Mud snails (Ilyanassa obsoleta) use chemical signals to organize breeding aggregations. It is hypothesized, as is the case for other mollusks that the egg laying hormone (ELH) serves both as a hormone and as an aggregation pheromone. We evaluate the response of reproductive and non-reproductive snails to the odors of egg cases and ELH. We find that the odor of ELH, whelk egg cases, and parchment worm tubes are attractive to snails preceding the breed season, and that the odor of whelk egg cases, tulip snail egg cases, and worm tubes are attractive to reproductive male snails. Snails do not respond to the odor of ELH during and following the breeding season. Our results support previous findings that attractive odors are released from egg cases and other glues, and show that ELH could function as an aggregation pheromone

 • Aggregation

• Kairomones

• Pheromones

• Gastropods; Egg laying hormone (ELH)

• Ilyanassa obsolete

: Laub E, McClellan-Green P, Rittschof D (2017) Mud Snail (Ilyanassa Obsoleta) Response to Reproductive Odors across Seasons. Ann Mar Biol Res 4(2): 1023.

Marine gastropods use chemicals in foraging [1,2], prey detection [3,4], predator avoidance [5-8], formation of breeding aggregations [9-11], and trail following [12-16]. Mud snails, Ilyanassa (=Nassarius=Nasa) obsoleta are an excellent model for understanding gastropod chemosensory behavior due to their abundance on the North Carolina shores, amenability to laboratory research, and extensive prior research [5,9,10,12,13,17-31]. In North Carolina usually from February through May, mud snails form vast breeding aggregations involving tens of thousands of snails where copulation and egg deposition occur, initially around oysters on the mud flats [9,32]. Breeding aggregations, copulation, and egg deposition are synchronized by temporal and environmental peptide chemical cues [9,10,33]. Rittschof found that breeding mud snails in the early part of the breeding season aggregate and deposit the majority of their eggs on live oysters and whelk egg capsules, and that mud snails follow the odors of live oysters. The cases of parchment worm cases (Chaetopterus sp.) are also attractive sites for egg deposition (Rittschof, personal communication). Moomijian determined that mud snails follow the odor of sexually active snails of the opposite sex, copulating snails, egg depositing snails, and oysters, suggesting that at least three sources of pheromones and one kairomone mediate breeding aggregations.

Although these studies begin to describe how environmental chemicals synchronize breeding behavior, questions remain about the specificity of chemical cues. Environmental chemicals have conserved structures that are often co-opted to serve multiple functions in the ecosystem [34-36]. Although it has not been isolated from mud snails, the neuropeptide egg-laying hormone (ELH) has been extracted from sea hares (Aplysia californica) and the giant triton snail (Charonia tritonis), and is shown to initiate egg laying behaviors [37-39]. Pheromones released from egg-laying individuals and egg strings maintain sea hare breeding aggregations [38,40,41]. Rittschof hypothesized that a peptide similar to ELH plays a role in assembling mud snail breeding aggregations and is incorporated into the egg capsules of mud snails and that oysters release a similar peptide to ELH that attracts snails as oysters reproduce concurrently with mud snails. Further supporting this hypothesis, genes encoding ELH have been found in the oyster species Pincata fucata and Crassostrea gigas, and ELH induces spawning in the oyster species Saccostrea glomerata [42,43]. We will investigate the role of ELH in mediating mud snail behavior through assays that determine response to ELH, whelk egg capsule, parchment worm tubes, and tulip snail egg capsule odors. We hope to contribute to understanding of pheromones in mollusks, the multi-functionality of environmental peptides [36], and seasonal impact on response to peptide pheromones and kairomones. We address these questions through behavioral assays that evaluate mud snail response to water born odors proceeding the breeding season, during the breeding season, and after the breeding season

 Snail collection spring 2011

Sexually receptive mud snails, Ilyanassa obsoleta, were collected from the Town Marsh embayment near oyster beds (34.711078, -76.668304) in February. Town marsh embayment was selected because imposex and parasitism, confounding factors for chemoreception, are in low percentage [9,27,29]. Snails were aggregating around oysters and mating. Sexually receptive males and females were tested separately. Pairs of snails found mating in the field were pulled apart and divided into males, with penis, and females. About 50 pairs of snails were collected. Snails were sorted into the categories in the field and maintained in separate tanks in with low flow in the laboratory. Snails were tested once each day until all odors were tested and then returned to the wild.

