of the plate. Feeding rate was examined after the time indicated in the text and figure legend.

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To determine feeding rate, we examined C. elegans using an Olympus IX73P2F microscope with a

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DP-80 camera (Olympus Microscopes, Center Valley, PA) using 4X objective lens (with an

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additional 2X magnification via a zoom function) using the bright field setting. We counted the

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number of pharyngeal pumps during a 10 s period, which was converted to pumps/second. The

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number of worms counted per condition was rounded to the nearest whole number and is presented

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in the figure legend.

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2.3 Modified learning assay

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We grew overnight cultures of the cry5B/luxI expressing bacteria. These cultures were

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washed and resuspendend in fresh M9 medium. 50 µL was spread across 5 mL of M9 medium with

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0.5% agar housed in 6 well plates. Unless otherwise indicated, the medium contained 1 mM IPTG

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and various concentrations of atc. After growing the bacteria at 37oC for 16 hours, C. elegans were

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inoculated in the center of each plate. After feeding on bacteria for various time periods, the worms

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were isolated by rinsing the plate with 1 mL of dH2O and centrifuging the worms for 2 minutes at

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15 x g. The worms were washed three times with dH2O. Thirty minutes prior to the addition of C.

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elegans, 5 µL of 20 µM AHL (3-oxohexanoyl-homoserine lactone (3OC6HSL, Sigma-Aldrich, Saint

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Louis, MO) and 5 µL of ethanol (carrier solvent for AHL, not an attractant [21]) were placed on

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randomly assigned and opposite sides of 5 mL of M9 medium with 0.5% agar in a 6 well plate (3 cm

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apart and 1.5 cm from the initial position of the worms). C. elegans were then placed in the center of

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the agar plate and was incubated at room temperature for one hour. Using a Leica M80 with a V-lux

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1000 light source at 60X magnification (Leica Microsystem, Buffalo Grove, II), we counted the

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number of worms located on the side of the plate that contained AHL or ethanol (control). Only C.

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elegans that had moved away from the point of inoculation were counted and worms that were within

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0.25 cm of the center of plate were not included. Choice index was calculated using the following

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equation.

168

ℎ



=

(





)

(Eq. 1)

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Attraction was characterized by movement towards AHL, which resulted in a positive choice index

170

(Eq. 1, (21)). A lack of attraction was characterized by a choice index that was not different from

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zero, or that was negative.

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2.4 Media switching assay

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We grew overnight cultures of cry5B/luxI expressing bacteria. We inoculated 50 µL of

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bacteria on agar plates containing 1 mM IPTG and 100 ng/mL of atc, 100 ng/mL atc or without

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inducers (control). After growing bacteria under these conditions for 16 hours at 37oC, we

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inoculated C. elegans. After 24 hours, the worms were removed from the agar plates, and washed

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three times using ddH2O. Each population of worms was then placed on agar plates that contained

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cry5B/luxI expressing bacteria (grown for 16 hours at 37oC) and 100 ng/mL of atc or without

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inducers (control). Only worms that had been previously exposed to either the 1 mM IPTG/100

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ng/mL atc or the 100 ng/mL atc conditions in the previous step were placed on plates containing

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100 ng/mL of atc. After 24 hours, C. elegans were removed, washed three times in ddH2O and

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subjected to our modified learning assay. Choice index calculated as per Eq. 1.

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2.5 Quantifying Intoxication

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A Leica M80 with a V-lux 1000 light source was used to observe C. elegans at 60X

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magnification. We examined each nematode for 10 seconds. A lack of movement, which often

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coincided with a highly vacuolated intestinal tract, during this period resulted in classifying the worm

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as intoxicated [32].

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2.6 Statistical analysis

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Statistical tests are specified in the figure legends along with corresponding P values;

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statistical significance was assumed if P ≤ 0.05. To compare choice indices to zero, we used a

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student’s one-tailed t-test ((un-paired, unequal variance, using Microsoft Excel (Redmond, WA)). To

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compare between choice indices, we used a student’s two-tailed t-test (un-paired, unequal variance).

