Animal Behaviour 82 (2011) 459e465
Contents lists available at ScienceDirect
Animal Behaviour
journal homepage: www.elsevier.com/locate/anbehav
Mucus trail following as a mate-searching strategy in mangrove littorinid snails
Terence P. T. Ng a, *, Mark S. Davies b, Richard Stafford c, Gray A. Williams a
a
The Swire Institute of Marine Science and The School of Biological Sciences, The University of Hong Kong, Hong Kong SAR, China
Faculty of Applied Sciences, University of Sunderland, Sunderland, U.K.
c
Department of Natural and Social Sciences, University of Gloucestershire, Cheltenham, U.K.
b
a r t i c l e i n f o
Article history:
Received 1 February 2011
Initial acceptance 18 March 2011
Final acceptance 9 May 2011
Available online 1 July 2011
MS. number: 11-00104
Keywords:
Littoraria
littorinid
mangrove
mate discrimination
mate search
mucus trail
polarity
trail following
Mate searching often involves chemical cues and is a key process in determining fitness in most sexually
reproducing animals. Effective mate-searching strategies are, therefore, essential for individuals to avoid
wasting resources as a result of misrecognition of mating partners. Marine snails in the genus Littoraria
are among the most successful molluscan groups that live closely associated with mangroves. Their
population densities are often low, and finding a mate within the complex three-dimensional habitat of
tree leaves, branches and trunks requires an effective searching strategy. We tested whether males of
L. ardouiniana and L. melanostoma located females by following their mucus trails. In the laboratory, male
tracker snails followed mucus trails laid by conspecific female marker snails at a higher intensity
compared with other markeretracker sex combinations in the mating season, but not in the nonmating
season, and this was more pronounced in L. ardouiniana. Male trackers did not move faster when
following the trails of conspecific female markers compared with other sex combinations; however,
tracker snails moved faster in the mating than in the nonmating season, although this might be related to
temperature. In both species, males tracked females regardless of trail complexity, and the majority of
male trackers were able to detect the direction (polarity) of the trails of conspecific females. Together
with previous studies on rocky shore Littorina species, these findings suggest that sex pheromones are
incorporated into mucus trails to facilitate the reproductive success of these snails. Mucus trail following
is, therefore, an adaptive mate-searching strategy in intertidal gastropod molluscs, and potentially in
other gastropod groups in which trail-following behaviour is prevalent.
Ó 2011 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Along with foraging or predator avoidance, finding a mate is
a key behaviour that determines the fitness of an individual (Krebs
& Davies 1993). In most internally fertilizing dioecious species,
reproductive success is strongly determined by the ability to search
for mates of the right species and sex (Parker 1978). Selection for
reproductive traits that will facilitate successful mate location is,
therefore, likely to reduce the time and effort wasted in locating
and ‘copulating’ with organisms of the wrong species and sex
(Andersson 1994). mate-searching mechanisms vary among
species, but are generally energetically costly activities that involve
physical movement and other forms of behaviour such as the
production of chemical cues (Kokko & Wong 2007). Chemical cues
involved in mate searching (sex pheromones) have been extensively studied and reported across animal taxa, especially in insects
(Mayer & McLaughlin 1990; Landolt 1997), and are the subject of
increasing attention in other animal groups such as molluscs
(Susswein & Nagle 2004). Apart from releasing pheromones
* Correspondence: Terence P. T. Ng, The Swire Institute of Marine Science, The
University of Hong Kong, Cape d’Aguilar, Shek O, Hong Kong SAR, China.
E-mail address: [email protected] (T.P.T. Ng).
directly into air or water, animals often incorporate their pheromones into excretory fluids such as urine, sweat and mucus (Law &
Regnier 1971; Wyatt 2003; Tirindelli et al. 2009). The study of how
animals utilize different media to transmit their sex pheromones is,
therefore, fundamental to increasing our understanding of sexual
communication in animals (Cardé & Baker 1984; Tirindelli et al.
2009).
Gastropod molluscs are important components of intertidal
communities (Underwood 1979). Mate searching for these animals
can be difficult, however, as intertidal habitats often support
substantial numbers of co-occurring species, the activity windows
of which are usually synchronized to specific tidal conditions (Little
et al. 2009). Snails in the family Littorinidae are among the most
common intertidal molluscan groups. They are widely distributed
throughout tropical and temperate regions, and play a significant
role in the ecology of intertidal communities (Reid 1989; McQuaid
1996). Their abundance and ubiquitous distribution make these
snails excellent models to study mate-searching strategies in
intertidal gastropod molluscs.
It has been suggested that members of the rocky shore genus
Littorina incorporate pheromones in their mucus trails to facilitate
mate searching (Struhsaker 1966; Dinter 1974; Saur 1990;
0003-3472/$38.00 Ó 2011 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.anbehav.2011.05.017
460
T.P.T. Ng et al. / Animal Behaviour 82 (2011) 459e465
Erlandsson & Kostylev 1995; Johannesson et al. 2010). Johannesson
et al. (2010), for example, demonstrated that during the mating
season, males of three Littorina species (L. littorea, L. fabalis and
L. obtusata) followed mucus trails laid by conspecific females for
longer distances than mucus trails laid by conspecific males. Males
of another rocky shore species, Littorina saxatilis, were even able to
discriminate between mucus trails laid by conspecific females of
different ecotypes (Johannesson et al. 2008). Despite this extensive
work on the rocky shore genus Littorina, it is unknown whether
mucus trail following is an important strategy for mate searching in
other members of the family Littorinidae, particularly those from
different habitats.