Snail collection fall 2013

Mud snails were collected during October of fall 2013 at low tide from the mud flats of Bird Shoals/Carrot Island in the Rachel Carson National Estuarine Research Reserve (34.710643, -76.675001). Although snails were aggregated near the oyster beds in fall 2013, breeding and egg laying were not observed. Over 1000 snails were collected from several large aggregations. Snails were housed in an 80L aquarium with an inch of sandy mud and an aerator.

Snail collection summer 2015

Mud snails were collected in May from NOAA beach, Beaufort NC, (34.717987, -76.671088) and kept in an 80L aerated tank with an inch of sandy mud for the duration of the project. Snails were only used for one assay, and were returned to the beach. The snails were non-reproductive. There was no evidence of mating or egg lying and snails were in muddy sand away from the oyster beds. Snails were collected from NOAA Beach during summer 2015.There were no longer empty egg capsules, evidence of spring breeding at the site.

Snail collection winter 2016

Mud snails were again collected from the Town Marsh embayment near oyster beds (34.711078, -76.668304) in late January. The large expanse of shallow water in the embayment results in warmer conditions than elsewhere in the area and snails begin breeding there first. Snails were aggregating around oysters although egg capsules and mating were not observed. Snails were kept in an 80L aerated tank with an inch of mud at 24°C for the duration of the project.

Odor ring assays

Odor ring assays were used to determine snail response to odors and conducted in accordance with the methods described by [9,10,23,25]. Odor ring assays were conducted in 8.5cm diameter finger bowls. An approximately 4 cm diameter, 1 cm width ring of solution was drawn in the bottom of the finger bowl with a saturated Q-tip

(Figure 1 and Figure 2).

Figure 1 Photograph of ring assay experiment (left) and diagram of ring assay set-up (right). The black circle indicates the margin of the finger bowl, the grey band is the circle of odorant, and the blue teardrop indicates the snail placed in the bowl

Figure 2 Photograph of ring assay experiment (left) and diagram of ring assay set-up (right). The black circle indicates the margin of the finger bowl, the grey band is the circle of odorant, and the blue teardrop indicates the snail placed in the bowl

Five odor solutions were use: aged sea water (ASW) (control), knobbed whelk (Busycon carica) egg capsules, parchment worm tube tips (Chaetopterus sp.), tulip snail egg capsules (Fasciolaria tulipa),and synthesized sea hare (Pelagia California) egg laying hormone (ELH), contributed by Dr. McCellan- Green (NC State University). Whelk capsule, tulip snail egg capsule, and parchment worm tube tip solutions were generated by soaking 3.12 g dried material per 200 ml ASW for 24 hours at 4 C. After 24 hours, the solutions were divided into 5 ml aliquots and frozen. The ELH solution was generated by combining 4 micrograms of the synthesized hormone with 1 ml of sterile deionized water. From this stock solution, 100 microliter aliquots were frozen until the experiments were to be performed, upon which 10 ml of ASW was added (.0396 microgram ELH per 1 ml ASW). The ELH stock solution was created in 2013. Aliquots frozen at -20 C were thawed as needed for the 2015 and 2016 tests.

Snails were placed inside the ring of test solution and their response was recorded upon contact with the odor ring (Figure 1and Figure 2). Follow was recorded for snails turned and followed the ring. No response was recorded for snails passing through the ring without changing direction. Snails that did not crawl into contact with the ring for longer than five minutes were removed and replaced. Chi-squared tests were used to determine significance of response to experimental odors compared to the ASW control. In 2011, 30 snails were tested in each condition. For other years 60 snails were tested for with each odor

Following testing, samples of snails were snails were removed from their shells for sex determination. The removal technique consisted of cracking the shell of the snail, severing theattachment muscle and extracting the body with forceps. Snails were observed under a dissecting scope and sex identified by different colored gonads. Males were identified by orange gonads and a penis (Figure 3).

