The Regulation of Developmental Diapause is Coordinated with Molting and Olfaction

Developmental and behavioral plasticity allow animals to prioritize alternative genetic programs during fluctuating environments. Behavioral remodeling may be acute in animals that interact with host organisms, since reproductive adults and the developmentally arrested larvae often have different ethological needs for chemical stimuli. To understand the genes that coordinate development and behavior, we used the nematode model Pristionchus pacificus to characterize mutants that inappropriately enter developmental diapause to become dauer larvae (Daf-c). We found several key olfactory differences between P. pacificus and C. elegans Daf-c dauers. In addition, the two P. pacificus Daf-c alleles disrupt steroid synthesis required for proper regulation of the conserved canonical steroid hormone receptor DAF-12, whose dauer-constitutive and cuticle exsheathment phenotypes can be rescued by the feeding of Δ7-dafachronic acid. One allele, csu60, has a deletion in the sole HydroxySteroid Dehydrogenase (HSD) in P. pacificus. Both hsd-2(csu60) adults and dauers show enhanced attraction to a beetle pheromone, possibly due to the heterochronic activation of dauer-specific neuronal development in the adults. Surprisingly, this enhanced odor attraction acts independently of daf-12, revealing unexpected targets of steroid hormones regulating ecdysis and olfaction in P. pacificus. Author Summary The remarkable evolutionary success of nematodes can be attributed to their dispersal ability as stress-resistant dauer larvae and the equivalent parasitic infective larvae. The decision to enter dauer development is regulated by a conserved steroid hormone receptor that receives multiple external and internal cues, though the extent in which these cues also coordinate other physiological and behavioral processes is not well understood in divergent species. We used the insect-associated nematode Pristionchus pacificus to genetically dissect two mutants that form dauers inappropriately, and found that one mutation in a steroidogenic enzyme, Ppa-hsd-2, is predicted to abolish the biosynthesis of the hormones needed to negatively regulate dauer entry when food is available. Unexpectedly, Ppa-HSD-2 is also required to properly complete the dauer larval molt, known as exsheathment, as well as to confer differences in adult versus dauer larvae chemotaxis behavior towards a host pheromone. Given that dauers are the host-seeking stage of parasitic nematodes, hormonal disruption represents a tenable target for biological control.


Introduction
pheromone methyl myristate (Fig. 3A). Given this wild-type chemosensory 256 response by the Daf-c DL, we expanded the survey of DL chemosensation to the 257 other insect pheromones E-TDA, hexadecanal, and ß-caryophellene but found that 258 the Daf-c DL show no response to these odorants (SI Fig. 3) [39,66,67]. We also 259 examined the responses of Daf-c DL to a panel of C. elegans attractants: 2,3-260 butanedione (also called diacetyl), 2,3-pentanedione, and isoamyl alcohol (IAA) 261 [24,39]. We found that the DL do not respond to these chemicals; P. pacificus 262 adults also do not respond (SI Fig. 3). Thus, the Daf-c DL exhibited near wild-type 263 responses to two known host-associated odors but have a narrower overall odor 264 response profile compared to wild-type adults. It remains to be determined the 265 degree of concordance between wild-type and Daf-c DL chemosensory profiles. 