Sex differences across organisms have been an intense area of research over the last 50 years. Despite these efforts, the mechanisms underlying many behavioral differences, including sensation, perception of sensory cues and decision-making and how this occurs at the level of the brain is still not understood. Sex differences have been identified from humans to worms. A number of studies have demonstrated differences between males and females in processes, including, olfaction, thermoregulation, aggression, learning and mood (Bjorkqvist et al ., 1994; Doty et al ., 1985; Berger-Sweeney et al ., 1995; Halpern et al ., 2000; Kaciuba-Uscilko et al ., 2001; Bielsky et al ., 2005). Behavioral studies in vertebrates and invertebrates have suggested that behavioral sex biases might have biological underpinnings. The understanding of the differences in neural signals and circuit function, that result in the variation in decision-making processes across female and male systems is not fully understood.

The hermaphrodite nematode C. elegans is attracted to or repelled by an array of volatile odorants (Bargmann and Horvitz, 1991; Troemel et al ., 1997; Debono and Maricq, 2005; Chao et al ., 2004; Zhang et al ., 2005; Ghosh et al ., 2017). In addition, hermaphrodites are able to generate decisions when encountering conflicting cues that are presented simultaneously (Ishihara et al ., 2002; Ghosh et al ., 2016; Harris et al ., 2019; Yang et al ., 2022). Male C. elegans are able to sense a number of chemicals, including, pheromones, food associated cues, diacetyl and salt (NaCl) (White et al ., 2007; Sakai et al ., 2013; Ryan et al ., 2014; Wexler et al ., 2020; Loxterkamp et al ., 2021; Portman et al ., 2017). To address sex specific differences in worm behavior, we used C. elegans to understand whether wild type hermaphrodites and males behave differently when exposed to conflicting cues, such as, food and the repulsive odor cue, 2-nonanone (Troemel et al ., 1997; Harris et al ., 2019; Ellis et al ., 2020).

We examined the behavioral differences between hermaphrodites and males using a “multi-sensory behavioral paradigm”, where we compare wild type C. elegans hermaphrodites and males in a food leaving assay when exposed to a repulsive cue, 2-nonanone (Fig. 1A, See schematic diagram, Harris et al ., 2019). We examined any differences between males and hermaphrodite adults in a multi-sensory behavioral assay, where both hermaphrodites and male worms were examined for food leaving during exposure to 2-nonanone. A number of genes and neurons have previously been identified for 2-nonanone-dependent food leaving in hermaphrodites (Harris et al ., 2019). Wild type hermaphrodites typically leave the E.coli (OP50) food patch within 10-15 minutes as normally observed after 2-nonanone addition next to the food patch (Fig. 1B and C). In contrast, when examining male worms we observe that males show a significantly delayed food-leaving during exposure to 2-nonanone (Fig. 1Ci (graph) and 1Cii (images)) showing an example of N2 males with reduced food leaving after 10 minutes). Suggesting, males leave a food patch significantly slower than hermaphrodites when stimulated by the presence of a repulsive odor. In addition, we observe that when N2 males leave the food patch during this period it is significantly delayed, and many of the males do not leave the food patch at all during our assay period of 45-60 minutes, despite positioning at the edge of the food patch furthest from the 2-nonanone (Fig. 1). Interestingly, wild type male worms have been shown to spontaneously leave a food patch, multiple hours later after being introduced to a food patch. But in the first hour of incubation of males on new food patch prior to adding 2-nonanone we do not see this increased leaving (Lipton et al ., 2004).

To address if N2 male worms were sensing 2-nonanone similar to hermaphrodites, or showing an actual difference in decision-making to leave the food, we used multiple approaches to do this. We analyzed male worm avoidance behavior to 2-nonanone using the 2-nonanone avoidance assay (Troemel et al ., 1997). We examine wild type males versus hermaphrodites in multiple ways. We found that N2 male worms avoid 2-nonanone the same as wild type N2 hermaphrodites (Fig. 1G). This was evident by N2 males avoiding 2-nonanone as strongly as wild type hermaphrodites tested in parallel. We also analyzed the male worms during the exposure to 2-nonanone while on the assay food patch. This was done by analyzing male behavior during the assay (See Fig. 1Cii Image of N2 males during repulsion) and measuring the time to reach the edge of the food patch after 2-nonanone was added next to the E. coli (OP50) food patch (see schematic diagram, Fig. 1a). Male worms crawled to the edge of the food and remained at the furthest edge away from the 2-nonanone repellent like wild-type hermaphrodite animals, suggesting N2 males could sense and perform a repulsive response to 2-nonanone (measured time taken for 90% of worms to reach the edge of the food) (Fig. 1F). This suggests that there is a difference in the decision to leave food during repulsive odor exposure, based on actual decision to leave food and not a difference in the general sensation of the repellent, 2-nonanone (Fig. 1F).