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For the feeding rate experiments, we collected data from each plate at different time points so we

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took a repeated measures approach. We used a mixed model in JMP 14 [36] to test for differences in

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feeding rates (using pumping rates as a proxy) among nematode strains with time (0, 24, 48, and 72

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hours) as a repeated measure and a Tukey HSD test to conduct multiple comparisons. We explored

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the effects of increasing ATC on feeding rates with a General Linear Model (GLM) in JMP, again

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using the Tukey HSD test for multiple comparisons. For all other data, we used a student’s two-

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tailed t-test (un-paired, unequal variance) to determine significance.

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3. Results

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3.1 Increasing expression of luxI in engineered bacteria increases feeding rate of C. elegans.

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To determine if we could manipulate the feeding rate of C. elegans by changing the

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expression level of luxI, we used a GFP expressing strain of bacteria to determine if increasing the

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concentration of atc in the medium increased expression of elements downstream of the Ptet

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promoter. We observed, as previously reported [37], that as the concentration of atc increased in the

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growth medium, an increase in GFP was observed (Supplementary Fig. S1). We then allowed C.

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elegans to feed on luxI-expressing bacteria grown in the presence of various concentrations of atc.

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After 1 hour, we observed that as the concentration of atc in the medium increased, feeding rate

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increased (Fig. 1b). Overall, we could increase the feeding rate of C. elegans by increasing the

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concentration of atc in the growth medium.

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3.2 Decreasing expression of luxI increases the time required for C. elegans to avoid AHL

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Our previous publication found that concurrent expression of cry5B and luxI drives aversive

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learning in C. elegans such that the worms are repulsed by AHL [31]. Building on this previous

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finding, we sought to determine how the expression level of luxI would affect the time required for

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C. elegans to learn to associate AHL with Cry5B toxicity. We first allowed C. elegans to feed on

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engineered bacteria expressing cry5B/luxI grown in the presence of 100 ng/mL atc and 1 mM IPTG.

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The concentration of these inducers allows maximal expression from the Ptet and Plac promoters,

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respectively [37]. We then used a modified learning assay to quantify choice index (Eq. 1), and thus

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learning (Fig. 2a). In line with previous publications, a positive choice index (Eq. 1) indicates

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attraction to AHL, whereas a choice index that was negative or not statistically different than zero

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indicated learning. After 1 hour of feeding on the engineered bacteria, we observed that C. elegans

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remained attracted to AHL as indicated through a positive choice index (Fig. 2b). After 3 hours, C.

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elegans had a choice index that was not different than zero. After 5 hours, the choice index became

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negative. Overall, our results suggest that aversive learning towards AHL occurred in as little as 3

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hours when 100 ng/mL of atc was included in the growth medium.

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We sought to determine how aversive learning would be impacted as the concentration of

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atc, and thus expression of luxI, decreased in the growth medium. We performed the experiment

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described above and reduced the concentration of atc in the medium while keeping the

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concentration of IPTG (1 mM) constant. We observed that when the concentration of atc was

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reduced to between 40 and 80 ng/mL, aversive learning was observed after C. elegans fed on the

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engineered bacteria for 48 hours (Fig. 2c). When the concentration of atc was 10-20 ng/mL,

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learning, as measured using the choice index, was not evident after 72 hours of feeding. We note

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that transgene expression in our experimental setup was generally stable over the entire period of

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feeding (Supplementary Methods, Supplementary Results, and Supplementary Fig. S2). We chose

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not to measure feeding rate past 72 hours as this would likely require the addition of nutrients or

241

transferring the worms to a new agar plate. Both of these manipulations could result in death of the

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worms, or additional stressors, which could influence choice index, and thus our ability to quantify

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aversive learning.

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To confirm that the observed decrease in choice index was not primarily due to habituation

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to AHL, or expression of cry5B alone, we grew the engineered bacteria on agar plates that

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independently contained 100 ng/mL atc, 1 mM IPTG or did not contain inducers. We allowed C.

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elegans to feed on these bacteria for 72 hours whereupon we removed the worms and performed our

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learning assay. We observed that C. elegans remained attracted to AHL after 72 hours of exposure to

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bacteria independently expressing luxI and cry5B (Fig. 2d). This indicated that habituation to AHL,

250

or toxicity from Cry5B, could not be primarily responsible for the trends observed in Fig. 2c.