Within the Littorinidae, snails in the genus Littoraria are among
the most successful gastropods in mangrove forests in the IndoPacific region (Reid 1986; Reid et al. 2010), where they play an
important role in food web dynamics (Alfaro 2007, 2008). Littoraria
often mate in a defined season, and males actively search and
mount females (Gallagher & Reid 1974). Population densities of
these snails are often low in mangroves, however (Reid 1985; Lee &
Williams 2002b; Sanpanich et al. 2008; Torres et al. 2008), and
searching for mates in the complex three-dimensional habitat of
tree leaves, branches and trunks would appear to be extremely
difficult without any specific mate locating strategies.
In this study, we investigated mating behaviour in the most
abundant littorinids found in the tree canopy of Hong Kong
mangroves: Littoraria ardouiniana (Heude 1885) and Littoraria
melanostoma (Gray 1839) (Lee & Williams 2002b). Males of these
two species search for, and mate with, females most intensively
during the hot, wet summer, and the frequency of males forming
pairs with males or heterospecific females is very low (Ng & Williams, unpublished data). On this basis, it was hypothesized that
trail following could be an effective mate-searching strategy
adopted by males to identify and locate conspecific females. To test
this, experiments were conducted in the laboratory to investigate
various aspects of mucus trail-following behaviour in relation to
the mating and nonmating seasons, to determine the role of mucus
trail following in mate searching in these mangrove snails.
METHODS
Animal Collection
Large (21e26 mm) mature (>12 mm; Lee & Williams 2002a)
L. ardouiniana and L. melanostoma were collected from Kandelia
obovata trees at Tsim Bei Tsui (22 290 N, 114 000 E), in outer Deep
Bay, NW New Territories, Hong Kong. Snails were sexed in situ by
turning their shells to determine whether the snails had a penis,
and sexes were held separately to prevent mating.
In the laboratory, male and female snails were kept in separate
tanks at 27e29 C in the mating season (July to August 2009, during
Hong Kong’s hot and wet season; see Kaehler & Williams 1996) and
at 18e20 C in the nonmating season (December to January 2009,
during Hong Kong’s cool and dry season) without water and food
overnight. All experiments were conducted on the day after
collection. Similar to other littorinids (e.g. Littorina subrotundata;
Zahradnik et al. 2008), males of L. ardouiniana and L. melanostoma
frequently mate with females on consecutive days after collection
from the field (T. P. T. Ng, unpublished observations). Mating before
experiments was, therefore, not assumed to be a significant confounding factor for the mucus trail-following behaviour of the snails.
Trail Following
Various combinations of the same or different species, or sex, of
snails were tested to determine responses to different mucus trails.
Experimental conditions were based on the environmental conditions animals experienced in the field, where activity is mainly
elicited by rain and moisture brought by the tide and dew, and
where submersion by sea water is generally avoided (Reid 1985;
Yipp 1985; Lee & Williams 2002a). To achieve this, snails were
activated by spraying with double distilled water (Little & Stirling
1984). Light was primarily provided by overhead fluorescent
lights and laboratory windows were covered with blinds to minimize external stimuli. Experiments were conducted in the same
room, and therefore at the same temperature, as the snails were
stored.
Three temporal replicate series of experiments were conducted
for both L. ardouiniana and L. melanostoma in both the mating and
nonmating season. In each set of experiments, snails were
randomly assigned as markers and trackers. Markers were snails
used to crawl and lay a mucus trail, whereas trackers were the
snails that were placed onto the starting point of the marker’s
mucus trail to allow trail following. In total five markeretracker
combinations were tested for each species, namely MaleeMale;
FemaleeMale; MaleeFemale; FemaleeFemale and Heterospecific
FemaleeMale, with 10 replicate trials for each combination
(SN ¼ season sex combination replicate series replicate ¼
2 5 3 10 ¼ 300).
Experiments were conducted on a plastic board (60 60 cm)
with a test arena of 50 50 cm and a fixed, marked centre point. In
each trial, a clean acetate sheet was attached to the plastic board
and a mist of double distilled water sprayed onto it. The marker
snail was placed at the centre and allowed to crawl until it reached
the boundary, where it was removed. Using this method, the snail’s
trail was clearly visible. The acetate sheet was then sprayed with
double distilled water to homogenize the surface and the tracker
snail was then introduced. To avoid errors caused by body orientation after attachment, the tracker snail was allowed to attach onto
a small moist acetate disc and then positioned so that it would
crawl onto the starting point of the marker trail. The tracker was
allowed to crawl until it reached the boundary of the arena, where
it was removed. During the experiment, the trail-following time
(time moving on the marker snails’ trail) of the tracker snails was
recorded. The marker snail’s distance and the overlapping distance
of the marker and the tracker trails were traced using a digital map
measurer (Yorter Model No. V-930, accuracy: 1 mm). The net
displacement (direct line from the initial point to where the snail
left the arena) of the marker trail was measured using a ruler
(1 mm). The experimental order for each replicate trial was
randomized and each snail was used only once, with clean acetate
sheets for each experimental trial. The tortuosity index (TI; net
displacement/distance travelled) of the marker and the coincidence
index (CI, distance followed by tracker/total trail distance of the
marker) were scored following Davies & Beckwith (1999). TI ¼ 1
when the marker produces a straight trail, and the more complex
the trail becomes, the smaller the value of TI. Trackers that
completely follow a marker score a CI value of 1, and 0 when they
do not follow the marker at all.