266 To better define the genetic pathways that are responsible for the 267 chemosensory differences between DL and adults, we focused on the responses 268 of the Daf-c mutants to the Oriental beetle pheromone ZTDO [68]. We observed 269 that wild-type PS312 showed the strongest difference between the DL and adults, 270 with an almost threefold stronger attraction in DL than in adults (Fig. 3B). We also 271 observed significantly stronger attraction by the adults of both Daf-c alleles, such 272 that there were no significant differences between the responses of DL and adult 273 stages. The csu60 adults were significantly more attracted to ZTDO than both wild-274 type and tu391 adults, and despite the dye-filling defect in tu391 being more 275 pronounced in adults compared to DL, the attraction to ZTDO is stronger in tu391 276 adults than in wild-type adults. Because the passage through the dauer stage may 277 introduce lasting post-dauer changes that alter odorant receptor expression and 278 hence modulate behavior [7,69,70], we also examined the ZTDO response of wild-279 type post-dauer adults. We found that dauer passage did not result in enhanced 280 ZTDO response in adults (Fig. 3C). Thus, the enhanced ZTDO attraction in Daf-c 281 adults was not due to developmental experience, but rather the heterochronic 282 activation of a dauer-specific neuronal development. 283 To determine how the Daf-c mutants interact with chemosensory mutants 284 mediating the ZTDO response, we examined the chemotaxis behavior of the Daf-285 c mutants in the loss-of-function obi-1(tu404) background that is defective for its 286 cGMP-dependent responses to ZTDO [68]. We found that the obi-1 background 287 did not affect ZTDO attraction in both tu391 adults and DL (Fig. 3B). In contrast, 288 the loss of obi-1 significantly reduced the ZTDO response in csu60 adults as well 289 as DL, from strong enhanced attraction to weak repulsion and neutrality, 290 respectively (Fig. 3B). We were unable to perform chemotaxis assays on the DL 291 of obi-1(tu404) mutants due to its high DL mortality rate, especially in the presence 292 of ZTDO [68]. Interestingly, while Ppa-daf-12 is epistatic to csu60 for dauer 293 formation, csu60; daf-12(tu389) double mutant adults still exhibited the csu60 294 enhanced attraction to ZTDO (Fig. 3D). Thus, the regulation of chemotaxis 295 responses can be uncoupled from dauer formation and requires a steroid hormone 296 that does not act through a DAF-12 homolog. Taken together, our results suggest 297 that tu391 acts downstream of or in parallel with obi-1, while csu60 likely acts 298 upstream of obi-1 and independent of daf-12 to mediate the enhanced pheromone 299 attraction of young adults.
Since host odor attraction in IJs of entomopathogenic nematodes is 301 enhanced by carbon dioxide (CO2) [21], we next tested the chemotaxis response 302 of P. pacificus DL to CO2. In C. elegans, well-fed adults are repelled by CO2 while 303 DL are attracted to the same concentration [20,21]. In P. pacificus well-fed adults, 304 CO2 also elicits a strong avoidance response which, as in C. elegans, is mediated 305 by a pair of internal gas sensing neurons known as the BAG neurons [20]. To 306 determine if CO2 response valence also differs between P. pacificus DL and adults, 307 we examined the response to CO2 in Daf-c DL and adults using the modified 308 chemotaxis assay. We found that wild-type P. pacificus DL exhibited a slightly 309 stronger avoidance response than adults, while both Daf-c DL and adults showed 310 equally robust repulsion by CO2 (Fig. 3E). Thus, similar to the enhanced response 311 to the beetle host pheromone, wild-type P. pacificus DL displayed a slightly 312 enhanced negative response to CO2, not a valence change. Interestingly, iL3s of 313 the mammalian parasites S. stercoralis, S. ratti, and Nippostrongylus brasiliensis 314 are also repelled by CO2 [22]. It is currently unclear how the CO2 avoidance 315 response of P. pacificus DL, but not C. elegans DL, factors into the different life 316 strategies of these two species in the wild. 317 Finally, to determine if genes that affect dauer development also modulate 318 olfactory behavior in other nematodes species, we also