Trail-Following Behavior of Land Snails

國立中山大學生物科學系碩士論文

Department of Biological Sciences National Sun Yat-sen University Master Thesis

陸生蝸牛的痕跡追隨行為

研究生：梁師瑀 Shih-yu Liang 指導教授：張學文博士 Dr. Hsueh-Wen Chang

中華民國 105 年 1 月 January 2016

摘要

費洛蒙為生物體產生的化學物質，會以不同的形式排出體外形成痕跡，並

由同種及異種間利用。而陸生腹足綱痕跡追隨的目的，大致上來說具有群聚、

交配，捕食、回巢及節省能量等功能。我利用兩種壽山常見的陸生蝸牛，分別

為雌雄同體的小菱蝸牛(Satsuma succincta)，及雌雄異體的皺大山蝸牛

(Cyclophorus friesianus) 進行追隨實驗。本研究提出三個問題：(1) 這兩種陸生

蝸牛是否也具有同種間的追隨行為，(2)追隨行為是利用何種化學物質進行追

隨，以及(3)雌雄異體的物種是否可藉由此行為作為交配策略。每次測試皆由兩

隻同種之蝸牛組成：一隻作為遺留黏液之 marker，另一隻為追隨黏液之 tracker。結果發現兩物種皆出現追隨行為，且室內與室外的結果沒有顯著差

異。小菱蝸牛同時出現黏液及以氣味追隨的行為，但以氣味作為主要的化學訊

號；皺大山蝸牛則單純以黏液追隨。在性別差異的實驗中，皺大山蝸牛在異性

間與同性間的追隨頻率雖然沒有顯著差異，但出現異性高於同性的傾向，且雄

性追隨雌性的頻率略高於其他性別配對。雖然無法支持黏液追隨可作為交配策

略，但高程度的重疊比例下，推測黏液追隨的功能可為減少移動能量的耗損達

到省能的目的。

關鍵詞：費洛蒙、痕跡追隨、同種間、交配策略、氣味、黏液

Abstract

Pheromone is produced by animals and secreted in variously ways to form trails

which can be used by conspecifics or between species. The functions for trail- following in terrestrial gastropod are aggregating, mating, predating, homing and energy saving. In this study, I used two species of terrestrial gastropod, Satsuma succincta and Cyclophorus friesianus, which are abundance in Mt. Shoushan,

Kaohsiung, Taiwan to test three questions: (1) Do these land snails follow the chemical trail laid by conspecifics? (2) If followed, what kind of chemical trail would they follow? (3) Whether trail-following is a mating strategy for dioecious species?

Every following behavior test I used two snails, marker for leaving trail and tracker for following trail. Both of the two species showed trail-following behavior, and there

were no difference between two studying areas. S. succincta had trail-following

behavior both by mucus and odor, but odor was the main chemical cue for C.

friesianus. In test for sexual difference on trail-following behavior, although there

were no significant difference between the different and same sex groups, the

frequency was higher in different sex group and female followed by male was higher

than other sexual combining pairs. This test supported that mucus trail-following might be a purpose for energy saving due to the trail overlapped about half of the

marker’s trail.

Key words: pheromone, trail-following, conspecific, mating strategy, odor, mucus.

Contents

中文摘要………………………………………………………………….i

Abstract…………………………………………………………………..ii Contents………………………………………………………………….iii Introduction………………………………………………………………1 Materials and Methods…………………………………………………...5 Results…………………………………………………………………..10 Discussion……………………………………………………………….13 Literature Cited………………………………………………………….17 Tables……………………………………………………………………21 Figures…………………………………………………………………..24

iii

Introduction

Trail-following behavior, individuals following the tracks or paths previously laid by themselves or other individuals, occurs in a variety of animal taxa and likely has evolved several times (Alexy et al. 2001; Cafazzo et al. 2012; Dehnhardt et al.