Polarity
To determine whether males can detect and respond to the
direction (polarity) of conspecific females from their mucus trails,
another trail-following experiment was conducted during the
mating season. An acetate sheet was evenly sprayed with a mist of
double distilled water and a female marker snail was placed at one
end of the sheet and allowed to crawl towards the opposite end.
The marker snail was then removed and the acetate sheet was
randomly rotated in either direction through 90 and sprayed with
double distilled water to homogenize the surface. A male tracker
T.P.T. Ng et al. / Animal Behaviour 82 (2011) 459e465
was then placed at least a body length away from the trail and
allowed to move forward so that it encountered the marker trail at
an angle of w90 . The tracker snail was judged to have followed the
marker trail if its trail overlapped with the marker trail for 5 cm
(Clifford et al. 2003). A positive (þ) polarity was scored if the
tracker followed the trail in the same direction as the marker, and
negative (e) polarity if the tracker followed the trail in the opposite
direction. A clean acetate sheet and fresh snails were used for each
trial.
Statistical Methods
To examine variation in the CI and trail-following speed (trailfollowing distance/trail-following time) of the tracker between the
five markeretracker sex combinations, and between the mating
and the nonmating seasons (fixed factors), a three-factor mixed
model ANOVA was conducted, with the three series of experiments
being a random factor nested within season, followed by StudenteNewmaneKeuls (SNK) post hoc tests. The same statistical
model was also applied to test for variation in marker TI and marker
distance to determine whether there were any confounding effects
brought about by these factors. Data were checked for homogeneity
of variances (Cochran’s test). Where variances were heterogeneous,
data were transformed where possible (double square root transformations on CI and speed data and square root (X þ 0.5) transformation on marker TI data for L. melanostoma; and square root
(X þ 0.5) transformation of speed data for L. ardouiniana). Marker TI
and distance data for L. ardouiniana were not possible to homogenize using standard transformations; however, ANOVA was still
performed on untransformed data, as it is considered to be robust
when there is a large sample size and balanced design (Underwood
1997). A two-way ANOVA was performed to compare the intensity
of males of either species (species, fixed factor) following conspecific or heterospecific females (sex combination, fixed factor) in the
mating season. Student’s t tests were applied to examine differences in trackers’ trail-following speed between the two species in
both seasons (data were double square root transformed to meet
requirements of normality). Spearman rank correlation analyses
were used to investigate the relationship between TI and CI in all
experimental trials and, in particular, the female markeremale
tracker combination of both species in the mating season to
investigate whether males were able to track females, regardless of
the complexity of the trails. Binomial tests were performed to
investigate whether male snails showed polarity in following
females’ mucus trails in 50 replicate trials for each species. All
461
ANOVAs and SNK post hoc tests were performed using GMAV 5
(University of Sydney, Underwood & Chapman 1997) and all other
statistical analyses were performed using SPSS 16.0 (SPSS Inc.,
Chicago, IL, U.S.A).
RESULTS
Trail Following
All experimental snails were observed to make frequent contact
with the substratum using their tentacles, and this may suggest
that they are able to detect the surrounding environment, including
mucus trails, when they are crawling. When the tracker encountered a deviation in the marker trail, the snail had one tentacle
touching the mucus trail while the other tentacle reached outside
the trail. This behaviour may allow the tracker to recognize the trail
turning and help the snail continue to follow the trail.
Males tracked conspecific females at a higher intensity than any
other sex combinations in the mating season, but not in
the nonmating season, for both L. ardouiniana and L. melanostoma
(Tables 1, 2, Fig. 1). In the case of L. ardouiniana, there was
a significantly higher CI in the mating than in the non-mating
season for the markeretracker sex combinations of both malee
male and femaleemale combinations (Table 1). In contrast,
L. melanostoma showed a higher CI value only for the femaleemale
combination in the mating season (Table 2). These results were
consistent for all experimental repeats in both seasons (mixed
model ANOVAs: Set (Season): P > 0.05, Tables 1 and 2). In the
mating season, males of both species tracked conspecific females at
higher intensities (greater CI) than heterospecific females (Table 3).
L. ardouiniana, however, showed a significantly greater CI than L.
melanostoma when males tracked conspecific females but not
heterospecific females (Table 3).
Effect of Trail Complexity on Trail Following
All marker trails of both species scored similar TIs among the sex
combinations in both seasons (L. ardouiniana: mean 95%
confidence intervals ¼ 0.89 0.01, N ¼ 300; L. melanostoma:
0.88 0.01, N ¼ 300; mixed model ANOVAs: all factors P > 0.05). A
weak but significant positive correlation was recorded between the
CI and marker TI in all experimental trials for L. ardouiniana
(Spearman rank correlation: rs ¼ 0.167, N ¼ 300, P < 0.001), indicating that tracker snails of L. ardouiniana followed less complex
mucus trails at a higher intensity. No significant correlation was
Table 1
Variation in coincidence index for Littoraria ardouiniana
Source of variation
Season
Sex combination
Set (Season)
Season*Sex combination
Season*Set (Season)
Error
SNK tests (Season*Sex combination)
Season
Mating:
HM¼MM¼MF¼FF<FM
Nonmating: HM¼MM¼MF¼FM¼FF
df
1
4
4
4
16
270
Mean square
F
P
1.687
0.534
0.049
0.595
0.054
0.088
34.61
9.96
0.55
11.10
0.61
0.004
<0.001
0.698
<0.001
0.878
Sex combination
HM: NM¼M
MF: NM¼M
FF: NM¼M
MM: NM<M
FM: NM<M
Summary of comparisons using mixed model ANOVA to investigate variation in coincidence index between the five sex combinations (sex combination: fixed factor) in the
mating and nonmating season (season: fixed factor) with the three experimental repeats in each season (nested within season, set: random factor). Variances were
homogeneous (Cochran’s test: P > 0.05). Significant interactions (P < 0.05; in bold) were further analysed using StudenteNewmaneKeuls (SNK) post hoc tests. Abbreviations
for sex combinations: HM ¼ male follows heterospecific female; MM ¼ male follows male; MF ¼ female follows male; FM ¼ male follows female; FF ¼ female follows female;
NM ¼ nonmating season; M ¼ mating season.
