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Mating strategies in Caenorhabditis elegans populations are determined by male developmental history

Mating strategies, whether sexual or asexual, confer unique costs and benefits to populations and species that facilitate evolutionary processes. In wild isolates of Caenorhabditis elegans, mating strategies are dependent on developmental history. Outcrossing levels significantly increase when one or both parents have transiently passed through the stress-resistant dauer diapause stage. However, the molecular mechanisms of how life history alters mating strategies have not been systematically explored. Sex-specific responses to pheromones are a major driver of mating behaviors in C. elegans. We demonstrated previously that postdauer hermaphrodites exhibit a decreased avoidance of the pheromone ascr#3 due to the downregulation of the osm-9 TRPV channel gene in postdauer ADL neurons. Thus, we hypothesized that altered responses to pheromones in postdauer animals could contribute to increased outcrossing. We conducted mating assays using wild type N2 Bristol, as well as daf-3/co-SMAD and mut-16/Mutator strains that fail to downregulate osm-9 in postdauer hermaphrodite ADL neurons. First, we show that the outcrossing level of N2 Bristol correlated with the developmental history of males, and that postdauer males exhibited an increased ability to detect mates via pheromones compared to continuously developed males. In addition, DAF-3 plays a critical role in postdauer males to regulate mating, while playing a more minor role in hermaphrodites. Furthermore, the mut-16 strain exhibited negligible outcrossing, and attempts to rescue the outcrossing phenotype resulted in transgenerational sterility due to germline defects. Together, our results suggest a model whereby mating strategy is driven by developmental history under combinatorial control of TGF-β and RNAi pathways.

responses to pheromones in postdauer animals could contribute to increased 23 outcrossing. We conducted mating assays using wild type N2 Bristol, as well as daf-24 3/co-SMAD and mut-16/Mutator strains that fail to downregulate osm-9 in postdauer 25 hermaphrodite ADL neurons. First, we show that the outcrossing level of N2 Bristol 26 correlated with the developmental history of males, and that postdauer males exhibited 27 an increased ability to detect mates via pheromones compared to continuously 28 developed males. In addition, DAF-3 plays a critical role in postdauer males to regulate 29 mating, while playing a more minor role in hermaphrodites. Furthermore, the mut-16 30 strain exhibited negligible outcrossing, and attempts to rescue the outcrossing 31 phenotype resulted in transgenerational sterility due to germline defects. Together, our 32 results suggest a model whereby mating strategy is driven by developmental history 33 under combinatorial control of TGF-β and RNAi pathways. 34

INTRODUCTION 35
Mating strategies, whether sexual or asexual, confer unique costs and benefits to 36 populations and species that facilitate evolutionary processes. The theoretical cost of 37 sex and its disadvantages as a mode of reproduction are far higher than that of asexual 38 reproduction (Otto 2009). Indeed, sexual reproduction has a two-fold cost for the 39 existence of males due to having to share half of the offspring's genome with a mate 40 assays, six L4 stage hermaphrodites and six young adult males were allowed to mate 157 on a single plate (Table S2). The parents were placed on 60mm NGM plates and 158 allowed to mate for 23 hours, followed by removal of males from the plates. 159 Hermaphrodites were then placed on new plates daily until egg-laying ceased and the 160 number of progeny (hermaphrodites and males) per assay was determined. At least 161 three mating assays using biologically independent populations were performed for 162 each condition. 163 The mating frequency was determined using the proportion of males in the F1 164 population after mating. Since males are produced through either mating or 165 nondisjunction events of the X-chromosome, the mating frequency can be determined 166 by subtracting the frequency of X-chromosome nondisjunction in that strain from the 167 overall observed male frequency (mating frequency = ). To determine the X-chromosome nondisjunction 169 frequencies, we conducted brood size assays using at least three biological replicates 170 of each genotype, with 6 hermaphrodites tested for each trial. The proportion of males 171 in the progeny of each trial was averaged across replicates ( Figure S1; Table S3). 