2001; Ng et al. 2013). A trail can be visible like hydrodynamic trail, footprint, and mucus (Dehnhardt et al. 2001; Harmsen et al. 2010; Ng et al. 2013), or invisible like odor (Farkas and Shorey 1972). No matter the trail is visible or invisible, it contains chemical cue used to communicate with conspecifics or heterospecifics (Farkas and

Shorey 1972; Harmsen et al. 2010; Davis-Berg 2011; Ng et al. 2011). Pheromones was one of the chemical cue, which could be released for communication in animals.

For examples, male moth (Pectinophora gossypiella) uses olfactory cues provided by pheromone plume to identify their mates (Farkas and Shorey 1972). Gastropods utilize mucus laid by other individuals for mate-searching, predation and aggregation

(Johannesson et al. 2008; Davis-Berg 2011; Ng et al. 2011; Stafford et al. 2012b).

Function of trail-following in gastropod

Terrestrial gastropods are well known to adopt pheromone trail-following behavior. Functions of this behavior in gastropods include facilitating aggregation, mating, homing, foraging, and energy saving (McFarlane 1980; Erlandsson and

Kostylev 1995; Chapman 1998; Erlandsson 2002; Davies and Blackwell 2007;

Johannesson et al. 2008; Johannesson et al. 2010; Davis-Berg 2011; Stafford et al.

2011; Ng et al. 2012). Intertidal species prefer living in groups to form aggregation, which could reduce desiccation stress or reducing predation (Coleman et al. 2004;

Stafford et al. 2012a; Stafford et al. 2012b). For cross-fertilization species, trail- following may increase the chance to find an available mate. For example, male

Littorina fabalis, L. ardouiniana, and L. melanostoma have the ability to track female 1 by gender-specific mucus cue, which can avoid maladaptive mating (Johannesson et al. 2010; Ng et al. 2011). Moreover, mucus following-behavior was a mechanism of reproduction barrier for L. saxatilis to different ecotypes (Johannesson et al. 2008).

The terrestrial carnivorous snail, Euglandina rosea, not only can it follow the mucus laid by pray, but also distinguish conspecifics from prey by mucus (Shaheen et al.

2005; Davis-Berg 2011). Slug Limax pseudoflavus approached the home area relative to the wind direction, and was suggested to have the homing ability by tracking its odor (Cook 1980). In the study of saving energy, Davies and Blackwell (2007) suggested that movements of snails could save approximately 70% of energy cost for trail-following. It was believed that gastropods crawl on mucus laid previously could save energy on rough surface or move faster (Davies and Blackwell 2007; Davis-Berg

2011).

Chemical cue in trail-following

According to the respiratory, gastropod could be divided into two groups,

Prosobranchia, and Pulmonata. Marine and fresh water snail species are mostly

Prosobranchia while terrestrial species are mostly Pulmonata. Previous research on the mucus trail-following mainly focused on aquatic Prosobranchia (Ng et al. 2013).

Little is known about the trail-following behavior of terrestrial Prosobranchia.

Olfaction is the principal sensory modality for detecting and locating by the posterior tentacles in all terrestrial pulmonates for they have no auditory organs and only rudimentary eyes (Chase and Croll 1981). Previous studies supported that some land snails and slugs used odor information to track conspecific or home (Gelperin 1974;

Chase et al. 1978; Lemaire and Chase 1998). Cook (1992) suggested that mucus may be subordinate to that of airborne odors, and appeared to be a low priority for trail- following in most slugs. In terrestrial slug Limax pseudoflavus, it would perform

2 mucus trail-following behavior only when the individual was isolated from airborne odors (Cook 1980). In contrast, Chase et al. (1978) documented that terrestrial pulmonate snail Achatina fulica used trail-following to find sexual mates while airborne stimuli (not trails) contain only nonsexual natural information. Nevertheless, another snail species, Otala vermiculata, did not follow the mucus left by conspecifics in the same study. So far, most terrestrial trail-following behavior studies focus on large and hermaphroditic species, and little is known about small hermaphroditic or gonochoric species (Ng et al. 2013).