compared the odor 319 response profiles of young adults vs. DL in C. elegans [71], including the odor 320 response profiles of two well-studied Daf-c mutants, daf-2(e1370) and daf-321 7(e1372, ok3125) [56,72]. We found that DL of wild-type N2 and both the Daf-c 322 mutants showed weaker odorant responses than adults. Specifically, wild-type DL showed lower attraction than adults to 2,3-butanedione, 2,3-pentanedione, and 324 IAA, consistent with the results of a previous study that included 2,3-butanedione 325 [27] (Fig. 4A-C). Similar to the finding in P. pacificus, we also found dauer passage 326 did not result in enhanced IAA or 2,3-pentanedione attraction in post-dauer adults 327 (SI Fig. 4). Most unexpectedly, the response to IAA exhibited valence changes 328 between adults and DL: it changed from attractive to neutral in wild-type animals, 329 and from attractive to repulsive in both daf-7 mutants. To evaluate if the avoidance 330 response to IAA is due to hypersensitivity in the daf-7 DL, we also tested a 10-fold 331 lower IAA concentration (0.1%) and found that IAA attraction was still significantly 332 reduced in both daf-7 alleles compared to wild type (Fig. 4D). Thus, C. elegans 333 wild-type and daf-2 DL showed decreased attraction compared to adults to all three 334 odorants tested, but more dramatically, daf-7 alleles showed a valence change by 335 accentuating the avoidance response of DL to IAA, suggesting that the TGF-ß 336 pathway is involved in both dauer regulation and IAA sensing. 337 338

Molecular cloning of a Daf-c mutant 339
In C. elegans, the binding of the nuclear hormone receptor DAF-12 to the steroid 340 hormones ∆4-dafachronic acids and ∆7-dafachronic acids (DAs) restrains L2 341 larvae from undergoing the dauer fate [51]. To determine if csu60 or tu391 act 342 upstream of Ppa-daf-12, we tested a synthetic DA for its ability to ameliorate the 343 Daf-c phenotype. Previous work has shown that the constitutive dauer formation 344 phenotype of dfc-1(tu391) is rescued by feeding the worms E. coli OP50 containing 345 7.5 µM ∆7-dafachronic acid (∆7-DA) [8]. ∆7-DA has been shown to prevent dauer 346 formation in both P. pacificus and the ruminant parasite Strongyloides papillosus 347 [8]. We found that both the dauer-specific exsheathment defect as well as the 348 constitutive DL phenotype of dfc-1(tu391) and csu60 were rescued by the 349 availability of ∆7-DA in the food ad libitum (Fig. 5A). These results suggest that 350 neither mutant produces the P. pacificus dafachronic acid that is functionally 351 equivalent to the dauer-suppressing ∆7-DA. 352 To further confirm this genetic pathway for dauer regulation by epistasis 353 analysis, we constructed csu60; daf-12(tu389) double mutants and found that they 354 resembled the dauer formation defective phenotype of daf-12(tu389) [8] (Table 1), 355 consistent with the expected position of csu60 acting upstream of the nuclear 356 hormone receptor (see Methods). Similarly, the dfc-1(tu391); daf-12(tu389) double 357 mutants completely masked the Daf-c phenotypes of tu391, placing tu391 358 upstream of daf-12 for the regulation of dauer formation. Hence, daf-12 is epistatic 359 to both csu60 as well as tu391. A small percentage (3.7%) of the csu60; daf-360 12(tu389) double mutants exhibited an ecdysis defect in the molts between late 361 larval stages (J3-J4; J4-adult), which is a synergistic phenotype not found in either 362 single mutant (Table 1). These results suggest that both mutations disrupt the 363 steroid biosynthetic and signaling pathways critical for dauer formation and 364 possibly molting. 