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Published as doi: 10.1096/fj.06-6551com.
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The role of Caenorhabditis elegans insulin-like signaling in the behavioral avoidance of pathogenic Bacillus thuringiensis

Martin Hasshoff1, Claudia Böhnisch1,2, Daniela Tonn, Barbara Hasert and Hinrich Schulenburg1,3

Institute for Animal Evolution and Ecology, Westfälische Wilhelms-University, Münster, Germany

2Correspondence: Department of Animal Evolutionary Ecology, Zoological Institute, Eberhard-Karls University Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany. E-mail: hinrich.schulenburg{at}uni-tuebingen.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pathogens cause damage, and their elimination requires activation of the costly immune response. A highly economic defense strategy should thus be the behavioral avoidance of pathogens, as manifested in humans by all aspects of hygiene or revulsion at pathogen-rich material. Despite its potential importance, behavioral defenses have as yet received only little attention in biomedical research—in stark contrast to the physiological immune system. In the present study, the genetics of such behavioral defenses are elucidated in a simple model organism, the nematode Caenorhabditis elegans. We show for the first time that mutations in the insulin-like receptor (ILR) pathway lead to two distinct behavioral responses against pathogenic strains of the Gram-positive bacterium Bacillus thuringiensis (BT), including the physical evasion of pathogens and their reduced oral uptake. Since this pathway also contributes to nematode stress resistance, the results surprisingly reveal a genetic link between physiological and behavioral defenses. Considering that many signaling pathways have conserved their functions across evolution, including the ILR pathway, this signaling cascade may represent an interesting candidate regulator for behavioral defenses in more complex organisms, including humans.—Hasshoff M., Böhnisch C., Tonn D., Hasert B., Schulenburg H. The role of Caenorhabditis elegans insulin-like signaling in the behavioral avoidance of pathogenic Bacillus thuringiensis.


Key Words: immune system • host-pathogen evolution


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IN MANY ORGANISMS, DEFENSE AGAINST pathogens or parasites does not exclusively rely on the immune system. An extremely economic alternative may be provided by behavioral responses: They can decrease the risk of infection, while, at the same time, economizing on the animal’s resources, if they help to minimize up-regulation of the costly physiological immune defense (1 , 2) . It is thought that pathogen avoidance behavior has shaped the ecology, the general behavioral patterns, and also socio-ecological traits of animals, possibly to an as-yet-unknown extent. Many animals, including mammals, show diverse behavioral strategies to avoid parasitized con-specifics and potentially microbe-infested compounds. As such, these behaviors seem to play an important role in habitat choice, group formation, foraging behavior, and mate choice (1 , 3 4 5 6) . In humans, innate disgust and revulsion at unpleasant odors, specific compounds, or locations may similarly serve to reduce the risk of contracting disease (7 8 9) .

Despite its potential importance, almost no information is available as to the underlying mechanisms and genetics. We cite only three main exceptions to our knowledge. Two of these refer to work in the nematode Caenorhabditis elegans. In the first case, the evasion of pathogenic Serratia marcescens and its biosurfactant serrawettin W2 was found to require the only toll-like receptor (TLR) homologue of C. elegans, tol-1, and also the AWB chemosensory neurons, the latter involving at least the TAX-2/TAX-4 cGMP-gated channel, the G protein ODR-3, and the G protein receptor kinase GRK-2 (10 , 11) . Consistent with these results, a recent study could associate C. elegans avoidance of pathogenic Microbacterium nematophilum with the TAX-2/TAX-4 cGMP-gated channel (12) . In the second case, C. elegans was able to learn avoidance of pathogenic Pseudomonas aeruginosa and S. marcescens. This aversive olfactory learning behavior was mediated by ADF chemosensory neurons and the neuron-specific release of the neurotransmitter serotonin (13 , 14) . The third case refers to the avoidance of parasitized con-specifics in mice. The genetics of this social aversive behavior was found to depend on the neuropeptide oxytocin and two estrogen receptors. Oxytocin- and estrogen-signaling may interact in the hypothalamus and amygdala within the brain to enhance the recognition and avoidance of potentially contagious con-specifics (6 , 15 16 17) .

Here, we report on the reverse genetic analysis of behavioral defense in C. elegans. This analysis was motivated by our previous results on the natural variation of different defenses against the Gram-positive soil bacterium Bacillus thuringiensis (BT). In the previous and the current study, we have chosen BT as a model pathogen because it is likely to interact with C. elegans in nature (18 , 19) , so that the nematode should have evolved specific defenses against this pathogen, including behavioral and/or physiological responses. Our previous work revealed that survival in the presence of BT and the behavioral avoidance of BT are positively correlated (20) . One possible explanation for this is that both bear a common genetic basis (20) . We previously proposed that such a genetic link could be provided by the insulin-like receptor (ILR) pathway (19) .