462
T.P.T. Ng et al. / Animal Behaviour 82 (2011) 459e465
Table 2
Variation in coincidence index for Littoraria melanostoma
Source of variation
df
Season
Sex combination
Set (Season)
Season*Sex combination
Season*Set (Season)
Error
Mean square
1
4
4
4
16
270
0.004
0.072
0.052
0.059
0.015
0.022
SNK tests (Season*Sex combination)
Season
Mating:
HM¼MM¼MF¼FF<FM
Nonmating: HM¼MM¼MF¼FM¼FF
Sex combination
HM: NM¼M
MF: NM¼M
FF: NM¼M
F
0.08
4.67
2.32
3.81
0.69
P
0.787
0.011
0.058
0.023
0.805
MM: NM¼M
FM: NM<M
Summary of comparisons using mixed model ANOVA to investigate variation in coincidence index between the five sex combinations (sex combination: fixed factor) in the
mating and nonmating season (season: fixed factor) with the three experimental repeats in each season (nested within season, set: random factor). Variances were
homogenous after double square root transformation (Cochran’s test: P > 0.05). Significant interactions (P < 0.05; in bold) were further analysed using StudenteNewmaneKeuls (SNK) post hoc tests. Abbreviations for sex combinations: see Table 1.
found, however, when the analysis was performed only for the
female markeremale tracker combinations in the mating season
for both L. ardouiniana and L. melanostoma (Spearman rank correlations: P > 0.05), which suggests that males followed conspecific
females in the mating season irrespective of the complexity of their
trails.
1
0.8
Mating season
Nonmating season
L. ardouiniana
0.6
Coincidence index
0.4
Effect of Marker Trail Distance on Trail Following
Littoraria ardouiniana showed a longer marker trail distance
in the mating season (mean 95% confidence intervals ¼
324 11 mm, N ¼ 150) than in the nonmating season (310 7 mm,
N ¼ 150, mixed model ANOVA: Season: F1,270 ¼ 65.04, P < 0.01; Sex
combination and Set (Season): P > 0.05), indicating (given the
square arena and the similar TIs) that more marker L. ardouiniana
crawled towards the arena corners in the mating season. Even if two
tracker snails followed marker trails for a similar distance, based on
the equation for calculating CI (i.e. distance followed by tracker/total
trail distance of the marker), a smaller CI value would be scored for
an individual that followed a longer marker trail. To account for this
effect, an additional test using the same mixed model ANOVA and
SNK tests was conducted on the tracker trail-following distance
instead of CI, which gave a similar outcome to the original analysis
and confirmed that marker trail distance did not confound the
original analysis. Littoraria melanostoma showed no difference in
marker trail distance between sex combination or between season
(mean ¼ 318 6 mm, N ¼ 300, mixed model ANOVA: all factors
P > 0.05).
0.2
Trail-following Speed
0
1
0.8
Mating season
Nonmating season
L. melanostoma
0.6
0.4
Tracker snails moved significantly faster, approximately two times
faster, when following mucus trails in the mating season (L. ardouiniana: mean 95% confidence intervals ¼ 1.62 0.12 mm/s, N ¼ 150;
L. melanostoma: 1.30 0.12 mm/s, N ¼ 150) than during the nonmating season (L. ardouiniana: 0.85 0.07 mm/s, N ¼ 150; L. melanostoma: 0.61 0.05 mm/s, N ¼ 150) in both L. ardouiniana (mixed
model ANOVA: Season: F1,270 ¼ 55.93, P < 0.01) and L. melanostoma
(mixed model ANOVA: Season: F1,270 ¼ 61.17, P < 0.01) for all sex
combinations and experimental repeats (for both species, mixed
model ANOVAs: Sex combination and Set (Season): P > 0.05). In
general, L. ardouiniana tracked marker snails faster than L. melanostoma in both seasons (mating season, Student’s t test: t298 ¼ 3.85,
P < 0.001; nonmating season, Student’s t test: t298 ¼ 5.62, P < 0.001).
0.2
Polarity
0
HM
MM
MF
FM
Sex combination
FF
Figure 1. Mean coincidence indexes for the five markeretracker sex combinations in
the mating and nonmating seasons of both species. Abbreviations for sex combinations: see Table 1. Error bars: 95% confidence intervals.
