Chemosensory Adaptations in Caenorhabditis Males during the Establishment of Androdioecy

Chemosensory Adaptations in Caenorhabditis Males during the Establishment of Androdioecy | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Chemosensory Adaptations in Caenorhabditis Males during the Establishment of Androdioecy Harini Kannan , King L Chow doi: https://doi.org/10.1101/2025.05.24.655903 Harini Kannan 1 Division of Life Science, The Hong Kong University of Science and Technology , Clear Water Bay, Kowloon, Hong Kong, CHINA Find this author on Google Scholar Find this author on PubMed Search for this author on this site King L Chow 1 Division of Life Science, The Hong Kong University of Science and Technology , Clear Water Bay, Kowloon, Hong Kong, CHINA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: bokchow{at}ust.hk Abstract Full Text Info/History Metrics Preview PDF Abstract Caenorhabditis elegans has evolved from its dioecious ancestors to adopt an androdioecious reproductive strategy. In this process, ancestral female C. elegans acquired genetic modifications that enabled self-sperm generation, self-sperm activation, and a reduced reliance on sexual reproduction. However, how males have adapted during this transition from dioecy to androdioecy is less explored. Using respective Caenorhabditis species, we demonstrated that androdioecious hermaphrodites exhibit a reduction in sex pheromone potency, while androdioecious males show notably heightened olfactory habituation and diminished mate exploration capabilities. The behavior of androdioecious males can be reverted to resemble that of dioecious males by replacing the SRD-1 receptor with its dioecious orthologs. This intrinsic characteristic is contingent upon the cytoplasmic domain of the receptor. We propose a theoretical framework where C. elegans males have accumulated genetic variations in their pheromone receptor, leading to altered chemosensory perception of the opposite sex, which confer a selective advantage that favors the establishment of hermaphroditism. Our study provides insights into an overlooked male trait that was shaped by changes in chemosensory signaling. The findings underscore the capacity of chemosensory variations to influence how organisms perceive critical ecological factors and eventually facilitate the emergence and stabilization of hermaphroditism. 1. Introduction In the Caenorhabditis clade, evolutionary shifts have led to diverse mating strategies, with dioecy as the dominating mode. 1 , 2 Mutations allowed ancestral females to produce self-sperm, enabling self-reproduction and reducing male reliance. 3 – 5 Some lineages evolved into self-fertilizing hermaphrodites, 6 exhibiting weaker sex pheromone potency 7 and altered behaviors to avoid males 8 , 9 . Younger hermaphrodites resist male advances, while older and sperm-depleted ones show higher female-like receptivity. 10 Hermaphroditism was thought to have evolved in low-density environments as an adaptive response to ecological pressure 11 . Social interactions also shape reproductive strategies, such as cooperative breeding in birds fostering monogamy or polyandry, 12 , 13 underscoring the influence of social factors in mating system evolution. 12 Historically, hermaphroditism research focused on a two-stage model of female-to-hermaphrodite transition, emphasizing self-sperm production and activation. 14 , 15 However, male traits’ roles may have been overlooked. Our investigation elucidates male phenotypic characteristics associated with sex pheromones, including enhanced olfactory habituation and diminished mate exploratory behavior, thereby differentiating androdioecious males from their dioecious equivalents. The behavior of androdioecious males can be reverted to resemble that of dioecious males by replacing the SRD-1 receptor with its dioecious ortholog. This intrinsic characteristic is contingent upon the cytoplasmic domain of this receptor, implying that modifications in chemosensory signaling may have conferred a selective advantage to the establishment of androdioecy in Caenorhabditis species. Our study underscores the capacity of chemosensory variations to influence how organisms perceive critical ecological factors and eventually facilitate the emergence and stabilization of hermaphroditism. 