The ILR pathway has previously attracted particular interest because of its fundamental role in longevity, whereas its inactivation causes at least a two-fold life span extension (21 , 22) . The pathway consists of two main elements: the insulin-like receptor DAF-2, which is a transmembrane tyrosine kinase, and a forkhead/winged helix-related transcription factor of the dFOXO family, DAF-16, which is negatively regulated by DAF-2. Signal transfer from the activated DAF-2 proceeds via AGE-1 (a phosphatidylinositol-3-OH kinase), PIP3 (phosphatidyl-inositol trisphosphate), PDK-1 (a 3-phosphoinositide-dependent kinase 1), AKT-1, and AKT-2 (protein-Ser/Thr kinases), ultimately leading to the phosphorylation and thus cytoplasmic retention of DAF-16 (21 , 22) . If DAF-2 is inactive or down-regulated, then DAF-16 translocates into the nucleus, where it is involved in the regulation of antimicrobial, detoxifying, and other stress-response genes, subsequently conferring increased longevity and stress resistance (23 24 25 26) . These findings suggest that the ILR pathway contributes to nematode immunity, at least in the context of a general stress response (27) . Consistent with this idea, ILR mutants show increased survival in the presence of pathogens (27 28 29) . At the same time, the ILR pathway is known to respond to environmental stimuli (30) , and, thus, it possesses important requirements for the regulation of behavioral responses.

In the current study, we now tested the ability of C. elegans strains with different mutations along the ILR pathway to resist and avoid the infectious stages of BT. Note that throughout the whole paper, the term "resistance"/"to resist" is used in its broad sense, i.e., increased survival in the presence of pathogens. Nematode resistance was thus examined with the help of two survival assays, one on agar and one in a liquid culture medium. We further studied two different behavioral responses against pathogens: the reduction of ingestion rates in pathogen-rich areas, and the physical evasion of such areas. Our results demonstrate that mutations of ILR signaling affect both pathogen resistance and avoidance.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Nematode and bacterial strains
In the four main experiments, we tested six different C. elegans strains, including the wild-type strain N2 and five mutant strains with i) a partial loss-of-function mutation in the daf-2 gene (allele e1370; strain CB1370); ii) a strong mutation in daf-2 (allele e1368; strain DR1572); iii) a weak mutation in age-1 (allele hx546; strain TJ1052); iv) a complete loss-of-function mutation in daf-16 (allele mgDf50; strain GR1307); and v) the partial loss-of-function daf-2(e1370) mutation combined with a complete loss-of-function mutation in daf-16(mgDf47) (strain GR1309). In the additionally performed experiments (see below, the section on specific assessment of the role of daf-16), we compared four strains, including two of the above strains [N2 and the daf-2(e1370) mutant], and also a second daf-16 loss-of-function mutant (allele m26; strain DR26) as well as the corresponding daf-2(e1370);daf-16(m26) double mutant (strain DR1309). The regulatory relationship of the considered genes is depicted in Fig. 1 . All C. elegans strains were obtained from the Caenorhabditis Genetics Center (Minneapolis, MN, USA). Nematode maintenance on nematode growth medium (NGM) with a lawn of the food bacterium Escherichia coli OP50 followed standard protocols (31) .


Figure 1
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  		Figure 1. Regulatory relationship between the considered components of the insulin-like receptor (ILR) pathway. Phosphorylation of the DAF-2 ILR receptor on ligand binding activates the phosphatidylinositol-3-OH kinase AGE-1, ultimately leading to phosphorylation and thus cytoplasmic retention of the forkhead/winged helix-related dFOXO transcription factor DAF-16. In contrast, inhibition of daf-2 gene expression or activity ultimately results in the nuclear translocation of DAF-16, where this transcription factor regulates the expression of diverse genes involved e.g., in development, metabolism, longevity, dauer formation, and stress resistance.

The nematicidal B. thuringiensis strain NRRL B-18247 was provided by the Agricultural Research Service Patent Culture Collection (United States Department of Agriculture, Peoria, IL, USA) and the nonpathogenic BT strain DSM-350 was obtained from the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). For the experiments, we used a spore-toxin crystal mixture from these strains, which was produced as described previously (20) . Note that spores associated with crystal toxins represent the infectious stage in the pathogenic strains, whereas the oral uptake of the spores is required for a persistent infection to occur (20 , 32) . With the exception of the survival assay in liquid medium, Escherichia coli OP50 was provided as food ad libitum in all experiments, in order to ascertain that the effects observed are due to differences in the pathogenicity but not the nutritious value of the BT strains. We also consistently used hermaphroditic fourth instar larvae (L4) of C. elegans for the different tests.

Survival assay on Agar in wormballs
Survival was examined on peptone-free NGM Agar in small wormballs, following our recently developed protocol (33) . These wormballs are transparent plastic balls with a diameter of 5 cm, as available from standard handicraft shops (e.g., Basteltreff Müller, Reutlingen, Germany). They consist of two halves that can be opened and closed firmly. Before closure, the inside of the two halves is completely covered with the Agar medium and inoculated with the bacteria of interest. The wormballs have the advantage that they force the worms into continuous contact with the tested bacteria, whereas the nematodes may escape in standard Petri dishes by crawling to the plastic side of the dishes, especially if bacteria are pathogenic (33) . Since peptone-free NGM Agar inhibits germination of BT, the usage of this medium ensures exposure of the worms to the infectious BT stages. At the same time, it does not affect proliferation of the nematodes (33) .

One day before usage, the plastic halves were sterilized by UV irradiation (10 min 150 mJ) and filled with a thin layer of the Agar medium (NGM Agar without peptone). Thereafter, both halves were inoculated with E. coli (for each wormball, a total vol of 700 µl of 1.5x1010 cells/ml) and either the pathogenic or the nonpathogenic BT strain (final concentration of 1.5x108 spores/ml). At the start of the experiment, a group of 40 hermaphroditic L4 per nematode strain was transferred into the wormballs. The survival of worms was recorded daily. Every other day, surviving worms were transferred to new wormballs prepared as above. The different C. elegans strains were examined in parallel in different wormballs and in randomized order at 20°C in the dark. Two replicates of the experiment were performed. Each replicate run was terminated after 45 d. The animals that were still alive on this day [only the daf-2(e1370) mutant under control conditions] were included as censored cases in the Kaplan-Meier survival analysis. For each strain and treatment, the results from the two replicates did not differ significantly and were thus combined for subsequent analysis.