In 50 trials, 32 (64%) male L. ardouiniana trackers and 26 (52%)
male L. melanostoma trackers followed the mucus trails laid by the
female markers. In both species, most male trackers (L. ardouiniana:
28 out of 32 cases, binomial test: P < 0.001; L. melanostoma: 19 out
of 26 cases, binomial test: P ¼ 0.029) were able to follow the mucus
T.P.T. Ng et al. / Animal Behaviour 82 (2011) 459e465
463
Table 3
Variation in coincidence index between Littoraria ardouiniana and L. melanostoma
Source of variation
df
Mean square
F
P
Species
Sex combination
Species*Sex combination
Error
1
1
1
116
0.999
3.978
0.363
0.091
11.04
43.08
4.01
0.001
<0.001
0.048
SNK tests (Species*Sex combination)
Species
LM: HM<FM
LA: HM<FM
Sex combination
HM: LM¼LA
FM: LM<LA
Summary of comparisons using two factor ANOVA to investigate variation in coincidence index between the two species (species: fixed factor) when males tracked heterospecfic females and conspecific females (sex combination: fixed factor) in the mating season. Variances were homogeneous (Cochran’s test: P > 0.05). Significant interactions
(P < 0.05; in bold) were further analysed using StudenteNewmaneKeuls (SNK) post hoc tests. Abbreviations: LM ¼ L. melanostoma; LA ¼ L. ardouiniana. Abbreviations for sex
combinations: see Table 1.
trails in the same direction as the female markers laid the trail (i.e.
showed a þ polarity).
DISCUSSION
Males tracking conspecific females showed a significantly
higher trail-following intensity in both L. ardouiniana and
L. melanostoma in the mating season, but not in the nonmating
season. This provides strong evidence that males of these mangrove
littorinids actively search for females and are capable of discriminating between the mucus trails laid by conspecifics and heterospecifics, as well as between conspecific males and females during
the mating season. These results match observations on several
rocky shore Littorina species (e.g. L. littorea, L. fabalis and L. obtusata;
Erlandsson & Kostylev 1995; Johannesson et al. 2010), and illustrate
that utilizing mucus trails as a mate-searching mechanism is not
unique to littorinids in the genus Littorina living on rocky substrata,
but is also used by Littoraria species living in the canopy of
mangrove trees.
Although males of the two Littoraria species in this study and
several Littorina species can discriminate conspecific males and
females from mucus trails, there are exceptions where males
appear incapable of identifying females from mucus trails (e.g.
Littorina saxatilis, Erlandsson 2002; Littorina keenae and Littoraria
irrorata, Peters 1962). Johannesson et al. (2010) proposed a sexual
conflict model to address this issue, suggesting that when population density of a species is high, excessive mating may reduce
fitness of females, and hence selection might favour females to
mask their sex in their mucus trails. Apart from population density,
another factor that might indicate whether a male littorinid can
recognize conspecific females from their mucus trails is the
occurrence of in situ false mating pairs. Erlandsson (2002) found
only w5% of false pairs (maleemale) in the ‘mate discriminator’
L. littorea, whereas the ‘mate indiscriminator’ L saxatilis showed
over 30% false pairs. The two mangrove littorinids L. ardouiniana
and L. melanostoma also conform to the above suggested indicators,
in that their population density is low (Lee & Williams 2002b; T. P.
T. Ng, unpublished observations) and very few (<10%) false mating
pairs are observed in the field (Ng & Williams, unpublished data).
Male trackers of both species did not, however, always follow the
entire trails of conspecific females during the mating season:
a pattern that has also been reported in L. littorea, suggesting that
the ability to discriminate sex from a trail may vary between individual males (Erlandsson & Kostylev 1995). The use of artificial
substrata providing no food component (i.e. the plant tissues on
their natural substratum; Lee et al. 2001) in laboratory experiments
may also be a factor causing reluctance for individuals to commit to
long-distance trail following. In preliminary trials, when male snails
were allowed to follow trails laid by female conspecifics on sections
of natural mangrove tree branches, and therefore presumably had
access to food resources, they invariably followed the entire trails (T.
P. T. Ng, unpublished observations). In a few cases, males might be
reluctant to follow conspecific females that are infected by trematode parasites (Erlandsson & Kostylev 1995; Davies & Knowles
2001), but this is unlikely in the present species, as infection rate
is extremely low (Ng & Williams, unpublished data). Occasional
occurrences (w14% in L. ardouiniana and w8% in L. melanostoma of
all experimental trials including both seasons) of long-distance trail
following (i.e. CI 0.5) in other sex combinations (male follows
male; male follows female; male follows heterospecific female;
female follows female) were also observed. As the mucus of these
two Littoraria species is more viscous than that of the patellogastropods, European littorinids and pulmonate limpets (Lee & Davies
2000), this may imply more energy is allocated to mucus production
in Littoraria. Mucus trail following, therefore, may have other
functions in addition to mate finding, such as in saving energy by
utilizing the mucus trails laid by other individuals (Davies &
Blackwell 2007). Starvation may also increase the tendency of
mucus trail following, as energy can be obtained through mucus
ingestion (Edwards & Davies 2002; Hutchinson et al. 2007). This is
unlikely to be a confounding factor in this study, however, as
experiments were conducted the day after snails were collected, so
the snails were unlikely to be starving.
In the mating season, males of both species did not show
a difference in trail-following intensity (CI) when tracking heterospecific females, but male L. ardouiniana tracked conspecific
females at a greater intensity than male L. melanostoma. As L.
ardouiniana occurs at a higher level on the mangrove trees than L.
melanostoma (Yipp 1985; Lee & Williams 2002b), L. ardouiniana
might need to rely more on trail following to locate mates owing to
the increased spatial complexity high in the mangrove canopy due
to the greater intensity of branches and leaves and the reduced
frequency of wetting by sea water. The increased tendency of L.
ardouiniana to follow mucus trails may also imply greater affinity to
form mating pairs in L. ardouiniana, which matches field observations (Ng & Williams, unpublished data).