Figure 3 Identification of male (left) and female snails (right). Note orange gonads and penis on male and cream gonads and capsule gland (large white gland with pinkspot) on female

Females were identified by clear to creamcolored gonads and a capsule gland (Figure 4).

Figure 4 Identification of male (left) and female snails (right). Note orange gonads and penis on male and cream gonads and capsule gland (large white gland with pinkspot) on female

 Response to substrate odors

Dissection of 136 snails during fall 2013 revealed partial gonad development in snails. Females had cream colored gonads and males had orange gonads. However, not all males had developed penises and not all females had developed capsule glands. Due to the intermediate sexual organ development and lack of observed breeding in the wild, snails were characterized as reproductively recrudescent. Snails followed the odors of parchment worm tips, and whelk egg capsules (60%, Chi-squared (C) = 12.19, P < .001; 64.4%, C = 15.57, P < .001) (Figure 5).

Figure 5 Snail response to odors from parchment worm tube tips, whelk egg capsule, and tulip snail’s egg capsules across seasons. Blue bars indicate percentage follow response to ASW, yellow bars indicate percentage follow response to parchment worm tips, green bars indicate percentage follow response to whelk egg cases, and red bars response percentage follow response to tulip snail egg cases. Asterisks indicate level of significance (* = P <.05; ** =P<.001). Recrudescent (mixture of not sex sexually receptive males and females, collected fall 2013) n = 60, reproductive females n = 30 (collected spring 2011), reproductive males n = 30(Collected spring 2011

Snails were reproductively active during spring 2011. Sexual receptivity during spring 2011 was determined by separating and testing copulating snails. Reproductive males were very responsive o the odors of parchment worm tube tips, whelk egg capsulesand tulip snail egg capsules (60%, C = 10, P = .00157; 60%, C = 10, P = .00157; 60%, C = 10, P = .00157) (Figure 5). In contrast, reproductive females did not follow the any of the experimental odors (36.67%, C = .6932, P =.405; 30%, C = .0821, P = .7745; 36.67%, C = .6932, P = .405) (Figure 5).

Response to egg-laying hormone (ELH) Snails tested during the fall of 2013 were considered reproductively recrudescent due to intermediate gonad development and lack of reproductive behavior. Recrudescent snails significantly followed the odor of ELH (80%, C = 32.257, P < .00001) (Figure 6). Snails tested during winter 2016 were considered reproductive as dissections revealed fully developed capsule glands and penises. Although snails were not observed mating and laying eggs at the embayment, eggs were found in the holding tank indicating that the snails were reproductive.

Out of 660 snails dissected, 12 were parasitized by nematode worms. During the winter of 2016, neither female nor male mud snails significantly followed the odor of ELH compared to ASW (36%, C = .4028, P = .5256; 38.23%, C =.0951, P = .757) (Figure 6).

Figure 6 Snail response to ELH across seasons. Blue bars indicate percent follow response to ASW and purple bars indicate follow response to ELH. Asterisks indicate level of significance (** = P < .001). Recrudescent n = 60 (collected fall 2013), Reproductive Female n = 25, Reproductive Male n = 34 (collected winter 2016), Post-Reproductive n = 60 (collected summer 2015)

Snails were not reproductive during the summer of 2015. No snails were observed breeding and they had moved away from the oyster beds. Dissection of 59 snails yielded two snails with capsule glands, no snails with penises, and no snails with colored gonads, further indicating that the snails were not reproductive. Snails did not follow the odor of ELH significantly more than ASW following the breeding season in summer 2015 (10%, C = .3235, P = .5695) (Figure 6)

 Here we found mud snails in the process of reproductive recrudescence responded to odors that are attractive to mating and spawning snails. Mud snails followed the odors whelk egg capsules and parchment worm tube tips, substrates on which mud snail eggs are laid [35]. These structures are hypothesized to be preferentially selected along with oyster lips because they are off the substrate a location where it is unlikely that sediments will bury and smother embryos [35]. Recrudescing snails followed the odor of ELH, suggesting that ELH or similar molecules function as aggregation cues in mud snails [10,35,37,38,44]. It appears that behavioral responses to aggregation cues develop before the cues that synchronize breeding are present. Post-reproductive snails did not respond to the odor of ELH, suggesting that aggregation behavior is activated as gonads develop