To further understand how general or specific these male differences to hermaphrodite worms are, we next varied two assay conditions. First, we used a second volatile repellent, benzaldehyde (undiluted) and measured benzaldehyde-dependent food leaving in males versus hermaphrodites (Yoshida et al ., 2012; Harris et al ., 2019). Interestingly, males also showed delayed food leaving in response to undiluted benzaldehyde when compared to wild type hermaphrodite animals (Fig. 1D). In addition, we changed the food patch type to assess any effect on male worm food leaving when compared to hermaphrodite worms (See Fig. 1C). Wild type hermaphrodite worms have been shown to exhibit a variety of behavioral outputs in response to food and food associated cues, including, showing varied feeding rates, sensory-dependent locomotory behavior, behavioral preference and food leaving dynamics (Zhang et al ., 2005; Shtonda and Avery, 2006; Meisel et al ., 2014; Olofsson et al ., 2014). We chose Comamonas sp food lawns and assessed male food leaving during exposure to 2-nonanone when compared to hermaphrodites . Wild type hermaphrodite worms also leave Comamonas sp during exposure to 2-nonanone, as previously described for wild-type hermaphrodites (Harris et al ., 2019; Ellis et al ., 2020). Upon examining male worms for 2-nonanone-dependent food leaving when compared to hermaphrodite worms, male worms are also significantly delayed food leavers on Comamonas sp , suggesting male worms show this general delayed food leaving across multiple foods, including at least, the regular tested food source, E. coli (OP50) and also Comamonas sp during exposure to 2-nonanone (Fig. 1E).

Overall, this suggests that wild type N2 male worms show decreased food leaving when compared to N2 hermaphrodites when exposed to different volatile repellents, including, undiluted 2-nonanone and benzaldehyde, and across different food types. Wild type hermaphrodites and males are known to behave differently in specific odor-guided behavior, locomotory dynamics and conditioning associated with salt chemotaxis (Loxterkamp et al ., 2021; Sakai et al. , 2013). This provides an avenue for further investigation of the neural mechanisms that influence behavioral differences observed between sexes in a multi sensory behavior.

C. elegans strains were cultivated under the standard conditions (Brenner et al ., 1974). Wild type N2 hermaphrodite young adults, and wild type male adults were used in the 2-nonanone-dependent food leaving experiment for this study.

Worm culturing, plate preparation and multi-sensory assay. All hermaphrodite and male worms were grown to young adult under E. coli ( OP50) well-fed conditions prior to performing behavior assay (Brenner et al ., 1974; Harris et al ., 2019).

Multi-sensory assay to analyze male vs hermaphrodite adult worms. For the 2-nonanone-dependent food leaving assay. Wild Type worms (N2) were assayed as previously described (Harris et al ., 2019; Ellis et al ., 2020). For all worms, E. coli (OP50) was used as the food source (Brenner et al ., 1974). For the preparation of assay plates, the NGM (Nematode Growth Medium) agar was made and poured into small assay plates (6 cm) and then allowed to cool and solidify (using standard NGM plate preparation protocols). After two days, a liquid suspension of E. coli (OP50) was prepared; consisting of 40 mL of NGM media and E. coli (OP50) colonies added to the NGM media, this media was then placed at 26°C overnight. The next morning, the E. coli (OP50) culture was centrifuged at 3500 rpm for 15 min. 35 mL of the NGM was removed and discarded (Harris et al ., 2019; Ellis et al ., 2020). The E. coli (OP50) pellet was then re-suspended in 5 mL of NGM through mixing, and then 55 µL of this E. coli (OP50) culture was then added to the center of an NGM agar assay plate (as seen in Fig. 1A, Schematic of behavioral assay). All worms were grown at 20-23°C on NGM plates that contained a lawn of E. coli (OP50). Next 20-25 young adult worms (either hermaphrodite or male worms) are placed onto the 1 cm diameter food lawn and are allowed to acclimate for 60 minutes. After 1 hour, a drop of 2-nonanone repellent (1 µL) is placed approximately two worm lengths away from the food lawn. The number of worms on the lawn was counted every 5 minutes for a total of 45-60 minutes, and the number of hermaphrodite or male worms that left the food lawn was determined. Food leaving is analyzed during exposure to the repellent 2-nonanone (Harris et al ., 2019; Ellis et al ., 2020). Worms present on the food at each 5-minute time-point are counted (represented in y-axis). For each plate of male worms tested, there was a separate hermaphrodite assay plate examined in parallel. No males were mixed with hermaphrodite adult worms during the behavioral assay. For all 2-nonanone-dependent food leaving assays on Comamonas sp food lawn, the assay was prepared and tested using identical conditions to E. coli (OP50) assays tested above. For all data analysis, a student’s t test was performed when comparing wild type N2 adult hermaphrodite worms to wild type N2 male adult worms tested on the same day in parallel conditions. Mean±SEM, Student’s t- test, *p ≤0.05, **p≤0.01, ***p≤0.001. n = number of repeated days tested across 1A - G.