Male trackers of both species did not move faster when following
conspecific females (as opposed to following other snails), but
trackers of all sex combinations moved faster in the mating than in
the nonmating season. This is probably a result of the overall
increase in ambient temperature (7e11 C higher) in the mating
season compared with the nonmating season. Many snails, for
example L. littorea, move faster at higher environmental temperatures (Newell 1958; Erlandsson & Kostylev 1995). The difference in
the tracker trail-following speeds between the two species could be
associated with morphological constraints such as differences in
shell weight, as the shell of L. ardouiniana is relatively thinner than
that of L. melanostoma (Lee & Williams 2002b; T. P. T. Ng,
464
T.P.T. Ng et al. / Animal Behaviour 82 (2011) 459e465
unpublished observations), which may facilitate faster movement.
Being able to move faster, and hence locate mates more quickly
within the narrow activity window available in the mangroves
(when wetted by rain or dew, or occasionally the rising tide; Little &
Stirling 1984; Yipp 1985), may further account for L. ardouiniana
showing a higher affinity to form mating pairs in the field.
Many males of both species did not follow mucus trails laid by
conspecific females in the experiment to investigate response to
trail polarity. This may indicate that a choice is made by the males,
that females do not always provide a cue, or that some males
cannot detect polarity and therefore cannot discriminate between
sexes, as proposed to explain why some males did not follow the
whole trails of conspecific females. However, most males were
capable of detecting trail polarity, as male trackers usually followed
female markers in the direction the trail was laid in both species.
The ability to detect trail polarity is common in littorinids (e.g. L.
littorea, Gilly & Swenson 1978; Nodilittorina unifasciata, Chapman
1998; Littoraria irrorata, Stirling & Hamilton 1986) and improves
the chances of finding mates (Johannesson et al. 2008) or joining an
aggregation before emersion (Stafford et al. 2007; 2008). The
mechanisms involved in the detection of trail polarity are not
understood, but there is evidence for the presence of both chemical
and physical cues (Cook & Cook 1975; Stirling & Hamilton 1986).
Being one of the most successful and widely distributed intertidal molluscan groups (Reid 1989; Williams et al. 2003), it seems
reasonable to assume that successful adaptive reproductive traits
must have evolved in the family Littorinidae, and mucus trail
following is likely to be one of the mechanisms that these snails
adopt for mate searching in intertidal habitats. By increasing
reproductive success, mucus trail following may account for, or
defray part of, the substantial production cost of mucus (Davies
et al. 1992; Davies & Hawkins 1998). If this is the case, it would
be reasonable to expect other internal-fertilizing gastropods also to
use this strategy, as mucus trail following is a common behaviour
observed in many gastropod species (Townsend 1974; Croll 1983;
Cook 1985; Alfaro 2007; Takeichi et al. 2007). As mucus production is such an energetically costly process, it seems reasonable to
assume that it must play an important role in the fitness of
gastropods, as opposed to simply being a very expensive means of
locomotion (Denny 1980). Further studies on different gastropod
taxa, and to identify the sex pheromones in pedal mucus, may
provide insights into the driving force behind the evolution of such
a costly excretory product in gastropod molluscs, and other dioecious animals that exude mucus.
Acknowledgments
We thank Miranda Cheng and Kelvin Wong for assistance with
field sampling. We are grateful to colleagues of the Hard Rock
Ecology Group and Stella Wong, HKU for their valuable comments
and suggestions on the manuscript. Ms Cecily Law, The Swire
Institute of Marine Science, HKU is thanked for her technical
support. This research was funded by a HKU postgraduate
studentship and conducted in partial fulfilment of a PhD degree by
T. P. T. Ng.
References
Alfaro, A. C. 2007. Migration and trail affinity of snails, Littoraria scabra, on
mangrove trees of Nananu-i-ra, Fiji Islands. Marine and Freshwater Behaviour
and Physiology, 40, 247e255.
Alfaro, A. C. 2008. Diet of Littoraria scabra, while vertically migrating on mangrove
trees: gut content, fatty acid, and stable isotope analyses. Estuarine, Coastal and
Shelf Science, 79, 718e726.
Andersson, M. 1994. Sexual Selection. Princeton, New Jersey: Princeton University
Press.
Cardé, R. T. & Baker, T. C. 1984. Sexual communication with pheromones. In:
Chemical Ecology of Insects (Ed. by W. J. Bell & R. T. Cardé), pp. 355e383. New
York: Chapman & Hall.
Chapman, M. G. 1998. Variability in trail-following and aggregation in Nodilittorina
unifasciata Gray. Journal of Experimental Marine Biology and Ecology, 224, 49e71.
Clifford, K. T., Gross, L., Johnson, K., Martin, K. J., Shaheen, N. & Harrington, M. A.
2003. Slime-trail tracking in the predatory snail, Euglandina rosea. Behavioural
Neuroscience, 117, 1086e1095.
Cook, A. 1985. Functional aspects of trail following in the carnivorous snail
Euglandina rosea Ferussac. Malacologia, 26, 173e181.
Cook, S. B. & Cook, C. B. 1975. Directionality in the trail-following response of the
pulmonate limpet Siphonaria alternata. Marine and Freshwater Behaviour and
Physiology, 3, 147e155.
Croll, R. P. 1983. Gastropod chemoreception. Biological Reviews, 58, 293e319.