Although we expected ELH to function as an aggregation pheromone during the breeding season, neither male nor female snails followed the odor of ELH during the breeding season [38,40,44,45]. Moomjian found that egg capsule depositing snails are attractive to both male and female snails. One explanation for the lack of response was the presence of egg capsules and reproductive snails in the holding tank released ELH or ELH similar compounds, desensitizing the snails to ELH before testing [9,40,44,45].

In contrast to other studies, we found a sexually dimorphic response to odors of egg laying substrates from reproductive snails [9]. Although male snails were attracted to the odors of parchment worm tube tips, whelk egg capsules, and tulip snail eggcapsules, reproductive female snails were not. Male snails arrive at spawning locations, oyster beds, or other snail’s egg capsules before females [9]. Female mud snails’ aggregate in response to the odor of reproductive male snails [9,10].The follow response of sexually active male snails to whelk and tulip snail egg cases further supports the hypothesis that gastropod egg cases release similar attractive compounds [9]. It remains unclear how related these attractive compounds are to ELH. Painter reported that egg cordons, egg laying animals, and sexually mature conspecifics are highly attractive to sea hares, however biochemical analysis of the artrial gland revealed that several compounds may be attractants [44]

Synchrony of reproduction through chemosensing is critical for the reproductive success of mud snails as formation of breeding aggregations enables snails to both find mates and increase reproductive success through risk dispersal [46,47]. We have demonstrated that egg capsule odors from several species are attractive to mud snails before and during the breeding season, suggesting that attractive compounds may be conserved across species [48]. In addition, our results support that ELH functions as an attractive pheromone for marine gastropods. Re-testing reproductive snails with ELH would be valuable to evaluate if it functions as an attractive pheromone during the breeding season. Furthermore, it would be of value to determineif the attractive compounds emitted by live oysters are related to ELH in order to understand how reproductive pheromones of one species may be co-opted for aggregation by another

I would first like to acknowledge the contributions of Dr. Patricia McClellan-Green. Dr. McClellan-Green provided ideas, consultations, and materials for this project and she deserves recognition for her gracious assistance. She was unable to read the final manuscript for this paper due to her death, and deserves to be recognized for her contributions. Professor G. Nagle provided the ELH use in experiments. In addition, I would like to thank Dan Rittschof for his mentorship and assistance editing this manuscript. Lastly, I would like to thank Beatriz Orihuelaand Dr. Humberto Diaz for their generous help with laboratory maintenance and animal husbandry