251

Instead, concurrent expression of luxI and cry5B was required to drive aversion towards AHL in C.

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elegans.

253

To confirm that the decrease in choice indices observed in Fig. 2b and c was due to learning,

254

we used a learning deficient strain of C. elegans (lrn-1) [38], which has been previously shown to be

255

impaired in associative olfactory learning, but not in non-associative learning (e.g., habituation) [39].

256

We allowed this strain to feed on bacteria concurrently expressing cry5B (1 mM IPTG) and luxI

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(various concentrations of atc) for 72 hours. Using our learning assay, we observed that the lrn-1

258

worms remained attracted to AHL as indicated through a positive choice index at all concentrations

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of atc examined (Fig. 2e). Moreover, lrn-1 worms also remained attracted to AHL after feeding on

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bacteria that independently expressed luxI (100 ng/mL atc), and cry5B (1 mM IPTG, Fig. 2f).

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Finally, we allowed wildtype C. elegans to feed on bacteria expressing both cry5B and luxI (1

262

mM IPTG and 100 ng/mL of atc) for 24 hours (Fig. 2g). We then carefully transferred these worms

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to medium where bacteria were only expressing luxI (100 ng/mL of atc). After 24 hours, we

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performed our modified learning assay. We observed that C. elegans remained attracted to AHL

265

under these conditions as indicated by a positive choice index. Furthermore, when C. elegans were fed

266

on bacteria expressing only luxI (100 ng/mL atc through the experiment) or bacteria that were not

267

expressing either transgene (0 mM IPTG/ 0 ng/mL of atc), wildtype C. elegans remained attracted to

268

AHL.

269 270

3.3 C. elegans reduces its feeding rate as a result of aversive learning

271

Previous studies have demonstrated that C. elegans will reduce its feeding rate when

272

confronted with pathogenic bacteria [25] and repulsive odorants [24] as a result of aversive learning.

273

We sought to determine if C. elegans was reducing its feeding rate in response to our engineered

274

bacteria. We measured the feeding rate of C. elegans exposed to bacteria concurrently expressing

275

cry5B (1 mM IPTG) and luxI grown on plates containing increasing concentrations of atc. When the

276

medium contained 100 ng/mL of atc, we observed a significant decrease in the feeding rate at 24, 48

277

and 72 hours relative to the feeding rate measured after 1 hour (Fig. 3a). In contrast, when the

278

medium contained 50 ng/mL of atc, feeding rate was not reduced after 24 hours as compared to the

279

feeding rate observed after 1 hour. However, after 48 and 72 hours, we observed that there was a

280

significant decrease in feeding rate under this condition as compared to worms observed at 1 hour

281

and 24 hours. A reduction in feeding rate was not observed when the medium contained 10 ng/mL

282

atc after 24, 48 and 72 hours.

283

To test if the reduction in feeding rate was due to aversive learning, we performed the

284

experiment described above but used the lrn-1 strain (Fig. 3b)., as well as an additional learning

285

deficiency strain, mod-1 (Fig. 3c). We generally observed that, when atc was included in the medium

286

at various concentrations, there was no significant reduction in feeding rate after 1, 24, 48 and 72

287

hours of feeding on bacteria expressing both luxI and cry5B (1 mM IPTG). An exception to this

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trend was observed when we compared the feeding rate of lrn-1 worms after 1 hour and 48 hours on

289

medium containing 100 ng/mL atc. We observed a significant decrease in feeding rate after 48

290

hours. However, this reduction was not observed at 72 hours.

291

To determine if expression of cry5B alone could account for the reduction in feeding rate

292

observed in C. elegans (Fig. 3a), we allowed wildtype, lrn-1 and mod-1 worms to feed on cry5B-

293

expressing bacteria for 24, 48 and 72 hours (1 mM IPTG in the medium, Fig. 3d). We observed a

294

significant decrease in feeding rate at 48 hours, but not after 72 hours, for the wildtype C. elegans

295

strain. A reduction in feeding rate was not observed for the learning deficient lrn-1 and mod-1 strains.

296

We note that basal feeding rate (in the absence of IPTG or atc) of the wildtype, lrn-1 and mod-1

297

worms is statistically identical indicating that physiological differences in feeding rate cannot account

298

for our results (Supplementary Fig. S4).