172 There was no significant difference between CON and PD male frequencies; thus, CON 173 male frequency values were subtracted from either condition. To compare the mating 174 assay results of the different conditions, a two-way ANOVA followed by Tukey's multiple 175 comparisons test was performed using GraphPad Prism version 8.4.3 for macOS, 176 GraphPad Software, San Diego, California USA. 177 178 Measuring osm-9 expression levels 179 The expression levels of osm-9 were measured using an integrated transgene 180 array expressing GFP under the regulation of 350 bp of osm-9 upstream regulatory 181 sequences (pdrIs1) as previously described (Sims et al. 2016). The expression of GFP 182 was determined in control and postdauer adults for wild-type and mut-16(mg461) strains 183 24 hours after the L4 larval stage. Animals were dye-filled using DiD to identify the 184 location of ADL and mounted on agar pads with sodium azide. GFP expression was 185 visualized using a Leica DM5500B microscope with a Hamamatsu 741 camera 186 controller C10600 ORCA-R2. At least 44 animals over a minimum of three biologically 187 independent trials were examined for each condition and genotype. Statistical analysis 188 was performed using multiple t-tests followed by Holm-Sidak correction within 189 One-day old adult, control and postdauer males and hermaphrodites were tested 193 for avoidance behavior in response to 100 nM ascr#3 as previously described (Hilliard 194 et al. 2002;Jang et al. 2012;Sims et al. 2016). Avoidance of 1M glycerol and M13 195 buffer was tested as the positive and negative controls, respectively. Briefly, animals 196 were picked onto a 60mm NGM plate without food and allowed to acclimate for 10 197 minutes. The drop test acute avoidance response assay was performed using a mouth 198 pipette with a pulled glass needle. An avoidance response was recorded when a 199 forward-crawling animal reversed within two seconds after being exposed to the 200 stimulus. The proportion of animals responding to ascr#3 was normalized by subtracting 201 the proportion of animals responding to the negative control (M13 buffer) as previously 202 described (Sims et al. 2016 For the brood size assays, larval L4 hermaphrodites were placed on seeded 207 NGM plate and transferred to new plates daily until egg-laying ceased, and the number 208 of progeny per plate was determined. For the transgenerational brood size assay, the 209 mut-16 expression transgene was injected into mut-16(pk710) and N2 strains 210 independently, and strains were propagated for more than ten generations before being 211 used in experiments. A red fluorescent co-injection marker that is easily visualized was 212 used when generating these mut-16 overexpression strains; thus, array+ refers to 213 progeny that inherited the extrachromosomal mut-16/dsRed transgene array and array-214 refers to the animals that did not inherit the array (Bharadwaj and Hall 2017). Ten 215 hermaphrodites carrying the extrachromosomal array overexpressing mut-16 pan-216 neuronally were used to establish the parent plate (P0). Ten hermaphrodite progeny 217 (either array+ or array-) were then transferred to a new NGM plate to establish F1 and 218 14 tester animals over 18 trials were examined for this assay. To test if hermaphrodites 265 had an increased attraction to postdauer males, the quadrant assay was used with CON 266 or PD unc-54; him-5 males alternating as the targets. CON hermaphrodites were used 267 as the tester animals. A total of 160 animals over 16 trials were tested. 268 To examine ascr#3 attraction, the quadrant assay was performed using ascr#3 269 and ethanol diluted in water as the alternating targets, and CON and PD males as the fertilization of oocytes by sperm that lack the X-chromosome (Hodgkin et al. 1979). 289 Thus, the outcrossing frequency was calculated using the proportion of male progeny 290 resulting from the mating assay minus the X-chromosome nondisjunction rate as 291  compared to the mating assays using CON adults (p = 0.012) ( Figure 1A). We found 300 that the type of males (CON or PD), but not the type of hermaphrodites, had a 301 significant effect on outcrossing levels in N2 Bristol (p = 0.0016, two-way ANOVA) 302 (Table S4), indicating that the developmental history of males is the major determinant 303 of outcrossing in wild-type populations. However, when comparing the outcrossing 304 levels from assays where only males experienced dauer to the assays where neither 305 parent or both parents experienced dauer, no significant differences were observed 306  Table S2. Additional data are provided in Table S4. (B) Mean percentage ± S.E.M. of ADL neurons expressing GFP in N2 and mut-16(mg461) strains. n ≥ 44 animals from at least 3 independent trials. **** p < 0.0001, multiple t-tests followed by Holm-Sidak correction. n.s., not significant. (C) Normalized mean ascr#3 avoidance ± S.E.M. for hermaphrodites and males in N2 and mut-16(pk710) strains. n ≥ 93 animals from at least 3 independent trials. **** p < 0.0001, multiple t-tests followed by Holm-Sidak correction. Additional data are provided in Table S5. Expression of osm-9 is regulated in a sex-specific manner 315 Since our original