365 Using the latest genome assembly of P. pacificus El Paco, we performed 366 whole genome sequencing of csu60 and identified a ~95 kb deletion on 367 Chromosome II that contains 11 predicted protein-coding genes. Among these 368 candidates, the predicted protein sequence of PPA10139 is orthologous to the 3b-369 hydroxysteroid dehydrogenase/∆5-∆4 isomerases (3b-HSDs) that are key 370 steroidogenic enzymes in vertebrates (GO:0003854). PPA10139 shares 89% 371 similarity with C. elegans HSD-2, which encodes one of three hydroxysteroid 372 dehydrogenases in C. elegans involved in the biosynthesis of dafachronic acids 373 [73]. Consistent with its potential role in repressing dauer formation, PPA10139 374 transcripts are significantly lower in DL than in the J2, J3, J4, and adult stages 375 (gene prediction Contig6-snapTAU.237) [74]. To confirm that the deletion of Ppa-376 hsd is solely responsible for the Daf-c phenotype of csu60, we made a construct 377 containing a ~2 kb promoter of PPA10139 driving its full-length cDNA to rescue indicating that a single HSD is likely the ancestral state, whereas the C. elegans 387 genome contains three copies (Fig. 6A). Each hsd gene in C. elegans differs by 388 the number of transmembrane domains: hsd-1 encodes for two predicted domains, 389 hsd-2 encodes for one domain, while hsd-3 does not encode for any predicted 390 transmembrane domains (as predicted by SMART, which utilizes TMHMM to 391 predict transmembrane domains). Although the overall protein sequence of PPA10139 shares the highest similarity to C. elegans HSD-2, the two 393 transmembrane domains predicted for Ppa-HSD-2 argues for more functional 394 similarity to C. elegans HSD-1 (Fig. 6B). 395 We hypothesized that if Ppa-HSD-2 is the only hydroxysteroid 396 dehydrogenase in P. pacificus involved in dafachronic acid synthesis necessary 397 for suppressing dauer formation, then the removal of the cholesterol precursor 398 should not exacerbate the severity of the Daf-c phenotype in the presence of food. 399 In both C. elegans and P. pacificus, 5 µg/ml cholesterol (~13 µM) is routinely added 400 to NGM medium [75,76], whereas the removal of this exogenous cholesterol for 401 two generations leads to constitutive dauer formation in the presence of food [50]. 402 Indeed, we observed that wild-type P. pacificus form 68% active DL at 25°C when 403  Utilizing whole genome sequencing of tu391 and mapping lines, as well as 411 previous fine mapping data [8], we confirmed that the mutation responsible for the transcripts are also expressed at significantly lower levels in DL than in J2 and J3 419 larvae [74]. However, given the complexity of the PPA21795 exon-intron structure 420  To confirm that the increased Ppa-odr-3 promoter activity and ectopic cellular 443 expression correspond to increased Ppa-odr-3 mRNA transcripts in csu60, we 444 performed qPCR on mix-stage cultures and found that Ppa-odr-3 transcripts are 445 indeed ~10 fold higher in csu60 than in wildtype (SI Fig. 7). 446 To determine if the duplicated Ppa-odr-3p::rfp expression approximated the 447 frequency of Daf-c DL at 20°C, we also examined earlier developmental stages. In 448 P. pacificus, the J1 larva undergoes a pre-hatching larval molt inside the eggshell, 449 so the first active post-hatching stage is the J2 [77,78]. Surprisingly, ~41% of J2 450 larvae and ~14% DL in csu60 mutants, but 0% in wild type, also expressed ectopic 451 Ppa-odr-3p::rfp in a duplicated pair of amphid neurons ( Table 2 higher RFP expression in csu60 dauers, we observed the dual ciliated dendritic 458 ends of the AM3(AWA) neurons, which was not visible in wild-type dauers [60]. regulates the conserved nuclear hormone receptor DAF-12 ( Fig. 8). In C. elegans, 478 orthologous mutations in the HSD homologs on their own do not cause constitutive 479 dauer or molting defects, which suggests that steroid hormone signaling in P. 480 pacificus represents an ancestral state more in common with parasitic nematodes 481 than with C. elegans. Most notably, the complete loss of Ppa-hsd-2 resulted in host 482 pheromone attraction by adults in a daf-12-independent