Sensory regulation of C. elegans male mate-searching behavior

Abstract

How do animals integrate internal drives and external environmental cues to coordinate behaviors? We address this question by studying mate-searching behavior in C. elegans. C. elegans males explore their environment in search of mates (hermaphrodites) and will leave food if mating partners are absent. However, when mates and food coincide, male exploratory behavior is suppressed and males are retained on the food source. We show that the drive to explore is stimulated by male-specific neurons in the tail, the ray neurons. Periodic contact with the hermaphrodite detected through ray neurons changes the male's behavior during periods of no contact and prevents the male from leaving the food source. The hermaphrodite signal is conveyed by male-specific interneurons that are postsynaptic to the rays and that send processes to the major integrative center in the head. This study identifies key parts of the neural circuit that regulates a sexual appetitive behavior in C. elegans.

Fig 1. Mate searching behaviour is inhibited after contact with a hermaphrodite

(A) Contact with a hermaphrodite is required to retain males. The hermaphrodite vulva is not required to retain males. P_L values (probability of leaving food per worm per hour) and C_r (coefficient of retention) for males alone or in the presence of agar, with paralysed living or PFA-fixed males, with paralysed living or PFA-fixed hermaphrodites, PFA-fixed hermaphrodites covered in agar or paralysed vulvaless (lin-39 n1760) hermaphrodites. The number of worms assayed and the number of independent assays is indicated under each bar as n (exp). Replicas from living males, fixed males and fixed hermaphrodites were performed in parallel on the same day 2 different days. Fixation was performed in fresh 4% PFA at 4°C overnight and worms were rinsed in PBS before the assay. Assays with PFA-fixed hermaphrodites covered in agar were performed in parallel and compared with assays of males alone in the presence of agar. Retention levels, as the proportion in P_L reduction, are represented with the coefficient of retention, C_r = [P_L(alone) - P_L(with hermaphrodites)]/ P_L(alone). C_r=1 indicates full retention (P_L(alone) >0 and P_L(with hermaphrodites)= 0), C_r= 0 indicates no retention [P_L(alone)=P_L(with hermaphrodites)]. Males were fully retained by all hermaphrodites except by those covered in agar, and only partially retained by males. * indicates p< 0.001 when P_L values are compared to wild type males alone; indicates p< 0.001 when P_L values are compared to each-other. (B) Male behaviour with a hermaphrodite. The position is shown of a single male on a food patch during an hour interval. In the presence of hermaphrodites, a male alternates between bouts of contact and bouts on the food edge. The graph plots a male’s distance from one hermaphrodite in the center of the plate (in mm) against time (min). Position was scored every 5 seconds. The food edge and the frame edge are marked. (C) Male behaviour alone. A male alone spends most of its time on the food edge and beyond. Events scored as exits are indicated by the brackets. The graph plots a male’s distance from the center of the plate (in mm) against time (min). The food edge and the frame edge are marked. (D) Males spend a significant amount of time on the food edge when they are in the presence of hermaphrodites but less time than when they are alone. For each condition, four or five males were analysed during observation periods of 1 hour and 30 minutes. (E) Males produce significantly more exit events per minute on the food edge when alone than when in the presence of hermaphrodites. For each condition, four or five males were analysed during observation periods of 1 hour and 30 minutes. indicates p< 0.003 compared to each-other (t-test).

Fig 2. The male specific ray neurons stimulate male exploratory behaviour away from food and retention by hermaphrodites

(A) The rays, but not other tail sensilla, are required to stimulate exploratory behaviour and for retention by hermaphrodites. The decrease in retention observed in rays 1-6 ablated wild type males indicates that activity of rays 1-6 is required to inhibit exploratory activity induced by rays 7-9. This suggests that the exploration and retention signals are two different signals that can be produced by different ray neurons. P_L and C_r values for wt, mutant and operated males alone and in the presence of hermaphrodites. Mutant strains used were mab-3(mu15) and lin-32(e1926). Laser ablations of wt and ocr-2(ak47) males were performed at the L1 or L2 stage: rays 1 to 6 were removed by bi-lateral ablation of V5p L-R and V6p L-R; rays 7-9 were removed by bi-lateral ablation of Tap L-R; p9p and p10p were ablated to remove the hook sensillum; Y was ablated to remove the post-cloaca sensilla. * indicates p< 0.01 when P_L values are compared to controls alone (wt for mutants and mock ablated wt and ocr-2 for ablations); indicates p< 0.002 when P_L values are compared to controls (wt or mock ablated wt and ocr-2 males with hermaphrodites) (B) The spicule neurons are not required for male exploratory behaviour or for retention. P_L and C_r values alone and in the presence of hermaphrodites for males with intact, cauterized, or ablated spicule-associated sensory neurons (SPD, SPV and SPC). Ablations were performed in young adults in a syIs33( P(gpa-1)∷gfp) background to visualize the spicule sensory neurons. Mock animals underwent the same manipulations as ablated animals with the exception of not being shot with the laser microbeam. These manipulations slightly reduced the rate of leaving alone compared to non-manipulated males. (C) RnA and RnB neurons stimulate male exploratory behaviour away from food. P_L values, alone and in the presence of hermaphrodites, for males with and without RnA or RnB neurons and pkd-2(sy606);lov-1(sy582) double mutants. Only males in which all RnB neurons were dead were assayed. RnA death was not complete. Strains used: bxIs14( P(pkd-2)∷gfp); bxEx136 [P(pkd-2)∷ICE+P(unc-122)∷gfp] for RnB genetic ablations; bxEx137[P(trp-4)∷caspase3-NZ+P(grd-13)∷CZ-caspase3+P(elt-2)∷gfp] or bxEx138[P(trp-4)∷ ICE+P(elt-2)∷gfp] for RnA genetic ablations; and bxEx136;bxEx137 or bxEx136;bxEx138 for RnA,RnB genetic ablations. * indicates p< 0.001 when P_L values are compared to wt males alone; indicates p< 0.02 when P_L values are compared to each-other. (D) Loss of all RnB neurons and most RnA neurons results in a reduction of exit events at the food edge. Bars plot the average of exit events per minute spent on food edge per worm in 1h 30 min observation periods alone and in the presence of hermaphrodites; * indicates p< 0.05 compared to intact males alone (t-test). (E) DIC photograph of a male tail with the ventral side in focus; posterior is oriented toward the left. All the male specific sensilla are labelled. Dashed lines indicate the position of the spicules, which remain retracted inside the gubernaculum.