1. Carr WE, Derby CD. Chemically stimulated feeding behavior in marine animals. J Chem Ecol. 1986; 12: 989-1011.
2. Larcher M, Crane AL. Chemoreception of hunger levels alters the following behaviour of a freshwater snail. Behavioural processes. 2015; 121: 30-32.
3. Rittschof D, Williams LG, Brown B, Carriker MR. Chemical attraction of newly hatched oyster drills. The Biol Bull. 1983; 164: 493-505.
4. Williams LG, Rittschof D, Brown B, Carriker MR. Chemotaxis of oyster drills Urosalpinx cinerea to competing prey odors. Biol Bull. 1983; 164: 536-548.
5. Atema J, Stenzler D. Alarm substance of the marine mud snail, Nassarius obsoletus: biological characterization and possible evolution. J Chem Ecol. 1977; 3: 173-187.
6. Jacobsen HP, Stabell OB. Antipredator behavior mediated by chemical cues: the role of conspecific alarm signaling and predator labelling in the avoidance response of a marine gastropod. Oikos. 2004; 104: 43-50.
7. Xu W, Zhang J, Du S, Dai Q, Zhang W, Luo M, et al. Sex differences in alarm response and predation risk in the freshwater snail Pomacea canaliculata. J Molluscan Stud. 2014; 80: 117-122.
8. Ueshima E,Yusa Y. Antipredator behaviour in response to single or combined predator cues in the apple snail Pomacea canaliculata. J Molluscan Stud. 2015; 81: 51-57.
9. Rittschof D, Sawardecker P, Petry C. Chemical mediation of egg capsule deposition by mud snails. J Chem Ecol. 2002; 28: 2257-2269.
10. Moomjian, Lauren, Sarah Nystrom, Daniel Rittschof. "Behavioral responses of sexually active mud snails: kariomones and pheromones.  J Chem Ecol. 2003; 29: 497-501.
11. Seuront L, Spilmont N. The smell of sex: water-borne and air-borne sex pheromones in the intertidal gastropod Littorina littorea. J Molluscan Stud. 2014; 81: 96-103.
12. Trott TJ, Dimock Jr RV. Intraspecific trail following by the mud snail Ilyanassa obsoleta†. Marine & Freshwater Behav Physiol. 1978; 5:  91-101.
13. Bretz DD, Dimock RV. Behaviorally important characteristics of the mucous trail of the marine gastropod Ilyanassa obsoleta (Say). J Exp Mar Biol Ecol. 1983; 71: 181-191.
14. Carr WE, Gleeson RA, Derby CD. Stimulants of feeding behavior in fish: analyses of tissues of diverse marine organisms. Biol Bull. 1996; 190: 149-160.
15. Edwards M, Davies MS. Functional and ecological aspects of the mucus trails of the intertidal prosobranch gastropod Littorina littorea. Marine Ecology Progress Series. 2002; 239: 129-137.
16. Ng T, Saltin SH, Davies MS, Johannesson K, Stafford R, Williams GA. Snails and their trails: the multiple functions of trail following in gastropods. Biol Rev. 2013; 88: 683-700.
17. Carr WE, Derby CD. Chemically stimulated feeding behavior in marine animals. J Chem Ecol. 1986; 12: 989-1011.
18. Crisp M. Studies on the behavior of Nassarius obsoletus (Say)(Mollusca, Gastropoda). Biol Bull. 1969; 136: 355-373
19. Atema J, Burd GD. A field study of chemotactic responses of the marine mud snail, Nassarius obsoletus. J Chem Ecol. 1975; 1: 243-251.
20. Stenzler D, Atema J. Alarm response of the marine mud snail, Nassarius obsoletus: specificity and behavioral priority. J Chem Ecol. 1977; 3: 159-171.
21. Dimock Jr RV, Parno JR. BiMmodal sensitivity to monochromatic light by the mud snail Ilyanassa obsoleta. Mar Freshwater Behav Physiol. 1981; 7: 291-296.
22. Brenchley GA, Carlton JT. Competitive displacement of native mud snails by introduced periwinkles in the New England intertidal zone.  Biol Bull. 1983; 165: 543-558.
23. Duval MA, Calzetta AM, Rittschof D. Behavioral responses of Littoraria irrorata (SAY) to water-borne odors. J Chem Ecol. 1994; 20: 3321-3334.
24. Oberdörster E, Rittschof D, McClellan-Green P. Testosterone metabolism in imposex and normal Ilyanassa obsoleta: Comparison of field and TBTA Cl-induced imposex. Mar pollut bull. 1998; 36:144-151.
25. Rahman YJ, Forward Jr RB, Rittschof D. Responses of mud snails and periwinkles to environmental odors and disaccharide mimics of fish odor. J Chem Ecol. 2000; 26: 679-696.