2. Materials & Methods Worm strains N2 CB4088: him □ 5(e1490)V was used for wildLJtype C. elegans for high incidence of males; EM464 ( C. remanei ), AF16 ( C. briggsae ); JU1428 ( C. tropicalis ); ZZY0401 ( C. sinica ); JU1904 ( C. wallecei ); CB4018: fog □ 2(q71) ( C. elegans hermaphrodites lacks self-sperm); For experiments expressing SRD-1 orthologs in C. elegans srd-1 mutant males we used KC301: srd □ 1(eh1)II, him □ 5(e1490)V; KC1445: srd □ 1(eh1)II, him □ 5(e1490)V, wxEX[Psrd □ 1::srd □ 1:gfp unc □ 54 3 ′ UTR + Pcrm1b::rfp unc □ 54 3 ′ UTR]; KC1447: srd □ 1(eh1)II, him □ 5(e1490)V, wxEX[Psrd □ 1::Cre □ srd □ 1::gfp unc □ 54 3 ′ UTR + Pcrm1b::rfp unc □ 54 3 ′ UTR]; KC1449: srd □ 1(eh1)II, him □ 5(e1490)V, wxEX[Psrd □ 1::Ce □ srd □ 1 ⍰ CT::Cre □ srd □ 1 CT::gfp unc □ 54 3 ′ UTR + Pcrm1b::rfp unc □ 54 3 ′ UTR] . Sex Pheromone Extraction Synchronized L4 C. remanei and fog-2 mutant C. elegans were separated, and after 24 hours, five virgin adult C. remanei (EM464) or 100 virgin fog-2 C. elegans females were incubated in 100 μl of M9 buffer at 25°C for 6 hours. The supernatant was then assessed on wild-type C. elegans or C. remanei males for quality control in the chemoattraction assay. Chemoattraction Assay A microscopic slide was coated with 2 ml of chemotaxis agar (1.5% agar, 25 mM NaCl, 1.5 mM Tris-base, 3.5 mM Tris-Cl) and placed in a 5.5-cm Petri dish. Two 2 μl droplets of 1 M sodium azide were applied 3 cm apart and evaporated. Then, 2 μl of extract supernatant and control buffer were added to these spots. Twenty synchronized one-day-old adult males were placed at the slide’s center, equidistant (1.5 cm) from test and control sites. After 30 minutes, immobilized worms were counted based on their positions: C (control area), E (experimental area), or N (other regions). The Chemotaxis Index (CI) was calculated as CI = (E - C) / (E + C + N), with values near 1.0 indicating strong attraction and near zero showing no attraction. Each test included ≥20 assays with 20 males each, totaling 400 animals. Results are shown as CI ± SD, with significance assessed via one-way ANOVA with Bonferroni correction (***P<0.001). Mate Exploration Assay Mating exploration was studied by placing a fixed number of Caenorhabditis males in a culture dish, allowing 10 minutes for dispersal, then introducing the same number of C. remanei virgin females to the dish. Observations lasted 30 minutes under a microscope, with vulval prodding indicating successful exploration. The earliest vulval prodding time on each plate was recorded to create a time vs. density graph. Population density was altered by adjusting dish sizes (3.5 cm, 5 cm, 10 cm for high, medium, and) and animal numbers. Each test included 15 assays, and significance was assessed using one-way ANOVA (***P<0.001). 3. Results and Discussion The transition to androdioecy from dioecious progenitors is not exclusive to Caenorhabditis species; 16 similar such transitions have occurred in Datisca glomerata 17 , 18 and Eulimnadia texana . 19 Androdioecy is advantageous in ecological contexts with high rates of extinction and recolonization. 20 C. elegans , recognized for its fluctuating population dynamics experiences a persistent “boom and bust” cycle 21 where androdioecy adoption may be an adaptive strategy to mitigate extinction risk. C. elegans males exhibit lower sex pheromone sensitivity than C. remanei males, 10 , 22 suggesting that gonochoristic Caenorhabditis males could be more efficient in mate pursuit. Androdiecious males exhibit deficient mate exploration and strong sex pheromone habituation Using C. elegans and C. remanei males as a proxy for the respective mating systems in this study, we assessed the mate-seeking abilities of androdioecious and gonochoristic males. Using light microscopy, we measured the time males took to locate virgin females and documented mating behaviors, such as vulval prodding, 23 within 30 minutes. C. remanei females were chosen for their ability to attract males across Caenorhabditis species. 