hypothesis was that variable osm-9 expression altered 316 pheromone responses resulting in increased postdauer outcrossing rates, we next 317 examined the regulation of osm-9 in males. Using a strain carrying an integrated GFP 318 reporter transgene driven by ~350 bp of the osm-9 promoter (osm-9p::gfp), we 319 quantified the number of ADL neurons that express GFP in control and postdauer wild-320 type hermaphrodites and males. As expected, we observed that significantly fewer 321 postdauer hermaphrodites have osm-9p::gfp expression in ADL compared to controls 322 ( Figure 1B). However, in males, GFP is expressed similarly in control and postdauer 323 adult ADL neurons, indicating that regulation of osm-9 by developmental history is sex-324 specific ( Figure 1B). We showed previously that hermaphrodites carrying a mutation in exposure to ascr#3 did not elicit a difference in avoidance behavior in postdauer males 345 compared to control males ( Figure 1C). Together, these results indicate that the 346 differential regulation of osm-9 in ADL neurons due to passage through the dauer stage 347 is hermaphrodite-specific and is unlikely to be a significant driver of outcrossing in 348 postdauer males. that RNAi is also required for increased outcrossing in postdauer populations by 354 performing mating assays using the mut-16(pk710) mutant strain. In the mating assay of 355 mut-16 control hermaphrodites and control males, we found an average male progeny 356 frequency similar to what was observed for wild type (0.129 ± 0.028) (Figure 2A). 357 However, unlike wild type, we found that the male progeny frequencies from all mating 358 assay combinations of mut-16 control and postdauer adults were not significantly 359 different and were similar to the baseline outcrossing levels (Figure 2A). These results 360 indicate that MUT-16 is required for the increased outcrossing phenotype in postdauer 361

populations. 362
In previous work, we demonstrated that mut-16 animals were partially dauer 363 deficient in the presence of high pheromone levels due to decreased expression of 364 genes encoding G proteins required for neuronal signaling. In addition, we were able to 365 rescue the dauer deficient larval phenotype by expressing a high-copy transgene array 366 carrying wild-type mut-16 coding sequence either pan-neuronally or in individual, 367 pheromone-sensing neurons (Bharadwaj and Hall 2017). To determine if MUT-16 was 368 also functioning in neurons to regulate mating behaviors, we performed the mating 369 assay using a mut-16 strain carrying a mut-16 expression transgene driven by a pan-370 neuronal promoter (rab-3p). Interestingly, we found the frequencies of male progeny in 371 the mut-16 pan-neuronal overexpression strain mating assays were suppressed below 372 baseline outcrossing in mut-16 mutants, particularly for the mating assay including CON 373 hermaphrodites with PD males (Figures 2A and 2B). Next, we attempted to perform 374 mating assays using mut-16 strains that carry overexpression transgene arrays driven 375 by single-neuron promoters. We examined the male progeny frequencies of mut-16 376 strains carrying the mut-16 transgene expressed only in ADL neurons (sre-1p) and 377 found that this strain also exhibited suppressed outcrossing below baseline, especially 378 for mating assays including PD males ( Figure 2C). We observed similar suppressed 379  Table S2. Additional data are provided in Tables S6-S10. outcrossing levels for the mut-16 strains expressing the transgene in the pheromone-381 sensing ASI neurons (gpa-4p) ( Figure 2D). However, mut-16 overexpression in ADF 382 neurons (srh-142p) did not result in as severe of mating suppression as the other 383 neurons tested in assays that included postdauer hermaphrodites ( Figure 2E). These 384 results support the conclusion that mut-16 expression is required at endogenous levels While performing the outcrossing assays with the mut-16 pan-neuronal and 391 individual neuron overexpression strains, we observed that they exhibited a decrease in 392 total progeny compared to wild-type and mut-16 strains. To investigate if their 393 suppressed outcrossing levels were due to germline defects, we first examined the 394 ability of the mut-16 neuronal overexpression strain hermaphrodites to produce self-395 fertilized progeny. We conducted brood size assays of CON and PD hermaphrodite 396 adults with N2 Bristol, mut-16(pk710), and two independent lines of the mut-16 strain 397 carrying an extrachromosomal array of pan-neuronal mut-16 expression transgene. 