26. Oberdörster E, McClellan-Green P. Mechanisms of imposex induction in the mudsnail, Ilyanassa obsoleta: TBT as a neurotoxin and aromatase inhibitor. Mar Environ Res. 2002; 54: 715-718.
27. Straw J, Rittschof D. Responses of mud snails from low and high imposex sites to sex pheromones. Mar Pollut Bull. 2004; 48: 1048-1054.
28. Rittschof D, McClellan-Green P. Molluscs as multidisciplinary models in environment toxicology. Mar Pollut Bull. 2005; 50: 369-373.
29. McClellan-Green P, Romano J, Rittschof  D. Imposex induction in the mud snail, Ilyanassa obsoleta by three tin compounds. Bull Environ Contam Toxicol. 1996; 76: 581-588.
30. Sternberg RM, Hotchkiss AK, LeBlanc GA. The contribution of steroidal androgens and estrogens to reproductive maturation of the eastern mud snail Ilyanassa obsoleta. Gen Com Endocrinol. 2008; 156: 15-26.
31. Guidone M, Newton C, Thornber CS. Utilization of the invasive alga Gracilaria vermiculophylla (Ohmi) Papenfuss by the native mud snail Ilyanassa obsoleta (Say). J Exper Mar Biol Ecol. 2014; 452: 119-124.
32. Sastry AN. Effect of temperature on egg capsule deposition in the mud snail Nassarius obsoletus (Say). Veliger. 1971; 13: 339-341.
33. Scheltema RS. The relationship of temperature to the larval development of Nassarius obsoletus (Gastropoda). Biol Bull. 1967; 132: 253-265.
34. Rittschof D, Bonaventura J. Macromolecular cues in marine systems. J Chem Ecol. 1986; 12: 1013-1023.
35. Rittschof D. Body odors and neutral-basic peptide mimics: a review of responses by marine organisms. American Zoologist. 1993; 33: 487-493.
36. Rittschof  D, Cohen JH. Crustacean peptide and peptide-like pheromones and kairomones. Peptides. 2004; 25: 1503-1516.
37. Chiu AY, Hunkapiller MW, Helle E, Stuart D K, Hood LE, Strumwasser F. Purification and primary structure of the neuropeptide egg-laying hormone of Aplysia californica. Proceedings of the National Academy of Sciences. 1979; 76: 6656-6660.
38. Painter SD. Coordination of reproductive activity in Aplysia: peptide neurohormones, neurotransmitters, and pheromones encoded by the egg-laying hormone family of genes. Biol Bull. 1992; 183: 165-172.
39. Bose U, Suwansa-ard S, Maikaeo L, Motti CA, Hall MR, Cummins SF. Neuropeptides encoded within a neural transcriptome of the giant triton snail Charonia tritonis, a Crown-of-Thorns Starfish predator. Peptides. 2017.
40. Jahan-Parwar B. Aggregation pheromone from the egg-mass of Aplysia californica. Physiologist. 1976; 19: 240.
41. Audesirk TE. Chemoreception in Aplysia californica. III. Evidence for pheromones influencing reproductive behavior. Behav Biol. 1977; 20: 235-243.
42. Stewart MJ, Favrel P, Rotgans BA, Wang T, Zhao M, Sohail M. Neuropeptides encoded by the genomes of the Akoya pearl oyster Pinctata fucata and Pacific oyster Crassostrea gigas: a bioinformatic and peptidomic survey. BMC genomics. 2014; 15: 840.
43. Van In V, Ntalamagka N, O’Connor W, Wang T, Powell D, Cummins SF, et al. Reproductive neuropeptides that stimulate spawning in the Sydney Rock Oyster (Saccostrea glomerata). Peptides. 2016; 82: 109-119.
44. Painter SD, Chong MG, Wong MA, Gray A, Cormier JG, Nagle GT. Relative contributions of the egg layer and egg cordon to pheromonal attraction and the induction of mating and egg-laying behavior in Aplysia. Biol Bull. 1991; 181: 81-94.
45. Painter SD, Clough B, Garden RW, Sweedler JV, Nagle GT. Characterization of Aplysia attractin, the first water-borne peptide pheromone in invertebrates. Biol Bull. 1998; 194: 120-131.
46. Feare CJ. The adaptive significance of aggregation behavior in the dogwhelk Nucella lapillus (L.). Oecologia. 1971; 7: 117-126.
47. Magnhagen C. Predation risk as a cost of reproduction. Trends Ecol Evol. 1991; 6: 183-186.
48. Powell Jr EH, Gunter G. Observations on the stone crab, Menippe mercenaria (SAY), in the vicinity of Port Aransas, Texas. Gulf Caribbean Res. 1968; 2: 285-299.