10 Mating behaviors under different population densities at a 1:1 male-to-female ratio were evaluated. In a 10 cm culture dish with 20 androdioecious males and 20 virgin C. remanei females, androdioecious males took over 25 times longer to initiate copulation than dioecious males. Fifteen or ten or five androdioecious males against equal number of C. remanei females results in failure of locating the females within the 30 min period, while dioecious males took 1 ± 0.8, 4 ± 1.35 and 4 ± 2.13 minutes respectively to do so ( Fig 1B ). When only one pair of animals were tested, neither group mated within 30 min. When the same set of experiments were repeated in medium and higher density conditions in 5cm and 3.5cm dishes, improved performance of androdioecious males was observed, although dioecious males consistently outperformed their androdioecious counterparts and initiated active mating within a shorter period irrespective of their population density ( Fig 1B , grey and black bars). Inadequate mate exploration was identified in other androdioecious Caenorhabditis species as well ( Sup Fig 2A ), whereas dioecious Caenorhabditis species exhibited consistent and proficient mate searching behavior ( Sup Fig 2B ). Although androdioecious males can locally search and copulate with females or hermaphrodites in proximity (Kannan and Chow, unpublished), their prolonged mate location time may indicate a deceptive perception of prospective partners nearby, as if they were in a habitat with low-population density. Download figure Open in new tab Figure 1: (A) Olfactory habituation was evaluated by measuring the response of C. elegans and C. remanei males to C. remanei sex pheromones following pre-exposure to either pheromones or M9 buffer ( n = 400); (B) The minimum time required for C. elegans and C. remanei males to initiate vulval prodding on C. remanei females was recorded under varying density conditions (symbols: square = 1 pair, circle = 5 pairs, diamond = 10 pairs, star = 15 pairs, triangle = 20 pairs; n = 15). High, medium, and low-density tests refer to experiments conducted in 3.5cm, 5cm and 10cm culture dishes. Signal transduction components essential for C. elegans to detect sex pheromone cues overlap with sensory pathways that perceive nutritional signals; 24 – 26 prolonged exposure to these signals induce olfactory habituation, thereby diminishing the animals’ sensitivity to nutritional stimuli. 27 To determine if sex pheromones trigger similar habituation phenomena and whether patterns differ between species of different mating types, we exposed androdioecious ( C. elegans, C. briggsae, C. tropicalis ) and gonochoristic males ( C. sinica, C. remanei, C. wallecei ) to C. remanei sex pheromones for varying durations. We then measured their chemoattractive indices (C.I.) in response to the pre-exposed odor (sex pheromones) in contrast to the control group exposed to M9 buffer. Androdioecious males showed the strongest habituation with their responses drastically compromised after 2.5 hours of sex pheromone pre-exposure: C. elegans (CI: 0.003 ± 0.07) ( Fig 1A ), C. briggsae (CI: 0.2 ± 0.09), C. tropicalis (CI: 0.005 ± 0.06) ( Sup Fig 1A ). In contrast, gonochoristic males remained highly responsive: C. remanei (CI: 0.65 ± 0.09) ( Fig 1A ), C. sinica (CI: 0.60 ± 0.1), C. wallecei (CI: 0.602 ± 0.1) ( Sup Fig 1B ). Impaired mate-searching ability and robust sex pheromone habituation are distinctly observed in contemporary androdioecious males likely having offered them selection advantages while also differentiating them from their dioecious equivalents. Download figure Open in new tab Supplementary Figure 1: Olfactory habituation was evaluated by measuring responses of androdioecious (A) and dioecious (B) males to C. remanei sex pheromones following pre-exposure to pheromones or M9 buffer ( n = 400 males). Hermaphrodites continue to produce sex attractant but at a lower potency Sex pheromones from dioecious females are more potent than the attractants from hermaphrodites. Pheromone extracts from 5 one-day-old virgin C. remanei females attracted conspecific males and males from various other species ( Sup Fig 4D ), while extracts from 5 one-day-old C. elegans N2 hermaphrodites failed to elicit male response ( Sup Fig 4C ). Lower concentrations or chemical differences may explain the weaker allure of hermaphrodites, challenging the idea that they lack pheromones entirely. 