Survival assay and analysis of infection load in liquid medium
The survival of individual animals was assayed in liquid medium in 96-well microtiter plates, as described previously (20) . In detail, the wells of a microtiter plate were each filled with 50 µl PBS buffer and a single animal was transferred into each well with a worm picker, followed by addition of 50 µl BT solution, which consisted of PBS with either the pathogenic or the nonpathogenic BT strain (final concentration of 1x108 spores/ml). After 24 h at 20°C in the dark, individual survival was examined. Animals were considered dead if they did not respond to light touch with a worm picker. A total of 96 individuals of each C. elegans strain was analyzed per treatment. All treatment combinations were assayed in parallel and in randomized order.

For the analysis of infection load, animals from the pathogenic treatment were transferred onto microscopy slides after the 24 h BT exposure. The samples were immediately frozen to stop pathogens from replicating within the worms. After thawing, the worms were analyzed via differential interference contrast (DIC) microscopy to determine infection load. The extent of infection was characterized using five categories (20) : 1) ≤ 30 bacterial particles, exclusively spores, all in the front part of the digestive tract, anterior-most intestinal cells all intact; 2) 30–100 particles, exclusively spores, mainly but not exclusively in the front part of the digestive tract, anterior-most intestinal cells intact or with weak damage; 3) >100 particles, exclusively spores, throughout the whole digestive tract, usually highly concentrated in the anterior segments, anterior-most intestinal cells severely damaged or destroyed; 4) as in category 3, but with vegetative cells in the first third of the body; 5) as in category 3, but with vegetative cells throughout more than the first third of the body, animals often packed with vegetative cells and spores.

For each nematode strain, alive and dead worms were analyzed separately, including at least 33 dead (maximum 70) and at least 10 alive (maximum 52) individuals per strain. In each case, an infection index was calculated by adding the recorded infection categories, followed by division with the respective total number of animals included. Hence, an infection index of 1 would indicate a generally low and 5 a generally high infection level.

Ingestion assay
The ingestion rate was assessed on minimum agar (3.4% w/v) test plates (diameter of 3 cm), inoculated with 40 µl of a mixture of E. coli (1.5x1010 cells/ml) and BT (either the nematicidal or the nonpathogenic BT; concentration in both cases, 3.67x107 spores/ml) ~15 h before the start of the experiment. At the beginning of the test, 10 L4 were transferred onto each plate with a worm picker. After 1 h and 6 h, the ingestion rate was determined for five individuals, which were found inside the bacterial lawn, by counting the pharynx grinder movements within a 30 s period. Strain and treatment combinations were randomized and examined in parallel at 20°C. We pooled the data from 10 independent experiments, including a total of 50 worms per factor combination.

Evasion assay
Microtiter plates (24-well) were prepared with minimal agar (3.4% w/v) and a lawn of E. coli OP50 (10 µl of 1.5x1010 cells/ml), which was left to dry overnight at 20°C. Thereafter, a small "spot" of the test bacterium (0.5 µl of either the nematicidal or the nonpathogenic BT; concentration in both cases: 3.67x107 spores/ml, diluted with PBS buffer; diameter of spot ca. 3 mm) was placed in the middle of the E. coli lawn of each well and left to dry for ~2 h. An individual nematode was placed in the middle of each BT spot with a worm picker. We then recorded the time until the animals’ entire body had left the small spot. If they did not leave the spot within 10 min, the observation was stopped and 10 min was noted for this individual. The different strain and treatment combinations were randomized and assayed in parallel at 20°C. Data were pooled from 8 independent experiments, yielding a total of 64 individual data points per strain and treatment.

Specific assessment of the role of daf-16
We specifically tested the importance of daf-16 in pathogen defense. For this purpose, the survival assay on agar in wormballs and the evasion assay were repeated under pathogen conditions. We examined a different daf-16 loss-of-function mutation (allele m26) either alone (strain DR26) or in combination with the daf-2(e1370) partial-loss-of-function mutation (strain DR1309). Both strains were compared with the wild-type strain N2 and the daf-2(e1370) mutant (strain CB1370). The two assays were performed as described above.