Davies, M. S. & Beckwith, P. 1999. Role of mucus trails and trail-following in the
behaviour and nutrition of the periwinkle Littorina littorea. Marine Ecology
Progress Series, 179, 247e257.
Davies, M. S. & Blackwell, J. 2007. Energy saving through trail following in a marine
snail. Proceedings of the Royal Society B, 274, 1233e1236.
Davies, M. S. & Hawkins, S. J. 1998. Mucus from marine molluscs. Advances in
Marine Biology, 34, 1e71.
Davies, M. S., Jones, H. & Hawkins, S. J. 1992. Pedal mucus production in Littorina
littorea. In: Proceedings of the Third International Symposium on Littorinid Biology
(Ed. by J. Grahame, P. J. Mill & D. G. Reid), pp. 227e233. London: The Malacological Society of London.
Davies, M. S. & Knowles, A. J. 2001. Effects of trematode parasitism on the
behaviour and ecology of a common marine snail (Littorina littorea (L.)). Journal
of Experimental Marine Biology and Ecology, 260, 155e167.
Denny, M. 1980. Locomotion: the cost of gastropod crawling. Science, 208,
1288e1290.
Dinter, I. 1974. Pheromonal behaviour in the marine snail Littorina littorea Linnaeus.
Veliger, 17, 37e39.
Edwards, M. & Davies, M. S. 2002. Functional and ecological aspects of the mucus
trails of the intertidal prosobranch gastropod Littorina littorea. Marine Ecology
Progress Series, 239, 129e137.
Erlandsson, J. 2002. Do reproductive strategy and breeding season influence the
presence of mate recognition in the intertidal snail Littorina? Invertebrate
Reproduction & Development, 41, 53e60.
Erlandsson, J. & Kostylev, V. 1995. Trail following, speed and fractal dimension of
movement in a marine prosobranch, Littorina littorea, during a mating and
a non-mating season. Marine Biology, 122, 87e94.
Gallagher, S. B. & Reid, G. K. 1974. Reproductive behaviour and early development
in Littorina scabra angulifera and Littorina irrorata (Gastropoda: Prosobranchia)
in the Tampa Bay region of Florida. Malacological Review, 7, 105e125.
Gilly, W. F. & Swenson, R. P. 1978. Trail following by Littorina: washout of polarized
information and the point of paradox test. Biological Bulletin, 155, 439.
Hutchinson, N., Davies, M. S., Ng, J. S. S. & Williams, G. A. 2007. Trail following
behaviour in relation to pedal mucus production in the intertidal gastropod
Monodonta labio (Linnaeus). Journal of Experimental Marine Biology and Ecology,
349, 313e322.
Johannesson, K., Havenhand, J. N., Jonsson, P. R., Lindegarth, M., Sundin, A. &
Hollander, J. 2008. Male discrimination of female mucus trails permits assortative mating in a marine snail species. Evolution, 62, 3178e3184.
Johannesson, K., Saltin, S. H., Duranovic, I., Havenhand, J. N., Jonsson, P. R. &
Gemmell, N. J. 2010. Indiscriminate males: mating behaviour of a marine snail
compromised by a sexual conflict? PLoS One, 5, e12005.
Kaehler, S. & Williams, G. A. 1996. Distribution of algae on tropical rocky shores:
spatial and temporal patterns of non-coralline encrusting algae in Hong Kong.
Marine Biology, 125, 177e187.
Kokko, H. & Wong, B. B. M. 2007. What determines sex roles in mate searching?
Evolution, 61, 1162e1175.
Krebs, J. R. & Davies, N. B. 1993. An Introduction to Behavioural Ecology. 3rd edn.
Oxford: Blackwell Science.
Landolt, P. J. 1997. Sex attractant and aggregation pheromones of male phytophagous insects. American Entomologist, 43, 12e22.
Law, J. H. & Regnier, F. E. 1971. Pheromones. Annual Review of Biochemistry, 40,
533e548.
Lee, O. H. K. & Davies, M. S. 2000. Mucus production and morphometrics in the
mangrove littorinids, Littoraria melanostoma and L. ardouiniana. In: The Tenth
International Marine Biological Workshop: The Marine Flora and Fauna of Hong
Kong and Southern China Vol. V (Ed. by B. Morton), pp. 241e253. Hong Kong:
Hong Kong University Press.
Lee, O. H. K. & Williams, G. A. 2002a. Locomotor activity patterns of the mangrove
littorinids, Littoraria ardouiniana and L. melanostoma, in Hong Kong. Journal of
Molluscan Studies, 68, 235e241.
Lee, O. H. K. & Williams, G. A. 2002b. Spatial distribution patterns of Littoraria
species in Hong Kong mangroves. Hydrobiologia, 481, 137e145.
Lee, O. H. K., Williams, G. A. & Hyde, K. D. 2001. The diets of Littoraria ardouiniana
and L. melanostoma in Hong Kong mangroves. Journal of the Marine Biological
Association of the United Kingdom, 81, 967e973.
Little, C. & Stirling, P. 1984. Activation of a mangrove snail, Littorina scabra scabra
(L.) (Gastropoda: Prosobranchia). Australian Journal of Marine and Freshwater
Research, 35, 607e610.
Little, C., Williams, G. A. & Trowbridge, C. D. 2009. The Biology of Rocky Shores. 2nd
edn. Oxford: Oxford University Press.