7 , 10 To verify this notion, we extracted pheromones from different numbers of C. elegans N2 hermaphrodites. Pheromones from 100 one-day-old C. elegans N2 hermaphrodites were about half as attractive (CI: 0.31 ± 0.1) as those from 5 virgin C. remanei females (CI: 0.72 ± 0.08) ( Sup Fig 4C ). Since hermaphrodites differ from females primarily by their ability to produce self-sperm, we further tested whether presence of sperms could impact the attractiveness. Using C. elegans fog-2 mutants, which produce pseudo-females lacking self-sperm, 28 , 29 we found pheromone extracts from 5 to 40 pseudo-females were unattractive to C. elegans males (CI: <0.20 ± 0.07) while extracts from 100 pseudo-females (CI: 0.72 ± 0.07) matched the potency of those from 5 C. remanei females ( Sup Fig 4C ). If concentration differences explain this disparity, female-derived pheromones should exceed the threshold for robust attraction. Titration of C. remanei pheromones showed attractiveness (CI: 0.49 ± 0.08) persisted even after a 1000-fold dilution ( Sup Fig 4E ). GC-MS analysis of pheromones from C. remanei females and C. elegans hermaphrodites revealed similar chemical profile with a key molecule contributing significantly to potency in both blends (Chan and Chow, unpublished). The weaker sex appeal, compared to C. remanei females, is primarily due to reduced pheromone potency. These findings contradict the earlier reports that C. elegans hermaphrodites have lost the sex pheromone production ability. 7 , 10 The chemosensory traits observed in androdioecious Caenorhabditis males converge on SRD-1 Apart from conservation in the sex pheromone blend, con-specific attraction elicited by female and hermaphrodite sex pheromones among Caenorhabditis males ( Sup Fig 4D ) may also be ascribed to the conservation of the sex pheromone receptor expressed in males. The amino acid compositions of receptor orthologs from C. elegans and C. remanei SRD-1 exhibited high conservation within their N-terminal region with notable polymorphisms primarily located in their cytoplasmic domain ( Sup Fig 4A ). Similar observation was noted in SRD-1 orthologs across various Caenorhabditis species, where N-terminal regions are highly conserved and distinct polymorphic variations were identified in the cytoplasmic domain ( Sup Fig 3 , Sup Fig 4A ). Notwithstanding the polymorphism observed within the cytoplasmic domain, the SRD-1 ortholog from C. remanei demonstrated an effective functional integration with the sex pheromone sensory circuitry of male C. elegans. C. elegans srd-1 mutant males, when expressing C. remanei srd-1 transgenes regulated by the 3 kb long native srd-1 promoter, resulted in a restoration of sex pheromone perception that was marginally more pronounced than that observed in C. elegans srd-1 mutant males expressing the native receptor ( Sup Fig 4B ). Assessments conducted on 3-4 independent transgenic lines suggest that the observed phenotypic traits were not confounded by variations in transgene copy number. Conservation in the sex pheromone communication mechanism among androdioecious and dioecious Caenorhabditis species, at the pheromone, its receptors, and the functional integration in androdioecious males, prompted us to test whether substitution of the sex pheromone receptor would alter male sexual behavior. Download figure Open in new tab Supplementary Figure 2: Minimum time for androdioecious (A) and dioecious (B) males to initiate vulval prodding on a C. remanei female was recorded under varying density conditions (square: 1 pair; circle: 5 pairs; diamond: 10 pairs; star: 15 pairs; triangle: 20 pairs; n = 15). Download figure Open in new tab Supplementary Figure 3: Comparative protein sequence alignment of SRD-1 orthologs was performed using Clustal Omega and visualized with BioRender. Download figure Open in new tab Supplementary Figure 4: (A) Protein sequence alignment of SRD-1 orthologs was performed using Clustal Omega, with polymorphisms highlighted by red circles and visualized using Protter; (B) C. elegans srd-1 mutant males expressing SRD-1 orthologs from C. elegans and C. remanei under the native srd-1 promoter were tested for their response to sex pheromones extracted from C. remanei females and C. elegans fog-2 pseudo-females ( n = 400); (C) Sex pheromones were extracted from varying numbers of one-day-old N2 and fog-2 C. elegans hermaphrodites and tested against C. elegans N2 males ( n = 400); (D) Males from various dioecious and androdioecious Caenorhabditis species were assessed for their behavioral responses to sex pheromones extracted from C. rem anei females and C. elegans fog-2 pseudo-females ( n = 400); (E) Sex pheromones extracted from five one-day-old