Statistical analysis
Statistical analysis was used for the pair-wise comparison of strains and treatments, in order to determine i) whether exposure to either pathogenic or nonpathogenic BT led to a different result in any of the C. elegans strains; ii) whether any of the mutants differed from the wild-type N2 within each BT treatment; and iii) whether any of the C. elegans strains differed from the daf-2(e1370) mutant within each BT treatment. For the survival assay on Agar, the kinetics of survival were plotted using the Kaplan-Meier method and the difference between strains and treatments was assessed using the log rank test (log), as implemented in the program JMP IN 5.1.2 (SAS Institute Inc., Cary, NC, USA). For the survival assay and infection load in liquid medium, statistical comparison was based on a likelihood ratio test (LRT). The ingestion rate was analyzed with an independent sample t test. The standard t test was used whenever the condition of homogeneity of error variances was met. Otherwise, the alternative t test was used, which does not assume variance homogeneity. Since the data of the evasion assay were not continuous (all measurements were terminated after 10 min), pair-wise comparisons were made with the rank-based Mann-Whitney U test (MWU). For the main experiments, we also used a meta-analysis-like approach following Fisher (34 , 35) , in order to test for an overall effect of ILR signaling on either survival or early behavioral responses. For this approach, the P-values inferred from different assays are combined using –2{Sigma}ln(P). The significance of this value is established by comparing it to a {chi}2 distribution with d.f. = 4. The LRT, t-test, MWU, and the meta-analysis-like test were performed with the program Statistical Packages for the Social Sciences (SPSS Version 12, SPSS Inc., Chicago, IL, USA). To account for multiple testing (i.e., multiple pair wise comparisons), the critical significance level was lowered to P = 0.0021 for the main experiments and to P = 0.0083 for the two additional experiments, following the Bonferroni method (35) . Differences with a probability of P < 0.05 or P < 0.1 are still indicated as a trend.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Survival on Agar in wormballs
The survival rate of the animals was determined with a newly developed assay, in which worms were exposed to BT on Agar in wormballs. These wormballs ensure continuous contact of the worms to BT and also high nematode recovery rates. Both factors may be affected in the commonly used survival assays in Petri dishes, where worms may try to escape pathogens by crawling to the plastic edge of the dish, where they often die due to desiccation.

On the nonpathogenic control, the daf-2(e1370) mutant produced the highest mean longevity, followed by the daf-2(e1368) and the age-1 mutants (Table 1 , Fig. 2 A). In fact, the daf-2(e1370) mutant survived significantly longer than all other strains under these conditions (for all pair-wise comparisons: log, {chi}2df=1≥19.92, P<0.001). In comparison with the wild-type N2, the daf-2(e1368) mutant also survived significantly longer (log, {chi}2df=1=11.48, P=0.001), the age-1 mutant showed a trend to survive longer (log, {chi}2df=1=5.19, P=0.023), and the daf-16 mutant survived significantly shorter (log, {chi}2df=1=31.33, P<0.001; Table 1 ).


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  		Table 1. Summary of results of the main experiments1


Figure 2
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  		Figure 2. Survival on Agar plates in the presence of either (A) nonpathogenic BT or (B) pathogenic BT. The survival rate (y-axis) is shown for each C. elegans strain (different colors as indicated) over a period of up to 45 d (x-axis). The survival curves were inferred with the Kaplan-Meier method implemented in JMP IN 5.1.2 (SAS Institute Inc.).

All C. elegans strains produced a significantly reduced survival rate on pathogenic BT when compared with the nonpathogenic control (all pair-wise comparisons: log, {chi}2df=1≥39.89, P<0.001; Table 1 , Fig. 2 ). On pathogenic BT, the daf-2(e1370) mutant survived significantly longer than all other strains (all pair-wise comparisons: log, {chi}2df=1≥34.90, all P<0.001; Table 1 ; Fig. 2B ). In comparison with N2, the daf-16 and the age-1 mutants showed a trend for reduced survival (log, {chi}2df=1=5.21, P=0.023, and {chi}2df=1=3.52, P=0.061, respectively).

Survival and infection load in liquid medium
As an alternative test, the proportion of surviving C. elegans was determined in liquid medium after 24 h of exposure. This assay similarly forces the worms into constant contact with the pathogens, i.e., physical evasion is not possible. All animals of the control treatment survived (Table 1) . However, the survival rate of each C. elegans strain was significantly reduced in the presence of pathogenic BT compared with the control (LRT, {chi}2df=1≥58.29, all P<0.001; Fig. 3 A, Table 1 ). Under pathogen conditions, the daf-2(e1370) mutant showed a significantly higher survival rate than the other strains (all pair-wise comparisons: LRT, {chi}2df=1≥29.90, P<0.001; Table 1 ). In comparison with N2, the age-1 mutant showed a trend of a higher survival rate (LRT, {chi}2df=1=2.73, P=0.098; Table 1 ).


Figure 3
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  		Figure 3. Survival assay in liquid medium, showing (A) the survival rate and (B) the infection load in the presence of the pathogenic BT strain. The different C. elegans strains are indicated along the x-axis. The infection index is given for surviving and dead animals separately (green and black color, respectively). The survival rate and the infection index are shown with the SEM.

Following the 24 h survival assay in liquid medium, the infection level was studied in surviving and dead animals using DIC microscopy and five categories of infection (1=low and 5=high). After exposure to pathogenic BT, the infection levels recorded for each C. elegans strain showed at least a strong trend to be higher in the dead animals than in the survivors (all pair-wise comparisons: LRT, {chi}2df=3–4≥11.77, P≤0.003; Fig. 3B , Table 1 ). Among the survivors, the daf-2(e1370) mutant exhibited a significantly lower infection level than all other strains (all pairwise comparisons: LRT, {chi}2df=4≥16.53, P≤0.002; Table 1 ). In comparison with N2, daf-2(e1370) was significantly different (see above) and daf-2(e1368) produced a trend of a lower infection rate (LRT, {chi}2df=4=8.29, P=0.078; Table 1 ).

Among the dead animals, the daf-2(e1370) mutant showed at least a trend of a smaller infection rate than the daf-16 and the daf-2;daf-16 mutants (LRT, {chi}2df=2≥6.91, P≤0.032). In comparison with N2, the daf-16 and the daf-2;daf-16 mutants produced at least a trend of a higher (LRT, {chi}2df=1≥4.31, P≤0.038), and the age-1 mutant of a smaller infection rate (LRT, {chi}2df=1=5.48, P=0.065).