Concentration in chemical cue

When pheromone is released from the gastropod to environment, its concentration may change due to diffusion, evaporation of water components of the mucus or change of environmental condition (e.g. rain, tide, wind and temperature).

The change of concentration may influence the signal strength of the chemical and, consequently, the trail-following behavior. For example, the intertidal species Cellana grata lives on high rocky shore, where the high temperature environment (> 50°C) may rapidly breakdown the chemical in the mucus. Therefore, the short persistence time of C. grata mucus serves no post-deposition function, i.e. homing, foraging, mate-searching and anti-predator, but it was only used for locomotion and adhering

(Davies and Williams 1995).

In this study, I tend to the following questions about trail-following behavior of land snail:

(1) Do land snails also follow the trail laid by conspecifics?

(2) If followed, under morphologic difference in tentacles, what kind of chemical cue

do they follow?

(3) Is trail-following a mating strategy adopted by gonochoric land snail species?

To answer these questions, I tested and compared the trail-following behaviour of two terrestrial gastropod species with distinctively different respirational and reproductive modes, Satsuma succincta (Pulmonata, hermaphroditc) and Cyclophorus friesianus

(Prosobranchia, gonochoric). Given the land snail also produced mucus while moving as other marine or intertidal species, I predicted that these two species would perform trail-following behavior as other snail species since trail-following may decrease the energy cost for the movement of the follower (or tracker) and provide benefits for aggregating or mating. If they follow the trail, I predicted the follower would move along or stayed closely to the mucus trail left by the previous individual. Given the pulmonate species was more sensitive to olfactory signals while prosobranch snails rarely used odor signals, I predicted the pulmonate species S. succincta, would follow both mucus and odor trail, while the prosobranch snail C. friesianus, would only follow the mucus trail. In other words, I predicted the follower’s trail would overlapped with the mucus trail laid by the previous individual more closely in the prosobranch species C. friesianus than in pulmonate species S. succincta. For gonochoric species, if trail-following provided benefits for mate-searching, the follower would gain additional reproductive benefits by following the individual of the opposite sex than same sex. I predicted that the gonochoric snail species C. friesianus would follow the individual of the opposite sex more frequently than same sex.

Materials and Methods

Organisms studied

Satsuma succincta is a lung breathing pulmonate gastropod and widely distributed in Southern Taiwan with two pairs of tentacle. The hermaphroditic land snail has coniform, smooth, dextral shell with 1-3 brown bands (Fig. 1). The shell is about 30 mm in width and 20 mm in height, with 5-6 layers of whorls without operculum.

Cyclophorus friesianus is a gill breathing prosobranch gastropod with one pair of tentacles and widely distributed in Southern Taiwan. The gonochoric land snail has campaniform, smooth, dextral shell with convex and growth lines on the whorl of the body (Fig. 2). The shell is about 20 mm in width and 17 mm in height, with 4-5 layers of whorls with operculum. Sexually mature snails could be determined by the presence of its thick lip around the aperture, and males have penis behind the right eye.

Collection of snails

I performed the study both in the field and laboratory. For the field study, I collected S. succincta and put them into a plastic container by hand along from the trails of Mt. Soushan and C. friesianus from those of Mt. Banpingshan both in

Kaohsiung, during July and August, 2015. I kept them active to facilitate rapid performances by spraying water inside the plastic container (Chase et al. 1978). For the laboratory study, I collected S. succincta from Mt. Soushan and Mt. Banpingshan and C. friesianus from Mt. Banpingshan during September and October, 2015. I maintained the snails in small transparent containers individually (size 7.5 x 5.5 x 8.5 cm) and provided water and carrots every three days. I kept the containers at room temperature, without particular light control, and maintained the moisture by adding 5 damp non-woven fabric at the bottom. Before each test, snails were gathered to a plastic container with water spraying to keep the snails active.