virgin C. remanei females were titrated and tested against C. elegans N2 males ( n = 400). We replaced the srd-1 gene of C. elegans males with the dioecious ortholog from C. remanei and evaluated the habituation trajectory with C. remanei sex pheromones and mate exploration pattern of the male subjects against 1-day-old virgin C. remanei females. After 2.5 hours of pheromone exposure, C. elegans srd-1 mutant males expressing the native SRD-1 completely lost the sensory response loss (CI: -0.01 ± 0.07), mirroring wild-type C. elegans ( Fig 2A ). In contrast, those expressing C. remanei SRD-1 retained ∼60% responsiveness (CI: 0.35 ± 0.09) ( Fig 2B ). Due to significant polymorphisms between C. elegans and C. remanei SRD-1 residing in their cytoplasmic domain, we created a chimeric receptor by substituting the C. elegans SRD-1 cytoplasmic domain with that of C. remanei SRD-1. C. elegans srd-1 mutant males expressing the chimeric receptor retained their sensory response (CI: 0.44 ± 0.08) after 2.5 hours of pheromone exposure resembling those expressing C. remanei SRD-1 orthologs ( Fig 2C ). Download figure Open in new tab Figure 2: Olfactory habituation was assessed by measuring responses of C. elegans srd-1 mutant males expressing (A) C. elegans SRD-1, (B) C. remanei SRD-1, and (C) Chimeric SRD-1 to C. remanei sex pheromones after pre-exposure to pheromones or M9 buffer ( n = 400 males). We further investigated the influence of SRD-1 on the animals’ ability to locate and participate in copulation. In a 10 cm culture dish containing 20 pairs of males and females, C. elegans transgenic males expressing C. elegans SRD-1 took an average of ∼26 ± 3.8 minutes to initiate mating. In contrast, those expressing C. remanei SRD-1 and chimeric SRD-1 averaged ∼3 ± 1.2 and ∼5 ± 1.7 minutes, respectively ( Fig 3A, B & C ). When the number of animals were reduced to 15 or fewer pairs, transgenic males expressing C. elegans SRD-1 could no longer locate the females, while males expressing C. remanei SRD- 1 or chimeric SRD-1 could still find mates within relatively short periods (∼3 ± 0.9 and ∼6 ± 2.1 minutes for 15 mating pairs; ∼3 ± 1.02 and ∼6 ± 3 minutes for 10 mating pairs; ∼4 ± 2.3 and ∼11 ± 3.8 minutes for 5 mating pairs). However, in experiments with only one male and one female, none of the C. elegans transgenic males were able to engage in mating, regardless of the receptor variants expressed. This suggests that SRD-1 plays a pivotal role in facilitating mating system-specific sexual traits in Caenorhabditis males, likely due to the polymorphisms within its cytoplasmic domain. Alternative receptor variants should have been eliminated over time, had they not aided in the stabilization of androdioecy. Subsequent investigations into the functional relevance of these polymorphic changes led us to confirm that the cytoplasmic domain variants alter the downstream interacting partner of the receptor (Kannan and Chow, unpublished). Characterizing the downstream interacting partners of the distinct polymorphic cytoplasmic domain of SRD-1 in each species may elucidate the varying cellular events associated with the distinct behavioral modifications. Download figure Open in new tab Figure 3: Minimum time for C. elegans srd-1 mutant males expressing (A) C. elegans SRD-1, (B) C. remanei SRD-1, and (C) Chimeric SRD-1 to initiate vulval prodding on a C. remanei female was recorded under varying density conditions (square: 1 pair; circle: 5 pairs; diamond: 10 pairs; star: 15 pairs; triangle: 20 pairs; n = 15). Critical facilitating factors for dioecy-androdioecy transition Ecological conditions favoring the establishment and maintenance of androdioecy are infrequent in natural environments. 30 , 31 We propose that contrary to the prevalent belief of ecological factors such as low population density favoring hermaphroditism; 11 evolution of hermaphroditism could have occurred even in community of higher population density if males and females did not frequently encounter each other. Ancestral female variants exhibiting reduced sexual allure, together with ancestral males that misperceive the availability of mating partners, lead to dramatic reduction of mating opportunities. As a result, a reproductive equilibrium of an emerging androdioecious population dominated by hermaphrodites can be assured. The emergence of hermaphroditism likely had cryptic female variants exhibiting aberrant spermatogenesis and/or oogenesis; mutations compromising self-sperm production and activation are insufficient to ensure a 100% probability of self-fertilization and offspring viability. 