manner, and is 483 accompanied by early neuronal fate changes that lead to ectopic Ppa-odr-3 G-protein expression in a pair of chemosensory amphid neurons. By testing two well-485 studied Daf-c mutants in C. elegans, we found that Daf-c alleles lowered the 486 already reduced wild-type DL response to adult attractants. This is in stark contrast 487 to the P. pacificus Daf-c alleles, which did not affect the response of DL but instead 488 increased the attraction of adults to host odorants. Thus, regulators of dauer 489 formation affect the olfactory system in developmentally distinct manners for 490 divergent species of nematodes. 491 492

Dafachronic acids control cuticle exsheathment and dauer entry 493
Retention of the previous cuticle, or ensheathment, is a hallmark of the infective 494 larvae of many parasitic nematodes as well as insect larvae. In insects, the pharate 495 larva forms a new cuticle while still within, but separated from, the old cuticle of the 496 previous stage [79]. In nematodes, the exsheathment of infective larvae in host cues for exsheathment [53]. In the pathogenic nematode of sheep, 505 Haemonchus contortus, exogenous application of an endogenous ∆7-DA 506 promotes the exsheathment of infective L3 larvae [10]. Although there are no candidate genes known to control dauer exsheathment in C. elegans-because its 508 dauers are 'naked'-conserved genes involved in molting may nevertheless 509 modulate the timing of cuticle shedding. Only one uncloned Daf-c allele, daf-510 13(m66), has been reported to display a dauer-specific cuticle defect [83]. 511 However, we could not observe either the Daf-c phenotype or the cuticle defect 512 from a revived stock. In all four C. elegans molts, the Zona-Pellucida domain 513 protein FBN-1 and integrins make up a part of the sheath between the old and new 514 cuticles [84]. We speculate that a similar mechanism may be involved in P. The ectopic expression of Ppa-odr-3p::rfp in csu60 J2 animals shows that steroid 524 hormones in P. pacificus are also responsible for maintaining proper cell fate in 525 putative olfactory neurons at or prior to the decision for dauer entry in J2 animals. 526 The homeotic transformation of the AWB to AWC neurons due to mutations in the 527 LIM-4 transcription factor was accompanied by changes in dendritic morphology 528 and chemotaxis behavior [85]. Based on a similar rationale, the loss of Ppa-hsd-2 529 could transform AM10(ASI) or AM6(ASG), which are normally involved in inhibiting dauer entry in C. elegans, into AM3(AWA) olfactory neurons. Coincidentally, AWA 531 and ASG are sister cells in the C. elegans cell lineage (www.wormatlas.org). 532 However, we did not observe fewer Ppa-che-1p::rfp expression in the amphid 533 neurons of Ppa-hsd-2(csu60) animals, as would be expected if one pair of the 534 neuron mis-expressing Ppa-odr-3p::rfp was derived from the AM6(ASG) or 535 AM5(ASE) neurons [60] (SI Fig. 6). Given the dynamic transcriptional profiles 536 associated with the dauer stage in P. pacificus [86], Daf-c mutants have the 537 potential to identify unknown molecular pathways involved in dauer commitment 538 outside of C. elegans. 539 540

Tributaries of DAF-12 flow into differing gene regulatory networks 541
Microarray and RNA-seq studies have shown substantial divergence of 542 dauer-specific genes between P. pacificus and C. elegans, as well as more 543 upregulated dauer-specific genes in P. pacificus than in C. elegans [74,86]. In P. 544 pacificus, the DAF-12/∆7-DA module is a shared regulatory target for both dauer 545 entry and mouth form dimorphism [37,87]. The dye-filling defect in tu391 J2 and 546 adults, but not in the DL, suggests that defects in the amphid neurons, possibly 547 those responsible for negatively regulating dauer entry, are remodeled during the 548 dauer stage. The C. elegans daf-19 gene, which encodes the