Ingestion behavior
Infection with BT must be preceded by the oral uptake of the respective infectious stages. Thus, a possible behavioral defense response may consist of reduced ingestion rates. Ingestion was quantified by counting the movements of the pharynx grinder within a 30 s period. After 1 h on nonpathogenic BT, the ingestion rate did not differ among C. elegans strains (all pair-wise comparisons: t-test, tdf=98≤|±1.54|, P≥0.128; Table 1 ; Fig. 4 A). The only exception refers to the daf-2(e1368) mutant, which showed a trend for a higher ingestion rate than N2 (t test, tdf=98=–2.00, P=0.048; Table 1 ; Fig. 4A ).


Figure 4
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  		Figure 4. Ingestion rate after (A) 1 h, and (B) 6 h exposure to either pathogenic or nonpathogenic BT (red and black color, respectively). The different C. elegans strains are indicated along the x-axis. The ingestion rate is given as the number of pharynx grinder movements within a 30 s period. The whiskers refer to the SEM.

In contrast, almost all strains had a significantly reduced ingestion rate after 1 h on pathogenic BT when compared with the results on the nonpathogenic control (all pair-wise comparisons: t test, tdf=80.2–98≥3.36, P≤0.001). The only exception was N2, where the difference was insignificant (t-test, tdf=98=1.18, P=0.240; Table 1 ; Fig. 4A ). Under pathogenic conditions, the daf-2(e1370) mutant ingested significantly less than N2 (t-test, tdf=77.7=3.15, P=0.002). It also showed a trend of a lower ingestion rate than the other four strains (all pairwise comparisons: t test, –2.59≤tdf=83.7–88.4≤–1.70, 0.010<P<0.093). No differences were found between N2 and the other four mutants (all pair-wise comparisons: t-test, tdf=93.6–98≤|±1.53|, P≥0.130; Table 1 ; Fig. 4A ).

After 6 h, all strains showed higher ingestion rates than after 1 h, irrespective of the treatment (Table 1 , Fig. 4 ). At this time point, exposure to pathogenic vs. nonpathogenic BT did not yield significant differences in ingestion rates (all pairwise comparisons: t-test, tdf=89.3–98≤|±1.50|, P≥0.137; Table 1 ; Fig. 4B ). The only exception was the daf-2(e1368) mutant, which showed a trend of reduced bacterial uptake on the pathogen (t test, tdf=96.8=1.85, P=0.067; Table 1 ; Fig. 4B ). Under both treatment conditions, daf-2(e1370) generated at least a trend of a smaller ingestion rate than the other strains (all pair-wise comparisons: t test, tdf=84.4–98≥|±2.27|, P≤0.026). In comparison to N2, daf-2(e1370) differed significantly under both treatment conditions (see above) and the daf-2(e1368) mutant showed a trend of a smaller ingestion rate on the pathogen (t test, tdf=96.1=2.13, P<0.036; Table 1 ; Fig. 4B ).

Evasion behavior
The physical evasion of pathogen-rich bacterial lawns should represent a powerful alternative behavioral defense response. Such pathogen evasion behavior was examined by recording the time required by an individual nematode to leave a small "spot" with either pathogenic or nonpathogenic BT. On the nonpathogenic control treatment, the daf-2(e1370) mutant showed at least a trend of a faster evasion response than the other strains (all pair-wise comparisons: MWU, Un=128≤1633.0, P≤0.048; Table 1 , Fig. 5 ). The other strains did not differ from N2 (all pairwise comparisons: MWU, Un=128≥1859.0, P≥0.365), with the exception of the daf-16 mutant, which showed a trend of a slower evasion rate (MWU, Un=128=1478.0, P=0.005; Table 1 , Fig. 5 ).


Figure 5
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  		Figure 5. Evasion time of the different C. elegans strains (x-axis) from either pathogenic or nonpathogenic BT (red and gray color, respectively). The results are shown as box-plots, where the horizontal black line gives the median, the boxes the interquartile range (25% of the data above and below the median), and the circles and stars the outliers.

In general, exposure to the pathogenic BT led to faster evasion than exposure to the nonpathogenic BT (Fig. 5) . However, this difference was only significant for daf-2(e1368) (MWU, Un=128≤1380.5, P=0.001), and it still showed a trend for daf-2(e1370) (MWU, Un=128=1571.5, P=0.023). None of the other strains produced significant differences (all pair-wise comparisons: MWU, Un=128≥1746.5, P≥0.148).

Under pathogenic conditions, the daf-2(e1370) mutant showed at least a trend of a faster evasion response than N2, the daf-16, and the daf-2;daf-16 double mutants (MWU, Un=128≤1420.0, P≤0.003), but neither the daf-2(e1368) nor the age-1 mutants (MWU, Un=128≥1706.5, P≥0.102; Table 1 , Fig. 5 ). In comparison with the wild-type strain N2, a trend of a faster evasion rate was inferred for daf-2(e1370) (see above) and also daf-2(e1368) (MWU, Un=128=1566.5, P=0.022). In contrast, the daf-16, and the daf-2;daf-16 double mutants showed at least a trend of a slower response (MWU, Un=128≤1612.5, P≤0.035; Table 1 ; Fig. 5 ).