Field experiments

Field experiments were conducted on the ground (i.e. soil, rock…) around the trails. A small, flat testing area of 21 x 21 cm2, clean without fallen leaves, branches or any obstacles that may potentially interfere the crawling of snails.

To facilitate the snails crawling over the testing area, I kept the ground wet by spreading reverse osmosis water before the test. At the beginning of the test, I placed one pair of S. succincta or C. friesianus with various sex combinations at the center of the 21 x 21 cm2 testing area simultaneously and kept the aperture of both individuals facing the same direction by hand. For the gonochoric C. friesianus, pairing included two males, two females, and one male and one female. I let the snail pair crawling freely until both individuals reached the boundary of the testing area. I designated the snail which crawled first as the “marker” and the other as “tracker”. I performed tests for 62 pairs of S. succincta with no sex pair, and 84 pairs of C. friesianus, consisting of 21 two-male (m-m), 21 two-female (f-f) and 42 opposite-sex pairs, of which each sex comprised half of the markers and trackers (m-f & f-m). Each pair was tested for only once. I also shifted the experiment to another testing area about 15 cm away after each test to avoid any mucus of pheromones left by the previous snail pair. All the field experiments were conducted between 6-9 a.m.

To record the crawling position, trail and time of the snails, I used a time-lapse camera (Brinno TLC200, Taiwan) shooting at a speed of one picture per second. The camera was set 60 cm right above the testing area, and the center of the lens was set perpendicular to the ground to avoid misjudging the snail position (Hutchinson et al.

2007).

Laboratory experiments

Test procedure in the laboratory was the same as in the field, except the ground testing area was replaced by a wet non-woven fabric on the table. I discarded the fabric after each trial, and used 70% alcohol to wipe the table to rid of mucus of pheromones left by the previous test. The time-lapse camera was set at about 40-70 cm above the fabric.

I performed the tests for 30 pairs of S. succincta, and 44 pairs of C. friesianus consisting of 11 two-male, 11 two-female and 22 opposite-sex pairs, of which each sex was accounted for half of the markers and trackers.

Parameters

I reconstructed the trail of the marker and tracker by connecting the positions of each snail recorded by the time-lapse photos at an interval of 30 seconds (Fig. 3). I examined the trail following behavior by measuring the parameters below:

(1) Trail angle: I used the angle between the moving directions of the marker and

tracker to examine whether the pair performed following behavior. I considered

the trails of the pair with an angle < 30° as following. I determined the trail angle

by drawing an imaginary, straight line connecting the start and end points of each

snail and calculated the angle between the lines using software Image J (1.48v,

NIH, U.S.A.).

(2) Body length: Due to the difficulty to measure the body length of the snail directly,

I used the software Image J (1.48v, NIH, U.S.A.) to measure the body length of

the crawling snail from the photo. I only measured the photo taken in the lab due

to the low contrast between the color of the snail and the background in the field,

which blurred the boundary of the image.

(3) Tracker’s inaction time: When moving the snail from the container to the testing

area, the snail usually retreated its body and head into shell at the beginning. It

usually took a short period of time before the snail stretched out its tentacles and

started to detect the surrounding environment. I determined the time period

between its landing on the testing area and stretching out the tentacles as the

stretching time. Then I defined the [tracker’s inaction phase] as [tracker’s

stretching time] – [marker’s stretching time]. The value could be negative if the

tracker extended its tentacles before the marker did, or 0 if both individuals did

not retreat their body into shell.

(4) Coincidence index (C.I.): The parameter was used to measure the degree of trail

overlapped between the two snails (Davies and Beckwith 1999). The formula

was: C.I. = Ol / Ml,

where Ol = length of trails overlapped, and Ml = length of the marker snail trail.

The value of C.I. ranged between 0 and 1, where 1 meant the trail of the marker

was completely followed (overlapping) by the tracker, and 0 meant no trail

overlapping between the marker and tracker.