RFX family of 549 transcription factors, is required for sensory neuron cilium formation and is one of 550 the few mutations that results in both Daf-c as well as dye-filling defective 551 phenotypes [62][63][64]. Although dfc-1(tu391) shares the Daf-c and dye-filling 552 defective (in the non-dauers), dfc-1(tu391) differs from C. elegans daf-19 mutants 553 in that they do not exhibit amphid sheath defects [88] and lack a chemosensory 554 defect. Moreover, mutations in the P. pacificus daf-19 ortholog did not result in a 555 dauer formation defect but did show a dye-filling phenotype [47]. The particular 556 combination of phenotypes in dfc-1(tu391) has no obvious candidates. 557 In C. elegans, genetic dissection of the relative contribution of the 558 steroidogenic enzymes is confounded by the unknown roles the HSD-2 and HSD-559 3 paralogs play in dafachronic acid biosynthesis. HSD-1 is a component in one of 560 the two branched pathways that includes the cytochrome P450 DAF-9 required for 561 dafachronic acid biosynthesis [89]. While HSD-1 and DAF-9 use cholesterol 562 precursors to synthesize ∆4-DA, the Rieske-like oxygenases DAF-36 and DAF-9 563 synthesize ∆7-DA, and together the dafachronic acid hormones converge to 564 promote reproductive development by binding to the DAF-12 receptor in the 565 inactive state [15,73,89]. Mutations in hsd-1 contribute to a nearly fully penetrant 566 Daf-c synthetic phenotype only when combined with a second mutation in NCR-567 1/cholesterol transporter, DAF-36/Rieske-like oxygenase, or DAF-28/insulin [73]. 568 In P. pacificus, the nearly fully penetrant constitutive dauer phenotype of the csu60 569 null allele at 25ºC suggests that the absence of Ppa-hsd-2, rather than a 570 temperature sensitivity of its gene product, can lead to an almost complete de-571 repression of dauer formation in a daf-12-dependent manner. Because the DNA 572 lesion for tu391 is not yet known, it is difficult to speculate if the tu391 mutation 573 acts in the same pathway as Ppa-hsd-2. With only a single hsd in the P. pacificus 574 genome, future studies will likely reveal the degree of functional overlap the P. pacificus ncr-1, daf-36, and daf-9 orthologs have with Ppa-hsd-2 in dauer 576 regulation and olfactory behavior. 577 578

C. elegans dauers have attenuated responses to adult odors 579
To achieve organismal plasticity, it is has been shown that C. elegans dauer 580 entry involves the remodeling of glia and neural circuitry, as well as differential 581 expression of chemosensory and neuropeptide receptors [3,4,6,7,19]. While 582 dauer-induced remodeling of the AWC dendritic ends that increase their surface 583 area could heighten sensitivity, our results show that wild-type, daf-2, and daf-7 584 DL were actually less attracted than adults to several odors, and the DL of two daf-585 7 alleles even displayed avoidance of isoamyl alcohol. Mutation of the daf-2 gene 586 also mostly eliminated acute CO2 avoidance [20], suggesting that Daf-c mutations 587 have multiple effects on chemosensation. Other dauer-specific changes in amphid 588 gene expression and function have been observed. Two pairs of amphid neurons, 589 the AWA and AWC, are the primary mediators of chemotaxis toward attractive 590 odorants [24]. In C. elegans adults, the AWA neurons detect 2,3-butanedione 591 (diacetyl), while the AWC neurons sense isoamyl alcohol and 2,3-pentanedione. 592 A significant upregulation of neuropeptide expression during dauer entry occurs in 593 both C. elegans and P. pacificus [86], some of which may correlate with stage-594 specific olfactory behavior, as well as dauer-specific behaviors such as nictation 595 and the change in CO2 response valence [6]. A combination of cellular identity and 596 gene expression changes may be responsible for stage-specific chemosensory 597 behaviors between adults and DL [69,70].