32 Unfit variants were likely purged over generations, a process where elimination of unfit female variants is counterbalanced by abundant progeny in each generation. As such, offsprings with optimal genetic combinations can support a healthy self-fertilizing hermaphroditic population. Despite producing a large number of progenies per generation, this process would have required more than hundreds of generations to fully restore the fitness of their ancestral outbreeding population. 33 Retention of a small number of males in these populations, due to the residual non-disjunction events, ensures low level of genetic variation created within a confined population thereby mitigating inbreeding depression. 7 Occasional mating with the residual males instead of complete elimination of males could have played a key role in sustaining a healthy hermaphroditic population as it expands and is stabilized. Our results imply that as androdioecy evolved from dioecious ancestors, androdioecious males continue to express downstream components of SRD-1, which may facilitate dioecious male-like behaviors, i.e., weak habituation and strong female exploration, as an evolutionary vestige. Based on our and others’ findings, we propose that establishment of hermaphroditism necessitates five key requirements: (1) self-sperm generation, (2) self-sperm activation, (3) diminished sex pheromone potency, (4) strong habituation to sex pheromones, and (5) decreased male mate-seeking behavior. Requirements 1, 2, and 3 are linked to physiological changes in female reproduction, while 4 and 5 pertain to modifications in chemosensory communication centered on males. All the above events very likely occurred in parallel to allow hermaphroditism to gain dominance in the ancestral population. Our data reveal that males from C. briggsae and C. tropicalis exhibit androdioecious male characteristics resembling C. elegans ( Sup Fig 1 & 2 ). SRD-1 receptors in these species also show significant polymorphisms in their cytoplasmic domain ( Sup Fig 3 ), suggesting that the observed male traits in C. briggsae and C. tropicalis could also converge on SRD-1. Nonetheless, our study indicates that the evolutionary transition towards self-fertilization in sparse populations 34 – 36 may not be obligatorily dependent on a context of low population density. This evolutionary process might also arise from alterations in chemosensory processing, resulting in behavioral adaptations associated with these androdioecious males. Author Contributions KLC conceptualized and supervised the entire project, while HK performed the experiments, conducted data analysis, and wrote the manuscript in consultation with KLC. Acknowledgements We thank former lab members Gus Chan and Olivia Wan for their GC-MS analysis of dioecious and androdioecious sex pheromone profiles and for constructing the SRD-1 clones used in this study. C. sinica, C. wallecei , and C. tropicalis were kindly provided by Dr. Zhongying Zhao from Hong Kong Baptist University. Other worm strains were sourced from the Caenorhabditis Genetics Center. This project was supported by Research Grants Council, Hong Kong CERG 660508 and 660513. We acknowledge the use of OriginPro, Version 2022b (64-bit), SR1. OriginLab Corporation, Northampton, MA, USA, for data analysis and visualization. Figures were created using BioRender.com. Funder Information Declared Research Grants Council, Hong Kong , CERG 660508 , CERG 660513 References 1. ↵ Fitch D H , B. Bugaj-Gaweda , S.W. Emmons ( 1995 ). 18S ribosomal RNA gene phylogeny for some Rhabditidae related to Caenorhabditis . Mol Biol Evol . doi: 10.1093/oxfordjournals.molbev.a040207 . OpenUrl CrossRef PubMed Web of Science 2. ↵ Blaxter , M.L. , De Ley , P. , Garey , J.R. , Liu , L.X. , Scheldeman , P. , Vierstraete , A. , Vanfleteren , J.R. , Mackey , L.Y. , Dorris , M. , Frisse , L.M. , et al. ( 1998 ). A molecular evolutionary framework for the phylum Nematoda . Nature 392 , 71 – 75 . doi: 10.1038/32160 . OpenUrl CrossRef PubMed Web of Science 3. ↵ Rine , J. , and Kenyon , C. ( 1989 ). 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