299 300

3.4 The percentage of intoxicated worms is maximized at intermediate expression of luxI

301

As the Cry5B protein intoxicates C. elegans through ingestion [32], we hypothesized that

302

changes in feeding rate would affect intoxication efficacy. To initially test this hypothesis, we allowed

303

a pumping deficient strain of C. elegans (eat-1) to feed on bacteria expressing cry5B for 48 hours. We

304

observed a significant decrease in the percentage of intoxicated eat-1 worms as compared to wildtype

305

C. elegans (Fig. 4a). This significant decrease was not apparent after the worms fed on the bacteria for

306

144 hours (Supplementary Fig. S5a), indicating that physiological differences between the strains, in

307

terms of the effectiveness of Cry5B, cannot account for this difference. In general, our results

308

suggest that reducing feeding rate reduces intoxication rate in the short term.

309

We then allowed wildtype C. elegans to feed on bacteria expressing cry5B (1mM IPTG) and

310

luxI (various concentrations of atc in the medium). After 24 hours, we observed that the percentage

311

of intoxicated worms tended to increase as the concentration of atc in the medium was increased.

14 312

After 48 hours, we observed that the percentage of intoxicated worms followed a biphasic trend

313

with increasing atc (Fig. 4b), where the percentage of intoxicated worms was highest at 60 ng/mL of

314

atc. This trend was largely maintained at 72 hours. To determine if this biphasic trend was owing to

315

aversive learning, we allowed the lrn-1 strain to feed on bacteria expressing cry5B (1 mM IPTG) and

316

luxI (various concentrations of atc, Fig. 4c). After 24 hours, we observed a similar trend to when

317

wildtype worms were used in the assay. However, after 48 and 72 hours, and contrary to the

318

biphasic trend observed with worms capable of learning, we observed that, in general, the

319

percentage of intoxicated worms increased as a function of atc concentration. We confirmed that

320

this difference was not due to strain specific susceptibility to intoxication by Cry5B. Indeed, we

321

observed that the percentage of intoxicated wildtype and lrn-1 worms was statistically identical when

322

fed on bacteria that only expressed cry5B (Supplementary Fig. S5b).

323 324

4. Discussion

325

We propose that interactions between AHL attractant driven feeding rate, and Cry5B driven

326

aversive learning maximized the percentage of intoxicated worms at intermediate concentrations of

327

atc (Fig. 5). At the highest examined expression rate of luxI (100 ng/mL), increased feeding allowed

328

C. elegans to quickly associate AHL with harm from Cry5B. In this scenario, C. elegans quickly reduces

329

its feeding rate, which serves to reduce the amount of Cry5B expressing bacteria consumed. This, in

330

turn, reduces the percentage of Cry5B-intoxicated worms. At low concentrations of atc (e.g., 10

331

ng/mL atc), AHL driven feeding rate was low, and thus the rate at which C. elegans was exposed to

332

Cry5B was also low. Subsequently, C. elegans does not learn to associate AHL with harm over 72

333

hours. Due to the reduced feeding rate, intoxication is also reduced under this condition. At

334

intermediate concentrations of atc (60 ng/mL) where the percentage of intoxicated worms was the

335

highest, the feeding rate of the worms was increased due to the increased amount of AHL. The

15 336

amount of AHL was sufficiently small so that aversive learning occurred at a reduced rate. As a

337

result, the worms experienced an extended time where they were exposed to Cry5B through

338

ingestion, which increased the percentage of intoxicated worms in the population. After 48 hours,

339

the worms reduced their feeding rate owing to aversive learning. These trends in feeding rate and in

340

the percentage of intoxicated worms were largely abolished when a learning deficient strain was

341

used, thus offering additional evidence that aversive learning plays an important role in this process.

342

We note that our use of mixed-stage populations of C. elegans might have added variability to our

343

data as previous studies have noted stage-dependent differences in response to crystal toxin proteins

344

[32]. Nevertheless, the trends presented in our manuscript appear to be robust according to our

345

analyses, and thus this additional variability does not appear to confound our results nor their

346

interpretation.