Fig 3. Male specific interneurons regulate retention by hermaphrodites

(A) Loss of EF/DX interneurons results in partial loss of retention by hermaphrodites. P_L and C_r values for intact and operated males alone and in the presence of hermaphrodites. EF and DX interneurons were removed by laser ablation of the U and F cells at L1 stage. DX interneurons are post-synaptic mainly to hook neurons and pre-synaptic to post-cloacal sensilla (PCS) neurons, and may be involved in sperm transfer (Male Wiring Project, Albert Einstein College of Medicine, http://worms.aecom.yu.edu/pages/male_wiring_project.htm), [23]. We have shown that, unlike the rays, hook and PCS are not required for retention, making the DX interneurons unlikely candidates for conveying the hermaphrodite signal. EF/DX ablation did not disrupt retention as severely as ablation of all rays. This difference could be explained by the fact that, unlike EF ablated males, males with no rays spend little or no time in contact with the hermaphrodite and therefore, the proportion of time available to explore away from the food lawn is increased in ray ablated males. * indicates p< 0.001 when P_L values are compared to mock ablated males with hermaphrodites. (B) A male without EF and DX interneurons moves beyond the food edge after bouts of contact with a hermaphrodite. The graph plots the male’s distance from the hermaphrodites in the center (in mm) against time (min). The food and the frame edge are marked. (C) EF/DX ablated males spend the same proportion of time on the food edge and in contact with hermaphrodites as intact males. Bars plot average percentage of time spent on food edge or in contact with a hermaphrodite per worm for intact and EF/DX ablated males. Same data for wild type male as in figure 1D and 2D; n=4 for each condition in 1h 30 min observation periods. (D) EF/DX ablated males in the presence of hermaphrodites produce the same frequency of exit events as intact males alone. Bars plot average of exit events per minute spent on food edge per worm. Same data for wild type male as in figure 1E; n=4 for each condition (n=5 for wt alone) in 1h 30 min observation periods * indicates p< 0.03 compared to intact males with hermaphrodites, (t-test). (E) Bouts of contact with a hermaphrodite are similar in length for EF/DX ablated and intact males. The duration (in minutes) of each contact bout produced by 4 individual intact or EF/DX ablated males in 1h 30 min observation periods is represented by a circle and the average is represented by a horizontal line. (F) Male neurons that regulate mate-searching behaviour. Drawing of a male worm, anterior is oriented to the left. Neurons are labelled in colours: A and B type ray sensory neurons in the tail (red and green); EF interneurons post-synaptic to ray neurons in the pre-anal ganglion (purple) send processes to the nerve ring in the head (integration center, pink); amphid sensory neurons in the head (blue).

Fig 4. Amphid neurons modulate male exploratory behaviour and contribute to retention

Mutants in food sensation display higher rate of exploratory behaviour but are fully retained by hermaphrodites; mutants defective for retention have defects in neurons involved in both food sensation and hermaphrodite detection. Unlike wild type hermaphrodites, which remain on food, osm-3(p802), tax-2(p691) and ocr-2(ak47) hermaphrodites left food at measurable rates, indicative of the role of these genes in food sensation. Examination of tax-2 and ocr-2 canonical reporter lines in males revealed no expression in ray neurons [18] [19]. Only osm-3 is expressed and disrupts the cilia of RnB neurons, as well as the other male specific ciliated neurons exposed to the outside [24]. Bars show the P_L and C_r values of hermaphrodites alone, males alone and males with hermaphrodites for wt and mutants in gustatory amphid and ray neurons. Dashed line indicates the rate of exploratory behaviour in wild type males. Strains used: wt, osm-3(p802), mab-3(mu15), mab-3(mu15);osm-3(p802), tax-2(p691) and ocr-2(ak47); * indicates p< 0.001 when P_L values are compared to wt males alone; indicates p< 0.001 when P_L values are compared to wt hermaphrodites; indicates p< 0.001 when P_L values are compared to wt males with hermaphrodites; * * indicates p< 0.03 when P_L values are compared to each other.