398 First, we observed that wild-type postdauer hermaphrodites showed a significantly 399 reduced brood size compared to controls, and that this difference is eliminated in the   Table S2. Additional data are provided in Tables S11-S13. fecundity is more severe in animals retaining the array compared to mut-16(pk710) 457 animals ( Figure 3A), we expected to observe a correlation of gonad morphology defects 458 with array+ animals that were not present in the array-counterparts. To test this 459 hypothesis, we examined the somatic gonad and germline morphology for defects in the 460 F1, F3, and F5 generations of mut-16 pan-neuronal overexpression strain 461 hermaphrodites that either retained (array+) or lost (array-) the extrachromosomal array. 462 As reported previously, the proportion of mut-16(pk710) animals displaying overall 463 gonad defects was significantly higher than that of N2 Bristol, including DTC migration 464 defects, decreased sperm number, and thin distal gonads ( Figures S3 and S4). 465 However, for small gonad defects, we detected a trend in the proportion of animals 466 exhibiting this defect and the observed brood sizes of these populations. Interestingly, 467 the array+ populations showed an increasing proportion of defects between the F1 and 468  To further characterize how the neuronal overexpression of mut-16 contributes to 473 decreased outcrossing, we examined whether small gonad defects were correlated with 474 fewer germ cells available for reproduction. Using DAPI-stained whole hermaphrodites, 475 we quantified the number of germ cells per gonad arm of array+ and array-progeny for 476 F1, F3, and F5 generations and compared it to wild-type and mut-16(pk710) strains. As 477 expected, we found that mut-16(pk710) hermaphrodites exhibited a significant decrease 478 in germ cell counts compared to wild type ( Figure 3F). Furthermore, we observed that 479 array+ generations exhibited a further decrease in total germ cells compared to mut-16 480 adults, and the array-animals exhibited an intermediate germ cell number between the 481 mut-16 and array+ populations ( Figure 3F). Upon further scrutiny, we found that the 482 differences in total germ cell number appear to be due to a decrease in cells in the 483 mitotic and pachytene regions of the germ line ( Figures S5 and S6) We previously showed that the osm-9 TRPV channel gene is regulated by the 493 TGF-β pathway in ADL neurons of postdauer hermaphrodites. Specifically, the DAF-494 3/co-SMAD transcription factor binds to the osm-9 promoter to promote its 495 downregulation after dauer, and mutations in daf-3 result in continued osm-9 expression 496 and ascr#3 avoidance in postdauer hermaphrodites (Sims et al. 2016). Thus, we tested 497 whether the increased outcrossing levels in postdauer populations were dependent 498 upon TGF-β signaling by performing mating assays using a daf-3(mgDf90) mutant 499 strain. For mating assays of daf-3 CON hermaphrodites and CON males, we observed  Table S2. Additional data provided in Tables S14-S16.
daf-3 mating assays included postdauer males, postdauer hermaphrodites, or both, the 504 male progeny frequencies were not significantly different from baseline ( Figure 4A; 505 Table S14). These results suggest that DAF-3 and the TGF-β signaling pathway may 506 play a minor role in the increased outcrossing in postdauer populations. Since osm-9 is 507 only downregulated in postdauer hermaphrodite ADL neurons, we next asked whether 508 DAF-3 was required in males or hermaphrodites to promote postdauer outcrossing. We 509 predicted that the continued expression of osm-9 and ascr#3 avoidance in postdauer 510 daf-3 hermaphrodites would decrease outcrossing. To test our hypothesis, we 511 examined male frequencies in the progeny of populations where only one sex carrying a 512 mutant allele of daf-3 was mated with wild-type animals of the other sex. In populations 513 where daf-3 males were mated to wild-type hermaphrodites, we found that the trends in 514 outcrossing were the opposite compared to wild type. Mating assays that included PD 515 males showed a trend of decreased male progeny frequencies compared to mating 516 assays that included CON males. Interestingly, mating assays that included PD wild-517 type hermaphrodites and CON daf-3 males exhibited significantly higher male progeny 518 frequencies compared to mating assays with CON wild-type hermaphrodites and PD 519 daf-3 males (p = 0.016; Figure 4B). These results are consistent with a requirement for 520 DAF-3 in postdauer males to promote outcrossing. 521 We further investigated a potential role of DAF-3 in hermaphrodites by examining 522 the male progeny frequency in mating assays of daf-3 hermaphrodites with wild-type 523 males. If hermaphrodites did not play a significant role in outcrossing levels, we would 524 expect that male progeny frequencies of these assays would be similar to wild type. 525 Instead, we observed that all combinations of CON and PD populations did not show 526 significant differences in male progeny frequencies ( Figure 4C). However, the mating 527 assays including PD wild type males trended towards increased male progeny 528 frequencies compared to assays with CON males similar to the daf-3 mating assays, 529 suggesting a more minor role for DAF-3 in hermaphrodites (Figures 4A and 4C). is a significant contributor to outcrossing rates, with hermaphrodites playing a more 538 minor role. We hypothesized two non-mutually exclusive mechanisms to explain how 539 postdauer males contribute to outcrossing levels. First, postdauer males may be more 540 efficient at finding mates than control males. A second possibility is that postdauer 541 males may produce a novel pheromone component that functions as an attractant to 542