347

It is important to note that it is challenging, if not impossible, to disentangle the effects of

348

cry5B-driven learning, and cry5B-driven intoxication, on feeding rate. As C. elegans progresses towards

349

a state of complete intoxication, it is inevitable that the worm will reduce, if not completely

350

eliminate, feeding. Nevertheless, on the one hand, there are several key findings in our data that

351

support the notion that C. elegans reduces its feeding rate as a result of aversive learning. First,

352

reductions in feeding rate are largely observed in wildtype, but not learning deficient (lrn-1 and mod-

353

1), C. elegans strains. Importantly, all three C. elegans strains have the same basal feeding rate

354

(Supplementary Fig. S4), and susceptibility to Cry5B intoxication (Supplementary Fig. S5b),

355

indicating that physiological differences alone cannot account for these trends. Second, reductions

356

in feeding rate in wildtype C. elegans coincide with the concentration of atc. That is, C. elegans reduces

357

its feeding rate more rapidly when exposed to higher amounts of AHL via increased luxI expression.

358

Third, when cry5B is expressed alone, the reduction in feeding rate is not sustained over 72 hours.

359

Finally, over the course of 72 hours, feeding rate is reduced by a large percent (68% 1 hour vs. 72

16 360

hours, 53% 24 hours vs. 72 hours) when wildtype C. elegans fed upon bacteria expressing both cry5B

361

and luxI (100ng/mL). In comparison, feeding rate is reduced by 36% (24 hours vs. 72 hours) when

362

wildtype C. elegans fed upon bacteria expressing only cry5B. However, on the other hand, there is

363

evidence that a reduction in feeding rate is occurring as a result of a physiological response to cry5B.

364

It is clear that exposure to cry5B alone can reduce feeding rate in C. elegans (Fig. 3d). Our results

365

presented in Fig. 2g suggest that sustained exposure to Cry5B is required to maintain aversion to

366

AHL. Previous work has indicated that learned aversion strategies can be reduced, or all together

367

eliminated, depending upon the pattern of exposure to aversive cues [40]. We also cannot exclude

368

the possibility that the observed effects are temporary or reversible once exposure to bacteria

369

expressing cry5B and luxI is discontinued. The mechanism of action of these toxins involves direct

370

binding with and piercing of gut membranes, which leads to vacuolization and pitting within gut

371

tissues [41]. The resulting pathogenic effects are persistent, and over time induce alterations

372

consistent with starvation, as well as other long term fitness consequences such as decreased brood

373

size and survival (e.g., [42]). Tissue repair mechanisms in nematodes remain poorly understood.

374

They have limited capacity to recover from and repair damage to their cuticle and epidermis [43], but

375

their ability to repair damage to gut membranes due to toxins such as Cry5B remain unknown and

376

require further study. Taken together, while we cannot exclude the notion that harm due to Cry5B is

377

also reducing feeding rate, evidence provided by learning deficient C. elegans indicates that aversive

378

learning is playing a large role.

379

Interplay between olfaction-mediated attraction and avoidance has been observed in

380

additional species. For example, in Drosophila, attraction to, or avoidance of, CO2 is context

381

dependent. In general, flies avoid CO2 as this gas is often emitted by stressed flies [44]. However,

382

several food sources of Drosophila, including ripening fruit, also emit CO2 and thus the flies must

383

discriminate between CO2 emitted due to stress, and CO2 emitted by a food source. To accomplish

17 384

this, additional odorants emitted by food sources attenuate the repulsive nature of CO2 at the neural

385

level [45], thus allowing flies to follow CO2 towards food sources [46]. In rats, neonatal odors are

386

attractive to parents but are avoided by sexually naïve individuals [47]. Bonobos (Pan paniscus) will

387

alter their feeding strategies using multisensory cues to reduce the incidence of infection if their food

388

source is contaminated with the feces of conspecifics, which may harbor pathogens [48]. In humans,

389

kissing or proximity to the mouth can be used to assess mate quality [49] through olfaction.

390

However, kissing also poses a risk for oral pathogen transmission [50]. Humans may risk pathogen

391

transmission from a mouth of someone who is considered a high-quality potential mate. Oral

392

pathogens that can ‘hide’ odorants, which are associated with aversion (or disgust) in humans, may

393

be more likely to be transmitted in the population.