Trail judgement

Previous studies used C.I. to determine whether a snail tracker follows a marker by mucus (Townsend 1974; Erlandsson and Kostylev 1995; Davies and Beckwith

1999; Ng et al. 2011). However, when the snail tracked the target by odor, the trail overlapping was not necessarily occurred (Gelperin 1974; Chase et al. 1978; Davis

2004). Therefore, I determined the tracker performing following behavior if (1) Ol > tracker’s body length, or (2) Ol < tracker’s body length and the trail angle < 30°.

Condition (1) indicated the snail following the trail by mucus (Cook 1992), and (2) by odor. Examples illustrating whether two trails were determined to be trail following or non-following were shown in Fig. 3.

Statistical analysis

Data were all analyzed using SAS software version 9.4 (SAS Institue Inc.,

U.S.A.). For trail following determined by condition (2), the expected frequency of trail angle < 30° would be 1/6 (30°/180°; Fig. 4) if both snails moved at random.

Therefore, I used the test of goodness of fit to examine whether the frequency of trail following by condition (2) was significantly higher than at random (1/6). I used Yates’ correction for continuity whenever one of the cells < 5, but did not perform test if more than one cells < 5. Test for independence was used to compare the difference between the study areas of the following behavior. Fisher’s exact test was used to compare the difference of sexual combining pairs. I used Mann-Whitney U test to determine whether the inaction phase were different between the pairs of trail following and non-following, and trail following by overlapping and angle.

Results

Tracking chemical cues laid by conspecifics

Mucus following

In Satsuma succincta, no pairs overlapped in laboratory (n= 30, 0%, C.I. = 0 ± 0,

Table 1), while only two marker-tracker pairs overlapped their trails in the field (n=

62, 3.23%, C.I. = 0.43 ± 0.04, Table 1). In Cyclophorus friesianus, five of the trails of pairs were overlapping in laboratory (n= 44, 11.3%, C.I. = 0.48 ± 0.28, Table 1) and field (n= 84, 14.2%, C.I. = 0.68 ± 0.21, Table 1). Although there were few pairs performed trail overlapping, the C.I. values were approaching to 0.5 in laboratory or over 0.5 in field. The C.I. value revealed that the tracker would follow the marker about half of the trail.

Odor following

The odor following frequency of both study areas were significantly higher than expected following ratio in S. succincta (goodness of fit test of 1:5, laboratory, χ2=

11.7, p< 0.001; field, χ2= 20.2, p< 0.001, Table 2), but not in C. friesianus (goodness of fit test of 1:5, laboratory, χ2= 3.74, p= 0.053; field, χ2= 0.90, p= 0.343, Table 2). In addition, no difference of odor following were found between study areas of either species (test for independence of 1:1, S. succincta, χ2= 0.02, p= 0.879; C. friesianus,

χ2= 0.77, p= 0.381).

In S. succincta, more trackers followed the markers by keeping their trail in a similar direction rather than completely overlapping the trail (goodness of fit test of

1:1, laboratory, 0 vs. 12, χ2= 8.33, p= 0.004; field, 2 vs. 23, χ2= 17.6, p< 0.001, Table

3). In C. friesianus, due to there was no significant difference between odor following frequency and expected following ratio, this species preferred followed the markers by keeping their trail completely overlapping the trail. 10

Trail forms

Most trails followed judging by angle were simple as Fig. 3c. However, in S. succincta, there were some complex trails which were similar in profile to each other but not overlapping (Fig. 5). This kind of trail was observed both in laboratory and field in S. succincta, but not in C. friesianus. In C. friesianus there were overlapping trails (Fig. 6) which were not shown in S. succincta.

Inaction time

For S. succincta, the trackers successfully followed the markers took less time to start following than those failed (Mann-Whitney U test, laboratory, Z= -1.67, p=

0.047; field, Z= -1.69, p= 0.045, Table 4). In C. friesianus, I recorded some negative inaction time because some trackers moved before the marker (Table 4). The inaction time of pairs that performed trail overlapping behavior took less time to start following than those failed though marginal significant in the laboratory (Z= -1.60, p=

0.055, Table 4). The successful trackers waited longer than those that failed to follow, though marginal significant in the field (Z= 1.63, p= 0.052, Table 4).