Meta-analysis-like approach
We pursued a meta-analysis-like approach in order to evaluate whether there is a general effect of ILR signaling on survival and/or early behavioral responses. For this purpose, the results of either the two survival assays or the two early behavioral assays (ingestion rate after 1 h and evasion rate) were jointly analyzed following the procedure of Fisher (34) . The comparison between pathogen and nonpathogen treatments revealed a significant overall effect on survival in all strains (Table 2 ). The daf-2(e1370) mutant produced significantly higher BT resistance than all other strains (Table 2) . In comparison with N2, daf-2(e1370) showed significantly higher (see above), the age-1 mutant a trend for higher, and the daf-16 mutant a trend for reduced overall resistance (Table 2) .


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  		Table 2. Results of the meta-analysis-like approach for the main experiments1

For the early behavioral responses, an overall difference between pathogen and nonpathogen exposure was only significant (P<0.0021) for three strains: the daf-2(e1370), the daf-2(e1368), and the age-1 mutants (Table 2) . The daf-2(e1370) mutant responded significantly faster to pathogens than N2, the daf-16, and the daf-2;daf-16 mutants. However, this strain produced only a trend of a faster response if compared to the second daf-2 and the age-1 mutants (Table 2) , in contrast to the findings for survival. In comparison with N2, daf-2(e1370) responded significantly faster and daf-2(e1368) showed a trend of a faster response (Table 2) . Moreover, the daf-16 and the daf-2;daf-16 mutants produced at least a trend of an overall difference to N2 (Table 1) . However, in these two cases, the inferred overall difference is a consequence of a faster response in ingestion behavior but a slower evasion rate (Tables 1 and 2) . Therefore, in comparison with N2, these two mutants do not show a consistent effect on the direction of the early behavioral response (fast vs. slow).

Specific assessment of the role of daf-16
In consideration of the structure of the ILR pathway (Fig. 1) , the daf-16 mutant and the daf-2;daf-16 double mutant should not differ in phenotype. Thus, it was a surprise that the double mutant generally appeared to perform better in pathogen defense than the daf-16 mutant. Although the difference was visible in most cases (Figs. 2 3 4 5) , it was significant only for the survival assay on Agar (log, {chi}2df=1=10.98, P=0.001) but insignificant for all other assays (P>0.1). We specifically tested the importance of daf-16 in pathogen defense by examination of a different loss-of-function allele of daf-16 (allele m26). We focused on the survival assay on Agar and the evasion assay, where the differences between the single and double mutant had been most pronounced (Figs. 2 and 5) .

In the survival assay on Agar, daf-2(e1370) survived significantly better than all other strains (all pair-wise comparisons: log, {chi}2df=1≥15.69, P<0.001; Table 3 ). In comparison to N2, daf-16(m26) showed a trend of a reduction in survival (log, {chi}2df=1=5.56, P=0.018). Importantly, the daf-2(e1370);daf-16(m26) double mutant survived significantly longer than daf-16(m26) (log, {chi}2df=1=15.35, P<0.001), thus confirming the results from the main experiments. In the evasion assay, daf-2(e1370) escaped the pathogen significantly faster than all other strains (all pair-wise comparisons: MWU, Un=144≤1747, P≤0.001; Table 3 ). In comparison to N2, the daf-16(m26) and the daf-2(e1370);daf-16(m26) mutants showed significantly reduced evasion rates (MWU, Un=144≤1882, P≤0.004). In this case, the daf-2(e1370);daf-16(m26) produced a trend of a faster evasion response than daf-16(m26) (MWU, Un=144≤2044.5, P≤0.019).


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  		Table 3. Results of the specific assessment of daf-16 in pathogen defence1


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Pathogen avoidance behavior is an extremely economic defense strategy, but, as yet, little attention has been paid to the underlying molecular genetics. Our study represents one of the first detailed genetic analyses of such behavioral defenses. The few previous examples refer to work in mice, and especially C. elegans, as pointed out in the introduction. In addition to these examples, our study now strongly suggests that the ILR pathway contributes to both resistance and avoidance behavior against a potentially natural pathogen. In particular, ILR down-regulation and concomitant activation of the DAF-16 transcription factor in the daf-2(e1370) mutant consistently enhance the resistance and the avoidance of pathogens. In contrast, inactivation of DAF-16 in either the daf-16 or the corresponding daf-2;daf-16 double mutant leads to a reversal of these effects.

Importance of ILR-pathway mutations in pathogen resistance
The results from both of our survival assays clearly demonstrate that mutations in the ILR pathway affect resistance against BT (see also results of the meta-analysis-like approach). As such, they are generally consistent with the previous finding by Garsin et al. (28) that ILR-mediated resistance is most efficient against Gram-positive bacteria. In addition to these main findings, the results provide important information as to our understanding of ILR signaling. In particular, survival on Agar under control conditions was highest for the daf-2(e1370) mutant, followed by the daf-2(e1368) and the age-1 mutants. This was expected in consideration of the structure of the ILR pathway (DAF-2 and AGE-1 both inhibit DAF-16). It was also not surprising that the two daf-2 alleles varied in their effect. They both differ to the wild-type allele by single nonsilent point mutations, and they define different daf-2 mutation classes, which vary in several phenotypic traits including embryonic and larval development, late progeny production, and brood size (36) . However, both the age-1 and the daf-2(e1368) mutations did not affect resistance against BT in the two survival assays. Similarly, previous studies reported that these two mutations had a much smaller effect on pathogen resistance than daf-2(e1370) or that the daf-2(e1368) mutation affected survival much stronger than the age-1 mutation (27 , 28) . These observations support the previous notion that the different mutations manipulate ILR signaling in distinct ways (28 , 36) . The underlying mechanisms for these effects still need to be determined.