394

Our system inherently examined pathogen transmission during nutrient acquisition via

395

predation, or consumption of a food source. Indeed, tradeoffs between various measures of

396

attraction (ease of catch, size of prey) and infection via consumption have been described. For

397

example, oystercatchers (Haematopus ostralegus) tend to consume intermediately sized cockles

398

(Cerastoderma edule) to avoid parasitism [6]. Larger cockles are considered more attractive as they

399

provide additional nutrients but are also more likely to contain parasites. In a more general sense,

400

some pathogens manipulate the behavior of the host to make it more attractive and easier to

401

capture, which facilitates transmission [51]. It is conceivable that evolutionary constraints, similar to

402

those observed in our study, have shaped these host-pathogen interactions. For example, prey

403

species that exhibit drastically abnormal pathogen-driven behavior relative to their conspecifics may

404

initially appear more attractive to predators, but could end up being more easily identified [52], and

405

avoided, thus reducing parasite transmission. In contrast, prey that is only marginally different from

406

their conspecifics in terms of ability to catch will likely be a proficient vector for pathogen

407

transmission. In our system, the production of a chemoattractant at a high-level likely results in a

18 408

less effective pathogen in the long term. Conversely, while one that produces chemoattractant at an

409

intermediate level will reduce the rate of initial consumption, it will also maximize its long-term

410

fitness by temporarily avoiding learning strategies, thus promoting infection. In congruence with our

411

results, pathogens likely walk a fine evolutionary line when tuning their behavior to become more

412

‘attractive’ to hosts.

413

As C. elegans is navigating its natural habitat, it uses chemoattractants to find bacterial food

414

sources. Bacterial [23, 26] and fungal pathogens [53] produce chemoattractants that attract C. elegans.

415

Previous studies using some of these pathogens have demonstrated that C. elegans will quickly learn

416

to avoid the chemoattractant, and thus the pathogen. This short-term response observed in the

417

laboratory setting could be a mechanism that drives the evolution of a more ingrained avoidance

418

strategy in C. elegans populations. Indeed, long-term evolutionary studies have shown that C. elegans

419

can evolve increased avoidance behavior to bacterial pathogens [54]. Recently, a study has

420

demonstrated that C. elegans can pass feeding based avoidance strategies to its young. C. elegans who

421

feed on the bacterial pathogen P. aeruginosa will quickly learn to associate it with harm. Through

422

epigenetic changes, this aversion to P. aeruginosa can be passed onto offspring and is apparently

423

stable for up to four generations [55]. Taken together, short term behavioral responses, or more

424

long-term evolutionary changes, are likely an important mechanism to increase the survival of C.

425

elegans in its natural environment. Interestingly, there appears to be natural variation in the degree of

426

initial attraction of C. elegans strains isolated from different geographic areas towards the pathogen

427

Serratia marcescens [56]. This variation in initial attraction could be shaped by variations in the

428

pathogens, and the amount of chemoattractants they produce, encountered in each geographic area.

429

Encountering pathogens that produce more chemoattractant may force C. elegans populations to

430

develop avoidance strategies more rapidly to ensure their survival.

431

19 432

Acknowledgements

433

This research is supported by a President’s Faculty Research and Development Grant

434

#335318 through Nova Southeastern University. Some strains were provided by the Caenorhabditis

435

Genetics Center, which is funded by NIH Office of Research Infrastructure Programs (P40

436

OD010440). The plasmid containing cry5B was a kind gift from Dr. Raffi Aroian.

437 438

Competing Interests

439 440

The authors have no competing financial interests.

441 442

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25 574

Figure legends

575 576

Fig. 1: Increasing the expression rate of luxI increases feeding rate.

577

a. Diagram of gene circuit housed in the engineered bacteria used in this study. An IPTG inducible

578

Plac promoter controls expression of cry5B, which encodes the Cry5B crystal toxin protein. The

579

atc inducible Ptet promoter controls the expression of luxI, which encodes the LuxI protein that

580

produces an AHL chemoattractant.