Sexual difference on trail-following behavior

Following frequency

I combined the same sex groups (m-m+f-f) and difference sex group (m-f+f-m) to examine the effect of sex in C. friesianus. The following frequency seems higher in different sex than in same sex in both studying sites (Fisher’s exact test, laboratory, same sex: 1 (4.6%), different sex: 4 (18.2%), p= 0.345; field, same sex: 4 (9.5%), different sex: 8 (19.1%), p= 0.352, Table 5). Sex combination had no significant affection the performing of following behavior in both study areas.

The male trackers performed trail-overlapping following in both laboratory and field while the females only did this in the field (Table 5). Nevertheless, the trail-

11 overlapping following frequencies were not significantly different between male and female trackers in field (goodness of fit test of 1:1, χ2= 3.00, p= 0.083, Fig. 7).

Inaction phase

For the same and different sex combination, the inaction time of trackers that successfully followed the markers was not different from that failed to follow in the laboratory (Mann-Whitney U test, same sex, Z= -1.58, p= 0.057; different sex, Z= -

0.3, p= 0.383, Table 6), and field (Mann-Whitney U test, same sex, Z= 0, p= 0.5; different sex, Z= 1.23, p= 0.109, Table 6).

Discussion

Whether these two species had the ability to track the chemical cue laid by conspecific

Comparing with Random following ratio

Trackers tracking conspecific markers showed a significantly higher trail- following intensity in Satsuma succincta, which is in agreement with the observations on terrestrial pulmonate gastropod, Achatina (Chase et al. 1978). However, this is not shown in Cyclophorus friesianus during the study period which is hot and wet.

Difference in following chemical

S. succincta performed trail-following by odor, and C. friesianus performed trail- following only by mucus. The results of following patterns and inaction time of S. succincta, agree with most terrestrial pulmonate gastropod which has the ability to detect the odor by the optic tentacles (Chase et al. 1978; Cook 1980; Chase and Croll

1981; Davis 2004). The comparatively low frequency in mucus following supported that this behavior is subordinate not only in slug but also in this pulmonate snail

(Cook 1992). As the uncommon terrestrial prosobranch, C. friesianus, the results nearly supported the hypothesis that this species behaves similarly as marine species mostly followed by mucus (Erlandsson and Kostylev 1995; Erlandsson 2002;

Hutchinson et al. 2007; Johannesson et al. 2008; Ng et al. 2011).

Test for sexual difference on trail-following behavior

In this study, although the results did not support that sexual difference might affect the ability to follow, it still had a tendency that male tracked conspecific individual at higher intensities in C. friesianus. Moreover, the higher frequency in female followed by male suggests that this terrestrial prosobranch species would use mucus trail-following as a mating strategy, which agreed with previous studies in 13 several marine prosobranch species (Erlandsson and Kostylev 1995; Erlandsson 2002;

Johannesson et al. 2008; Johannesson et al. 2010; Ng et al. 2011; Ng et al. 2012).

Following purpose

S. succincta

Although S. succincta was supported to have trail-following behavior, the following purpose was not clear yet. During the studying period, the individuals were put in the arena at the same time, however, the two individuals seldom moved around simultaneously or interacted with each other. It was hard to determine whether the following behavior was a way further to find a mate, but during the laboratory experiment period, two pairs of S. succincta mated when they were gathered in the container form isolated. It can be comprehend that aggregation may increase the opportunity to find a mate. In Mt. Soushan, the study area is full of Formosan macaque Macaca cyclopis, which is one of the predator for snails. Coleman et al.