Furthermore, the daf-2;daf-16 double mutant unexpectedly produced a higher survival rate than the corresponding daf-16 mutant in both survival assays. This difference was clearly significant for the survival assay on Agar, irrespective of the two daf-16 loss-of-function mutations examined. Similar observations can be made in the results of Garsin et al. (28) and Troemel et al. (27) . They strongly suggest that the DAF-2 ILR exerts its effects not exclusively through the transcription factor DAF-16 but also through at least a second currently unknown target. The presence of such an additional DAF-2 target is supported by the recent finding that DAF-2 mediates associative learning in C. elegans independent of DAF-16 (37) .

Importance of ILR-pathway mutations in pathogen avoidance behavior
The enhanced behavioral response toward pathogens of the daf-2(e1370) mutant relative to the other strains may have three alternative explanations: i) it represents an active defense response against pathogens mediated by the ILR pathway; ii) it results from a general difference between the daf-2(e1370) mutant and the other strains; and iii) it is caused by toxin-induced damage or paralysis that exclusively affects the daf-2(e1370) mutant in the ingestion assay or the daf-16 mutant in the evasion assay.

Of these, the latter point can be excluded. The daf-2(e1370) mutant is by far more resistant than the other strains tested, such that it should be less affected by the toxin. Furthermore, after the initial decrease in ingestion at the 1 h time point, all C. elegans strains produced higher feeding rates after 6 h exposure. This observation was made even for those strains with little resistance, which should be particularly prone to toxin-induced damage. Similarly, the evasion ability of the daf-16 mutant was unlikely affected by toxin-induced damage or paralysis, because the apparent lack of activity was observed under both pathogen and control conditions. Thus, it may represent a general consequence of the loss-of-function in DAF-16.

Furthermore, a general difference between the daf-2(e1370) mutant and the other strains (alternative explanation ii) is unlikely to be the main determinant of the recorded differences. In this context, note that a previous study found a significantly smaller ingestion rate of the daf-2(e1370) mutant on nonpathogenic E. coli if compared with the wild-type N2 (36) , suggesting that it may generally differ in feeding behavior. However, in our study, the feeding reduction and also the increased evasion response were clearly enhanced on the pathogen if compared to the nonpathogenic control. Moreover, for the ingestion assay, this mutant did not differ from the other strains after 1 h control conditions.

Consequently, the most parsimonious interpretation of our results is that the daf-2(e1370) mutant actively minimizes pathogen uptake and increases pathogen evasion. This pathogen-induced behavioral defense appears to be an early and possibly short-lived response. The evasion assay specifically examined a fast and early response. In the ingestion assay, the observed reduction on the pathogen is only significant after 1 h but not 6 h. Moreover, in this case, the significant difference between daf-2(e1370) and the other strains is only exclusive to the pathogen treatment after 1 h but not 6 h. A general effect of ILR-pathway mutations on an early behavioral response against pathogens is also strongly supported by the meta-analysis-like approach.

Such early responses may be crucial for controlling infection and survival until reproductive age. In fact, they may be responsible to a large extent for the observed increased survival and the decreased infection load of the daf-2(e1370) mutant in the two survival assays. These survival assays do not permit to distinguish between physiological and behavioral causes of increased survival, although at least physical evasion is unlikely important. At the same time, it seems improbable that behavioral defense is the only reason for increased survival, because the daf-2(e1370) mutation is known to associate with an up-regulation of putative immunity genes (e.g., lysozyme, antimicrobial nlp genes, saponins, c-type lectins; 23, 25–27).

In analogy with the survival assays, the second daf-2 mutation (the e1368 allele) and the tested age-1 defect differed in their impact on behavioral defense. This may again highlight that the different alleles and genes of the pathway vary in their effect on ILR signaling. Similarly, in the evasion assay, the daf-2;daf-16 double mutant showed a more pronounced behavioral response than the corresponding daf-16 mutant, irrespective of the daf-16 allele tested. Again, these findings point to a second output for ILR signaling.

To date, it is unknown how ILR signaling manipulates behavior. The previously inferred targets of the DAF-16 transcription factor include possible components of the nematode nervous system, e.g., two guanylyl cyclases (gcy-6 and gcy-18; 25). These genes are promising candidates for the regulation of behavioral responses. At the same time, it is also currently unknown how the ILR itself is regulated in response to pathogen exposure. Our results demonstrate that pathogenic and nonpathogenic strains of the same species are distinguished through this pathway. Pathogen recognition may be direct through perception of pathogenic BT factors or indirect through the incurred damage (danger model; 19 , 38 ). In this context, it should be particularly important to examine if and how ILR signaling relates to the previously identified mechanisms of pathogen avoidance, i.e., serotonin release through ADF chemosensory neurons as well as the activity of tol-1 or AWB chemosensory neurons (10 , 11 , 13) . Intriguingly, two recent studies demonstrated a two-fold link between serotonin and ILR signaling. In the one case, serotonin influenced DAF-16 activity in the nematode’s response to stress, whereby serotonin expression in NSM neurons enhanced and serotonin expression in ADF neurons suppressed DAF-16 nuclear translocation (29) . In the other case, DAF-16 itself was found to contribute to the regulation of serotonin synthesis (39) . The importance of these links for ILR-mediated pathogen avoidance clearly warrants further investigation.