Our study shows that dauers in two divergent nematodes can have either 599 attenuated or enhanced odor responses relative to reproductive adults, and that 600 the differences can be intensified in the Daf-c mutants. However, the genetic 601 mechanisms coordinating these developmental and behavioral changes in these 602 two species appear to evolve in separate gene regulatory networks. Several recent 603 C. elegans studies on the remodeling that occur after entry into dauer and in post-604 dauers adults show significant changes to olfaction and foraging behavior 605 [19,69,70]. Our study also revealed that Ppa-hsd-2(csu60); daf-12(tu389) mutants 606 have enhanced adult odor attraction, along with a mild but noticeable ecdysis 607 defect in non-dauer larval stages. This finding invigorates the suspicion that Ppa-608 HSD-2 may also be involved in the production of other steroid hormones, such as 609 ligands targeting the two putative P. pacificus ecdysone receptors [90]. Although 610 both Caenorhabditis and Pristionchus species co-occur on rotten vegetation as 611 both DL and feeding stages [91], only DL from Pristionchus species have been 612 isolated from beetles [32,34]. The robust chemoattraction to host pheromone 613 shown in this study supports the model that the DL is the preferred host-seeking 614 stage capable of locating beetle hosts. We expect we will have a better 615 understanding of how the decision for dauer entry is genetically coordinated with 616 host-associated remodeling of behavior when the cognate olfactory neurons for 617 the host pheromones are identified in P. pacificus. 618 Nematode Strains 622 P. pacificus and C. elegans strains were maintained at 20°C on NGM plates 623 seeded with OP50 as described previously [68]. The following P. pacificus strains 624 were used: PS312 (California wild-type, synonymous with RS2333), Ppa-obi-625 1(tu404) ChrI, Ppa-dfc-1(tu391) ChrI, and csu60 ChrII; Ppa-daf-12(tu389) ChrX. 626 For C. elegans, wild-type N2, as well as Daf-c mutants daf-2(e1370) and daf-627 7(ok3125, e1372) were used. A previous screen for Daf-c mutants using 50 mM 628 ENU or EMS yielded two highly penetrant alleles dfc-1(tu391) and Ppa-dfc-629 2(tu392), but only dfc-1(tu391) was viable for further characterization [8], while 630 csu60 was isolated as an off-target mutant from a CRISPR/Cas9-mediated 631 mutagenesis against a TGF-ß ortholog (Contig115-snapTAU.31)). Each allele was 632 Daf-c phenotype in the F2 progeny, which could be either heterozygous or 656 homozygous for the tu391 allele. We then examined the F3 progeny for dauer 657 formation defects guided by the following rational: If the daf-12 Daf-d phenotype is 658 epistatic to tu391, then no progeny of a non-DL F2 homozygous for daf-12(tu389) 659 will become DL. Alternatively, if the tu391 Daf-c phenotype is epistatic to daf-12, 660 then some F3 Daf-c progeny will be homozygous for daf-12(tu389). We found that 661 0 out of the 5 F2 lines from Daf-c dauers that exited were homozygous for the Ppa- 90 µl OP50 before seeding onto NGM plates. After two days, we picked 15-20 684 tu391 or csu60 gravid hermaphrodites onto each plate, which were allowed to lay 685 eggs for 5 hours before being removed for egg synchronization, or if the 686 hermaphrodites lay fewer than 50 eggs they were instead removed after 24 hours.