(1999) suggested that aggregating might reducing predation risk. Under these two situation, I speculate the following purpose for aggregating in S. succincta. According to the results, S. succincta also had the ability to follow mucus trail, but this behavior rarely appeared. However, some studies claim that mucus trail-following in terrestrial pulmonate gastropod is associated with mating (Chase et al. 1978; Cook 1992;

Shaheen et al. 2005). Chase et al. (1978) reported that Mediterranean terrestrial gastropod, Otala vermiculata, would not favor the evolution of mucus trail-following behavior because of dry air contributing to the rapid desiccation of trails. To sum up, odor following might be an evolution for terrestrial gastropod to adapt the dry environment because water is the major component of mucus (Davies and Hawkins

1998). For terrestrial species, mucus following might be replaced by odor following because their habitat has been changed from waterside to land, with limited water

14 mucus may dry up easily.

C. friesianus

C. friesianus, a terrestrial prosobranch, followed trail by mucus only, same as the marine prosobranch. In some Littorinidae species, males had significantly higher mucus trail-following intensity tracking conspecific females (Erlandsson and

Kostylev 1995; Ng et al. 2011). Terrestrial pulmonate Achatina fulica living in hot and wet region, can use trail following for finding sexual mates (Chase et al. 1978). It is considered evidence that mucus contains sexual pheromone. In my experiment, this was weak but nevertheless present, and mucus following as a mating strategy needs further study. For mucus trail-following, the function is not only for mating, but also for saving energy. Previous studies suggested that following over fresh trails can save mucus and energy (Davies and Blackwell 2007; Hutchinson et al. 2007). As the result did not support mucus trail-following as a mating strategy, saving energy may be the explanation.

Innovation parameters used in the study

Trails that non-overlapping

In the analysis, I use an angle < 30° as the indication of following. Comparing with previous studies in odor following, most of those focused on food finding

(Lemaire and Chase 1998), homing (Cook 1992), and conspecific aggregating (Chase et al. 1978). These studies focused on individual used odor to reach the target, which was quite different from a marker-tracker pair. This determination of angle < 30° as following is the first applied to odor trail-following, and further studies of the accurate determination while odor tracking would increase our understanding of how terrestrial gastropods track chemical cue in air.

Inaction time

Inaction time is also the first used for chemical trail-following. In S. succincta, this parameter explained the concentrations of chemical cue might diffuse as time passed reasonably. However, the results was quite different in the field experiments of

C. friesianus. I cannot exclude that this situation might correlate with mucus trail- following because the field experiment had higher overlap frequency among other three experiments (S. succincta in two studying areas, and C. friesianus in laboratory).

However, this uncertainty is still under suspicion and needs further work to resolve.

Further studies

In this experiment, two species preformed similarly in both studying areas, which supported that snails would not change their behavior away from their habitat. More sophisticated experiments investigating the trail-following behavior for the two species can be conducted in laboratory. For example, several previous studies investigated the differences conducted tentacle movements of odor tracking of snail in laboratory (Gelperin 1974; Lemaire and Chase 1998; Davis 2004) or mucus (Davis-

Berg 2011). Moreover, the wind direction can affect the odor trail-following because snails may not detect the odor at the up-wind side (Gelperin 1974; Cook 1992).

Related experiment can be conduct under laboratory condition. For most terrestrial species, mucus trail-following may not be a common behavior due to most of the species are pulmonate, however, other terrestrial prosobranch may retain this behavior, and can be conducted in further studies.

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Tables

Table 1. Summary of the mucus following behavior of Satsuma succincta and Cyclophorus friesianus at two study areas.

Species Area number of pairs Frequency of trail overlapping Coincident index (C.I.)