Main implications of the results
Our finding of the involvement of ILR signaling in pathogen avoidance is important for four main reasons. Firstly, since this pathway contributes to physiological resistance against diverse stressors (e.g., heavy metals, hypoxia), our data strongly support the previous notion that ILR signaling links different defense responses (24 , 25 , 40 , 41) . However, it has as yet been unknown that the ILR-mediated defense repertoire in C. elegans also includes avoidance behaviors. In fact, our data strongly suggest that the behavioral avoidance of the infectious stages of a potential natural pathogen represents a new function of nematode ILR signaling. The combination of different defenses is evolutionarily advantageous as part of a general stress response that helps the nematode to survive in an unfavorable environment. Interestingly, none of the previously identified pathogen avoidance mechanisms is as yet known to contribute to alternative defense responses and/or physiological immunity. At least for the C. elegans TLR gene tol-1, an involvement in immunity appears highly unlikely, since it does not confer pathogen resistance (10 , 42) , in contrast to TLRs in many other organisms (e.g., 43 , 44 , 45 ). Moreover, increased P. aeruginosa resistance could be associated with reduced serotonin signaling, most likely in NSM neurons (29) . However, this effect may be independent of the serotonin-dependent aversive learning behavior against the same pathogen, which relies on an increase of serotonin release in ADF neurons (13) .

Secondly, our results highlight the potential role of behavior in defense. Similar behavioral defenses were previously reported in C. elegans against BT (physical evasion and reduced ingestion; 20 ), S. marcescens (physical evasion; 10 , 11 , 13 ), P. aeruginosa (physical evasion; 13 , 14 ), Photorhabdus luminescens (physical evasion and reduced ingestion; 33 ), and M. nematophilum (physical evasion; 12 ). Reduced ingestion was additionally reported after exposure to P. aeruginosa, Salmonella enterica, and Burkholderia species, although in these cases it is likely to be due to pathogen-induced paralysis (46 47 48) . Since C. elegans inhabits the soil environment (49 50 51 52) and is likely to encounter potentially harmful pathogens alongside its food, the considered behavioral responses are expected to increase nematode fitness under natural conditions. They reduce either the general exposure to pathogens or their oral uptake. As such, they are likely to prevent damage and economize on resources, otherwise required for up-regulation of the immune response.

Thirdly, these behavioral defenses should play a pivotal role in several previously characterized phenotypes of the ILR pathway, e.g., the increased survival in the presence of pathogens or other stressors like heavy metals (28 , 53) . In these cases, increased survival is usually explained by physiological factors, i.e., the immune system or detoxification processes. However, the importance of behavior for these phenotypes is as yet completely unexplored. Importantly, the commonly used survival assays on Agar in Petri dishes do not permit comparative evaluation of behavioral vs. physiological effects. For instance, in these cases, high resistance of daf-2(e1370) could be caused by either enhanced physiological immunity, physical resistance, reduced bacterial ingestion, increased evasion rates, or a combination of them.

Finally, our findings may serve as a primer for the analysis of behavioral defenses in other organisms. Recent data in mammals and flies demonstrated that ILR signaling also contributes to both sugar metabolism and longevity, whereas longevity is most likely regulated in response to environmental signals and by activation of stress resistance genes (54 55 56 57 58 59) . Intriguingly, a recent study also provided evidence of a role of the Drosophila ILR pathway for the behavioral aversion of noxious food (60) . Multiple highly similar functions of this pathway in distantly related taxa strongly suggest that they share a single origin. Thus, it is tempting to speculate that the new functions of ILR signaling in C. elegans described here have also been conserved through evolution in other organisms. Behavioral defenses against pathogens are generally suggested to be of immense importance in animals (1) . In humans, they are likely to have shaped the evolution of hygiene and disgust (7 , 8) , possibly in concert with the immune response (9) . The determination of the currently unknown genetics of human defensive behaviors should provide important insights into our species’ defense portfolio and may be of applied value in medical disease prevention and risk assessment programs.


   ACKNOWLEDGMENTS
 
We thank Jonathan Ewbank, Danielle Garsin, Michael Habig, Andreas Lengeling, Nico Michiels, Nathalie Pujol, Thomas Roeder, Mathieu Sicard, Adrian Streit, Evi Wollscheid, and anonymous reviewers for valuable advice. All C. elegans strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources. This project was supported by a grant from the German Science Foundation, priority program 1110 on innate immunity (grant SCHU 1415/3).


   FOOTNOTES
 
1 These authors contributed equally to this work.

3 Current address: Department of Animal Evolutionary Ecology, Zoological Institute, Eberhard Karls University Tübingen, Tübingen, Germany

Received for publication May 31, 2006. Accepted for publication January 4, 2007.


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M. C. K. Leung, P. L. Williams, A. Benedetto, C. Au, K. J. Helmcke, M. Aschner, and J. N. Meyer
Caenorhabditis elegans: An Emerging Model in Biomedical and Environmental Toxicology
Toxicol. Sci., November 1, 2008; 106(1): 5 - 28.
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K. L. Styer, V. Singh, E. Macosko, S. E. Steele, C. I. Bargmann, and A. Aballay
Innate Immunity in Caenorhabditis elegans Is Regulated by Neurons Expressing NPR-1/GPCR
Science, October 17, 2008; 322(5900): 460 - 464.
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R. Rae, M. Riebesell, I. Dinkelacker, Q. Wang, M. Herrmann, A. M. Weller, C. Dieterich, and R. J. Sommer
Isolation of naturally associated bacteria of necromenic Pristionchus nematodes and fitness consequences
J. Exp. Biol., June 15, 2008; 211(12): 1927 - 1936.
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