581

b. Feeding rate of C. elegans feeding on luxI-expressing bacteria for 1 hour with increasing

582

concentration of atc in the growth medium. When all data points from experiments containing

583

atc are compared, P < 0.001 (GLM). Average number of worms per replicate = 27. Standard

584

deviation from three biological replicates

585 586

Fig. 2: The expression level of luxI influences the time required for C. elegans to develop

587

aversive learning towards AHL.

588

a. Schematic of the modified learning assay. On one side of the plate, we placed AHL. On the

589

opposite side of the plate, we placed ethanol (carrier solved control). After allowing the worms

590

move for one hour, the number of worms on either side of the plate was counted. Attraction to

591

AHL resulted in a positive choice index (Eq. 1). Aversive learning resulted in a choice index that

592

was not statistically different from zero, or that was negative. Our learning assay produces

593

qualitatively similar results when compared to a traditional learning assay that uses a large agar

594

plate and sodium azide (Supplementary Fig. S3).

595

b. Choice indices of wildtype (wt) C. elegans that fed on engineered bacteria expressing cry5B and

596

luxI (1 mM IPTG and 100 ng/mL atc) for the time indicated. When C. elegans fed on the

597

engineered bacteria for 1 hour, the choice index was significantly greater than zero (P = 0.005,

26 598

one-tailed t-test). After 3 and 5 hours of feeding, the choice index was not significantly different

599

from zero (P > 0.06, one-tailed t-test). Average worms counted per replicate = 16. For panels b-

600

g, standard deviation from a minimum of three biological replicates and all data points with * are

601

not different from zero (P ≥ 0.057, one-tailed t-test). For data points without the star, choice

602

index was significantly greater that zero (P < 0.048, one-tailed t-test).

603

c. Choice indices of C. elegans that fed on bacteria expressing cry5B (1 mM IPTG) and luxI

604

(increasing concentrations of atc). For a given concentration of atc, once learning occurred, we

605

no longer performed a learning assay resulting in different scales along the x-axis. Average

606

number of worms counted = 25. The 0 ng/mL atc condition does not contain IPTG.

607

d. Choice indices of C. elegans that fed on bacteria independently expressing cry5B (1 mM IPTG),

608

luxI (100 ng/mL atc) or neither (0 mM IPTG, 0 ng/mL atc) for 72 hours. A positive choice

609

index (attraction towards AHL) was observed in all cases (P < 0.03, one-tailed t-test). Average

610

number of worms counted = 47.

611

e. Choice indices of a learning deficient strain of C. elegans (lrn-1) that fed on bacteria expressing

612

cry5B (1 mM IPTG) and luxI (increasing concentrations of atc) for 72 hours. A positive choice

613

index (attraction towards AHL) was observed in all cases (P ≤ 0.043, one-tailed t-test). Average

614

number of worms counted = 86. The 0 ng/mL atc condition does not contain IPTG.

615

f. Choice indices of the lrn-1 strain fed on bacteria independently expressing cry5B (1 mM IPTG),

616

luxI (100 ng/mL atc) or neither (0 mM IPTG, 0 ng/mL atc) for 72 hours. A positive choice

617

index (attraction towards AHL) was observed in all cases (P ≤ 0.03, one-tailed t-test). Average

618

number of worms counted = 64.

619

g. Choice indices of wildtype (wt) C. elegans fed on bacteria expressing cry5B and luxI (1 mM IPTG

620

and 100 ng/mL of atc), luxI (100 ng/mL atc) or neither gene (control) for 24 hours. Worm

621

feeding on bacteria expressing cry5B and luxI or luxI were subsequently transferred to bacteria

27 622

expressing only luxI (100 ng/mL of atc) or neither gene (control) and allowed to feed for 24

623

hours. A choice assay was then performed. A positive choice index (attraction towards AHL)

624

was observed in all cases (P ≤ 0.03, one-tailed t-test). Average number of worms counted = 40.

625 626

Fig. 3: A reduction in feeding rate is observed in C. elegans at an intermediate concentration

627

of atc after feeding on cry5B and luxI- expressing bacteria for 48 hours.