Lab 30 0 0 ± 0 Satsuma succincta Field 62 2 0.43 ± 0.04 Lab 44 5 0.48 ± 0.28 Cyclophorus friesianus Field 84 12 0.68 ± 0.21

Table 2. Comparison of the odor following behavior and expected following ratio of Satsuma succincta and Cyclophorus friesianus at two study areas by chi-square goodness of fit test of 1:5. Observed frequency of odor following Expected following ratio Species Area p-value Trail angle < 30° Trail angle > 30° Trail angle < 30° Trail angle > 30° Lab 12 18 5 25 < 0.001 Satsuma succincta Field 23 37 10 50 < 0.001 Lab 11 28 6.5 32.5 0.053 Cyclophorus friesianus Field 15 57 12 60 0.343

Table 3. Comparison of the mucus and odor following behavior of Satsuma succincta by chi-square goodness of fit test of 1:1. Species Area Frequency of trail overlapping Trail angle < 30° p-value Lab 0 12 0.004 Satsuma succincta Field 2 23 <0.001

Table 4. Comparison of inaction time between individuals successfully following and those not in Satsuma succincta and Cyclophorus friesianus at two study areas by Mann-Whitney U test. Inaction phase for Inaction phase for Species Area n n p-value following non-following Lab 25.4 ± 67.8 12 111.7 ± 163.4 18 0.047 Satsuma succincta Field 176.9 ± 441.1 25 240.8 ± 498.0 37 0.045 Lab -12.2 ± 29.4 5 49.2 ± 115.0 39 0.055 Cyclophorus friesianus Field 397.8 ± 584.3 12 228.0 ± 311.3 72 0.052

Table 5. Summary of the following frequency of C. friesianus in different sexual combining pairs at two study areas. The pairs are male followed by male (m-m), female followed by male (f-m), female followed by female (f-f), and male followed by female (m-f). n for sample size, total represented following frequency which was combined with trails overlapping and non-overlapping. Area Pair n Overlapping Non-overlapping m-m 11 1 10 f-m 11 4 7 Lab f-f 11 0 11 m-f 11 0 11 m-m 21 2 19 f-m 21 7 14 Field f-f 21 2 19 m-f 21 1 20

Table 6. Comparison of the inaction phase of C. friesianus in different sexual combining pairs at two study areas by Mann-Whitney U test. Same sex was combining with m-m and f-f, and different sex was combining with f-m and m-f. Inaction phase for Inaction phase for Area Pair n n p-value following non-following Same sex -60 1 87.6 ± 145.9 21 0.057 Lab Different sex -0.3 ± 14.2 4 4.3 ± 24.1 18 0.383 Same sex 93.8 ± 47.7 4 115.4 ± 106.8 38 0.5 Field Different sex 549.8 ± 675.5 8 357.6 ± 407.9 34 0.109

Figures

Fig 1. The shell of Satsuma succincta. Left image: lateral view showing the spire and aperture of the shell. Central image: dorsal view of the shell, showing the apex. Right image: basal view showing the umbilicus.

Fig 2. The shell of Cyclophorus friesianus. Left image: lateral view showing the spire and aperture of the shell. Central image: dorsal view of the shell, showing the apex Right image: basal view showing the umbilicus.

Fig. 3. Trails for each pair placed at the center of the testing area. Triangle represent position of a marker and circle a tracker. The number is the angle of the two trails. (a) Tracker did not follow marker for the angle > 30°. (b) Tracker followed marker by mucus trail as two trails were overlapping. (c) Tracker followed marker by odor trail for the angle < 30° to form non-overlapping.

Fig. 4. The symbol of random following ratio. The semicircle is divided into six sectors, which is 30° in each. Aligning the trail of marker on the straight line under the arrow, if the angle of the two trails falls in the gray region, it would be defined as follow. If the angle falls in the white region, it would be defined as not follow. The gray region: white region is 1:5.

Fig. 5. Examples of complex but similar profile trails formed by S. succincta markers (triangle) and trackers (circle).

Fig. 6. Examples of overlapped trails formed by C. friesianus markers (triangle) and trackers (circle).The dotted line is the tracker’s body length.

10 N.S. 9 8 7 6 5 4 Frequency 3 2 1 0 Laboratory Field Area

Male tracker Female tracker

Fig. 7. Comparisons of observed male tracker and female tracker (male tracker: m-m, and f-m, female tracker: f-f, and m-f) in overlapping following frequencies of C. friesianus in the lab and field. “N.S.” represents p-value >0.05.