Assay to measure the ability to reach the edge of a food patch during 2-nonanone exposure

Wild type hermaphrodites and male worms were examined for their ability (Time to reach the edge) to reach the furthest edge of the food patch during exposure to undiluted 2-nonanone. 20-25 hermaphrodites and males were examined per replicate. Wild type N2 hermaphrodites and wild type N2 males were examined for the time taken for 90% of the worms to reach the edge of the food patch furthest from the 2-nonanone drop placement (last sector of the food patch). Time was determined as time taken (sec) for worms to reach the edge of the food from the point of 2-nonanone addition next to the E. coli (OP50) food patch. The 1 cm food patch was divided into 5 sectors (0.2 cm each). Sectors A on food patch being closest to the 2-nonanone, Sector E on food patch being furthest away from 2-nonanone drop (As previously described in Harris et al ., 2019). Wild type N2 male worms were measured and compared to wild type N2 hermaphrodites tested in parallel on separate behavior assay plates. Wild type N2 hermaphrodite and male worms were not mixed on behavioral assay plates. Mean±SEM, student’s t-t est, *p≤0.05, **p≤0.01, ***p ≤0.001. n=number of repeated days tested for hermaphrodite and male worms.

2-nonanone avoidance assay

To examine the avoidance of 2-nonanone in hermaphrodites and males, chemotaxis assays were performed essentially as previously described (Troemel et al ., 1997; Harris et al ., 2019). Briefly, animals were placed in the center of a square plate that was divided into sectors A, B, C, D, E and F and 2 drops of 1 µl of undiluted 2-nonanone was added to one side and 2 drops of 1 µl ethanol was added to the opposite side of the plate as a control. Approximately 75-100 hermaphrodite or male worms were used in each assay. 2-nonanone avoidance was analyzed by counting the number of worms in the sectors A-B, C-D, and E-F with E-F being furthest away from the 2-nonanone point sources. The avoidance index was calculated as the number of animals in sectors A and B (at 2-nonanone) minus the number of animals in the sectors E and F (at control) and normalized with the total number of animals in all 6 sectors on plate (Avoidance Index between 0 and -1.0). For all data analysis, a Student’s t test was performed when comparing wild type N2 hermaphrodite worms to wild type N2 male worms tested on the same day in parallel identical conditions. Mean±SEM, Student’s t -test, p ≤0.05*, p≤0.01**, p≤0.001***.

List of worm and bacterial strains used in experiments in the present study

1) Wild type N2 Bristol hermaphrodite C. elegans were purchased from CGC,

2) N2 wild type male C. elegans were purchased from CGC,

3) E. coli (OP50) bacterial strain was purchased from CGC,

4) DA1877 Comamonas sp bacterial strain was purchased from CGC,

All strains were provided by the CGC (Caenorhabditis Genetics Center) at the University of Minnesota, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).

This project was funded through lab start-up (Harris G), California State University Channel Islands and Mini-Grant awarded, California State University Channel Islands, 2020 - 2021.

Chemicals used in study. All chemicals used in this study, including, 2-nonanone (Cas # 821-55-6) and Benzaldehyde (Cas # 100-52-7) were purchased from Sigma Aldrich.