Plates were incubated at 20°C or 25°C for 4-5 days and then scored for dauer 688 larvae and non-dauer stages. 689 690

Behavioral assays 691
Pharyngeal pumping rates were observed through a Leica DM6000 upright 692 microscope with a 40x objective, without any anesthetic. J3 larvae, dauer larvae, 693 and incarcerated dauer larvae were mounted onto M9 buffer and 2% agar pads on 694 microscope slides. We counted at least four 15-second intervals per animal, which 695 were then summed to obtain pumps/minute. 696 The population chemotaxis assay for P. pacificus were performed on 697 covered 10 cm Petri dishes for ~16 hours at 22°C as previously described [39,68]. 698 However, because P. pacificus DL is coated with a long-chain polyunsaturated 699 wax ester (nematoil) that makes them highly hydrophobic [35], we modified the 700 chemotaxis assay by adopting a chemotaxis medium containing a detergent (10 701 mM MOPS pH 7; 2.5% Tween 20; 1.5% Bacto-agar) [65]. This MOPS/Tween 702 medium promotes both adult and DL dispersal, which when 5% rather than 10% 703 ZTDO was used as an attractant, likely contributed to the slightly higher CI values 704 for PS312 and obi-1(tu404) adults than expected when compared to previously 705 reported [68]. DL were loaded to the center of the plate by placing and removing 706 immediately a 1 cm 2 agar chunk from a culturing plate containing either wild-type 707 dauers from starved cultures or Daf-c DL from densely populated cultures. Young 708 adult worms from nearly-saturated cultures were washed twice with M9 buffer and 709 collected by centrifuging at 2500 rpm for 2 minutes. Approximately 100 worms were loaded onto each assay plate. Assays with obi-1(tu404) DL could not be 711 successfully performed due to their fragility and higher mortality. To anesthetize 712 animals at the odor sources, we place 1.5 µl of 1 M sodium azide on both sources 713 and used 100% ethanol as the counter attractant. All odorants were diluted with 714 ethanol to the following concentrations: 5% (Z)-7-tetradece-2-one (previous 715 studies used 10% ZTDO, which resulted in more DL paralysis at loading), 1% 716 methyl myristate, 1% (E)-11-tetradecenyl acetate, 1% (E)-11-hexadecenal, 10% 717 ß-caryophyllene, 1% 2,3-butadione(diacetyl), 1% 2,3-pentadione, and 1% isoamyl 718 alcohol. Interestingly, we found sodium azide to be superfluous when using ZTDO 719 as the attractant due to ZTDO's paralyzing effects on DL. Chemicals were 720 purchased from Fisher Scientific, Sigma-Aldrich, or Bedoukian Research 721 (Danbury, CT). 722 The chemotaxis assays for C. elegans were set up in a quadrant with worms 723 loaded onto the center of 6 cm plates containing the same MOPS/Tween agar as 724 described above, along with two opposing pairs of odors and controls [92,93] Young adults were washed off plates using M9 buffer and collected in a 65 mm-734 Syracuse watch glass. Animals were then washed 2X with M9 buffer followed by 735 1X with sterile water and immediately transferred to the center of 6 cm 736 MOPS/Tween agar plates. Excess water was removed with Whatman paper. DL 737 were directly transferred to the 6 cm MOPS/Tween agar plates on chunks of agar 738 from starved plates. A CO2 gradient was generated by delivering specific 739 compositions of gasses through holes in the plate lids as previously described [94]. reading frames. One of the predicted genes in this interval, PPA10139 (UMM-S10-1.4-mRNA-1), encodes a 376 amino acid homolog of the C. elegans 802 hydroxysteroid dehydrogenases 2 (HSD-2). 803 To make the Ppa-hsd-2 cDNA rescue transgene, 2063 bp of the Ppa-hsd-2 804 promoter was amplified from PS312 genomic DNA using RHL1012 and RHL1013. 805 The full-length Ppa-hsd-2 cDNA sequence was amplified from PS312 mixed stage 806 cDNA using RHL1014 and RHL1015. The Ppa-hsd-2 promoter and cDNA were 807 combined with Ppa-rpl-23 3' UTR to make pHC12. The mix injected into PS312 808 adult hermaphrodites contains the following: 2.5 ng/µl pHC12, 4 ng/µl Ppa-egl-809 20p::rfp, 80 ng/µl csu60 gDNA (all digested with PstI). The transgenic line was then 810 crossed into the csu60 mutant background using PCR to genotype for the deletion 811 (RHL1006 and RHL1007 will amplify 693 bp if there is a deletion; RHL1043 and 812 RHL1044 will amplify 1008 bp if the same locus is wildtype) and scored as a 813 percentage of dauer or non-dauers (J3/J4/adult) containing the rescue transgene. 814 The csuEx54 transgene transmission rate was approximately 27%. 815         SI  Fig. 8). Images of wild-type samples were taken at 700 ms exposure, and csu60 samples were taken at 700 ms exposure or lower due to much stronger expression level. Sample sizes are shown in Table 2. Anterior is left and dorsal is up. Scale bar: 25 µm.