hermaphrodites. 543
To test these possibilities, we first performed mate preference assays using 544 genetically immobilized hermaphrodites to determine male preference and their ability to 545 locate mates (Fagan et al. 2018). Each quadrant contained immobilized control 546 hermaphrodites that can (Phe ON : unc-54) or cannot (Phe OFF : unc-54; daf-22) secrete 547 pheromones into their environment. In each assay, either control or postdauer wild-type 548 males were placed in the center of the plate and scored for their location in the 549 32 551 552 quadrants after 30, 60, and 90 minutes ( Figure 5A). Both control and postdauer males 553 exhibited preference towards hermaphrodites that were able to secrete pheromones 554 ( Figure 5B). Interestingly, we observed that postdauer males were better able to locate 555 the pheromone-producing hermaphrodites compared to control males ( Figure 5B). 556 These results support the hypothesis that postdauer males are more efficient at locating 557 mates compared to control males. To test if the increased postdauer male efficiency at 558 locating mates was facilitated by ascr#3 detection, we also conducted chemotaxis 559 quadrant assays with 10 nM and 100 nM ascr#3 (Fagan et al. 2018). Males were tested 560 for their preference to four quadrants containing either ascr#3 or ethanol ( Figure 5C). 561 Postdauer males exhibited a slight, but statistically insignificant, increase in chemotaxis 562 towards both 10 nM and 100 nM ascr#3 concentrations compared to control males 563 ( Figure 5D). This result was not surprising given that males are attracted to a blend of 564  Table S17. pheromones secreted by hermaphrodites, and ascr#3 detection may be just one 565 contributor to the increased mate finding efficiency of postdauer males. 566 To test the possibility that postdauer males secrete a pheromone for 567 hermaphrodite attraction, we again performed the mate preference quadrant assays 568 using immobilized (unc-54) control or postdauer males in each quadrant. The location of 569 control wild-type hermaphrodites was scored every 30 minutes, up to 90 minutes 570 ( Figure 5E). In contrast to the assay for male preference, hermaphrodites did not exhibit 571 any preference between control and postdauer males ( Figure 5F). Together, these 572 results suggest that postdauer males promote outcrossing through their increased 573 ability to locate potential mates using pheromone cues. 574

DISCUSSION 576
Phenotypic plasticity plays a significant role in regulating reproductive modes. 577 Here, we showed that the change in reproductive strategies to increase mating in C. 578 elegans following dauer diapause requires sexually dimorphic changes in behavior. 579 First, the developmental history of males was the major contributor to outcrossing 580 levels, potentially due to the increased ability of postdauer males to detect mates using 581 pheromone cues compared to control males. In addition, we uncovered a minor role for 582 the developmental history of hermaphrodites in regulating mating levels. These 583 phenotypes depend on RNAi and TGF-β pathways, which potentially act to regulate 584 pheromone production and detection. Furthermore, we demonstrate that 585 overexpression of MUT-16 pan-neuronally or in individual neurons negatively regulates 586 hermaphrodite fecundity by decreasing germ cell number transgenerationally. Together, 587 our results illustrate some of the genetic determinants required for increased 588 outcrossing behaviors in postdauer populations. 589 590

Contributions of males and hermaphrodites towards outcrossing frequency 591
Our original hypothesis driving this work was that downregulation of osm-9 592 expression in postdauer hermaphrodite ADL neurons facilitated the opportunity for 593 increased mating due to their failure to avoid high concentrations of ascr#3. However, 594 our results indicated that the developmental history of males was the primary 595 determinant of outcrossing rates, with hermaphrodite life history playing a more minor 596 role ( Figure 1A). C. elegans males balance their drive for reproduction and survival by 597 alternating between foraging (food-seeking) and exploration (mate-seeking) behaviors. 598 foraging and exploration behaviors more efficiently or more frequently than control 603 males, resulting in the increased outcrossing rate we observed (Gray et al. 2005). This