628

a. Feeding rate (pharyngeal contractions) of wildtype (wt) C. elegans that fed on bacteria

629

expressing cry5B (1 mM IPTG) and luxI (various concentrations of atc). Wildtype worms fed

630

on medium containing 100 ng/mL had a significantly lower feeding rate after 24, 48 and 72

631

hours as compared to one hour (P ≤ 0.0008, GLM with Tukey’s HSD, *). Worms fed on

632

medium containing 50 ng/mL atc did not show a reduced feeding rate at 24 hours as

633

compared to one hour (P = 1.0, GLM with Tukey’s HSD) atc. After 48 and 72 hours, this

634

feeding rate was reduced as compared to 24 hours (P ≤ 0.0007, GLM with Tukey’s HSD,

635

**). For all panels, standard deviation from individual feeding rates from a minimum of three

636

biological replicates. Average number of worms counted in each condition = 61.

637

b. Feeding rate (pharyngeal contractions) of learning deficient C. elegans (lrn-1) that fed on

638

bacteria expressing cry5B (1 mM IPTG) and luxI (various concentrations of atc). There was

639

no significant difference in feeding rate across concentrations of atc with the exception of a

640

decrease between one and 48 hours on medium containing 100 ng/mL atc (P = 0.008,

641

GLM, *). Average number of worms counted in each condition = 58.

642

c. Feeding rate (pharyngeal contractions) of learning deficient C. elegans (mod-1) that fed on

643

bacteria expressing cry5B (1 mM IPTG) and luxI (various concentrations of atc). There was

644

no significant decrease in feeding rate across concentrations of atc (GLM with Tukey’s

645

HSD). Average number of worms counted in each condition = 80.

28 646

d. Feeding rate (pharyngeal contractions) of wildtype (wt) and learning deficient (lrn-1 and mod-

647

1) C. elegans fed on bacteria expressing cry5B (1 mM IPTG/0 ng/mL atc). There was a

648

reduction in feeding rate for the wildtype strain between 24 and 48 hours (P = 0.014, GLM),

649

but this did not continue for 72 hours. A reduction was not observed in the learning

650

deficient strains (P > 0.202, GLM with Tukey’s HSD). Average number of worms counted

651

in each condition = 67.

652 653

Fig. 4: A biphasic relationship between expression of luxI and the percentage of intoxicated

654

worms.

655

a. The percentage of intoxicated wildtype (wt) and pumping defective (eat-1) C. elegans fed on

656

cry5B-expressing (1 mM IPTG) bacteria. The percentage of intoxicated worms was higher in

657

the wildtype strain as compared to the eat-1 strain after 48 hours (P =0.047, two-tailed t-test).

658

When the medium lacked IPTG, the percentage of intoxicated worms was reduced (P <

659

0.011, two-tailed t-test). Average number worms per assay = 29. In all panels, standard

660

deviation from a minimum of three biological replicates.

661

b. The percentage of intoxicated worms fed on bacteria expressing cry5B (1 mM IPTG) and

662

luxI (various concentrations of atc). The highest percentage of intoxicated worms was

663

observed at 60 ng/mL and after 48 (60 ng/mL vs. 100 ng/mL, P = 0.05, 60 ng/mL vs. 10

664

ng/mL, P =0.004) and 72 hours of feeding (60 ng/mL vs. 100 ng/mL, P = 0.079, 60

665

ng/mL vs. 10 ng/mL, P =0.018, two-tailed t-tests). Average number of worms counted at all

666

time points = 131.

667

c. The percentage of intoxicated worms when learning deficient (lrn-1) C. elegans fed on

668

engineered bacteria that expressed cry5B (1mM IPTG) and luxI (various concentrations of

669

atc) for 48 hours. Average number of worms counted at all time points = 118.

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Fig. 5: We hypothesize that the biphasic relationship between intoxication efficacy and

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attraction (atc concentration) is due to interactions between feeding rate and aversive

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learning. As the amount of attractant increases, feeding rate increases. This increases the rate at

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which the worms are exposed to Cry5B through ingestion, which increases the rate of aversive

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learning. As a result, they reduce their feeding rate, which decreases the rate at which they are

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intoxicated. The interaction between these two trends results in the percentage of intoxicated worms

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being maximized at intermediate amounts of AHL attractant (atc concentration) after 48 and 72

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hours of feeding. We note that we cannot rule out that the intoxication from ingestion of Cry5B is

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also reducing the feeding rate in addition to aversive learning.

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