T.P.T. Ng et al. / Animal Behaviour 82 (2011) 459e465
McQuaid, C. D. 1996. Biology of the gastropod family Littorinidae. II. Role in the
ecology of intertidal and shallow marine ecosystems. Oceanography and Marine
Biology: Annual Review, 34, 263e302.
Mayer, M. S. & McLaughlin, J. R. 1990. Handbook of Insect Pheromones and Sex
Attractants. Boca Raton, Florida: CRC Press.
Newell, G. E. 1958. The behaviour of Littorina littorea (L.) under natural conditions
and its relation to position on the shore. Journal of the Marine Biological Association of the United Kingdom, 37, 229e239.
Parker, G. A. 1978. Searching for mates. In: Behavioural Ecology: An Evolutionary
Approach (Ed. by J. R. Krebs & N. B. Davies), pp. 141e166. Sunderland, Massachusetts: Sinauer.
Peters, R. S. 1962. Function of the cephalic tentacles in Littorina Philippi (Gastropoda: Prosobranchiata). Veliger, 7, 143e148.
Reid, D. G. 1985. Habitat and zonation patterns of Littoraria species (Gastropoda,
Littorinidae) in Indo-Pacific mangrove forests. Biological Journal of the Linnean
Society, 26, 39e68.
Reid, D. G. 1986. The Littorinid Molluscs of Mangrove Forests in the Indo-Pacific
Region: the Genus Littoraria. London: British Museum (Natural History).
Reid, D. G. 1989. The comparative morphology, phylogeny, and evolution of the
gastropod family Littorinidae. Philosophical Transactions of the Royal Society B,
324, 1e110.
Reid, D. G., Dyal, P. & Williams, S. T. 2010. Global diversification of mangrove
fauna: a molecular phylogeny of Littoraria (Gastropoda: Littorinidae). Molecular
Phylogenetics and Evolution, 55, 185e201.
Sanpanich, K., Wells, F. E. & Chitramvong, Y. 2008. Reproduction and growth of
Littoraria (Gastropoda: Littorinidae) at Ang Sila, Thailand. The Raffles Bulletin of
Zoology, 18, 225e233.
Saur, M. 1990. Mate discrimination in Littorina littorea (L.) and L. saxatilis (Olivi)
(Mollusca: Prosobranchia). Hydrobiologia, 193, 261e270.
Stafford, R., Davies, M. S. & Williams, G. A. 2007. Computer simulations of high
shore littorinids predict small-scale spatial and temporal distribution patterns
on rocky shores. Marine Ecology Progress Series, 342, 151e161.
Stafford, R., Davies, M. S. & Williams, G. A. 2008. Self-organization of intertidal
snails facilitates evolution of aggregation behavior. Artificial Life, 14, 409e423.
Stirling, D. & Hamilton, P. V. 1986. Observations on the mechanism of detecting
mucus trail polarity in the snail Littorina irrorata. Veliger, 29, 31e37.
465
Struhsaker, J. W. 1966. Breeding, spawning periodicity and early development in
the Hawaiian Littorina: L. pintado (Wood), L. picta Philippi and L. scabra (Linné).
Proceedings of the Malacological Society of London, 37, 137e166.
Susswein, A. J. & Nagle, G. T. 2004. Peptide and protein pheromones in molluscs.
Peptides, 25, 1523e1530.
Takeichi, M., Hirai, Y. & Yusa, Y. 2007. A water-borne sex pheromone and trail
following in the apple snail, Pomacea canaliculata. Journal of Molluscan Studies,
73, 275e278.
Tirindelli, R., Dibattista, M., Pifferi, S. & Menini, A. 2009. From pheromones to
behavior. Physiological Reviews, 89, 921e956.
Torres, P., Alfiado, A., Glassom, D., Jiddawi, N., Macia, A., Reid, D. G. & Paula, J.
2008. Species composition, comparative size and abundance of the genus
Littoraria (Gastropoda: Littorinidae) from different mangrove strata along the
East African coast. Hydrobiologia, 614, 339e351.
Townsend, C. R. 1974. Mucus trail following by the snail Biomphalaria gIabrata
(Say). Animal Behaviour, 22, 170e177.
Underwood, A. J. 1979. The ecology of intertidal gastropods. Advances in Marine
Biology, 16, 111e210.
Underwood, A. J. 1997. Experiments in Ecology: their Logical Design and Interpretation
Using Analysis of Variance. Cambridge: Cambridge University Press.
Underwood, A. J. & Chapman, M. G. 1997. GMAV 5 for Windows. Australia: Institute
of Marine Ecology, University of Sydney.
Williams, S. T., Reid, D. G. & Littlewood, D. T. J. 2003. A molecular phylogeny of the
Littorininae (Gastropoda: Littorinidae): unequal evolutionary rates, morphological parallelism, and biogeography of the Southern Ocean. Molecular Phylogenetics and Evolution, 28, 60e86.
Wyatt, T. D. 2003. Pheromones and Animal Behaviour: Communication by Smell and
Taste. Cambridge: Cambridge University Press.
Yipp, M. W. 1985. Tidal rhythms of Littorina melanostoma and L. scabra in a Hong
Kong mangal. In: Proceedings of the Second International Workshop on the Malacofauna of Hong Kong and Southern China: the Malacofauna of Hong Kong and
southern China Vol. II (Ed. by B. Morton & D. Dudgeon), pp. 613e621. Hong
Kong: Hong Kong University Press.
Zahradnik, T. D., Lemay, M. A. & Boulding, E. G. 2008. Choosy males in a littorinid
gastropod: male Littorina subrotundata prefer large and virgin females. Journal
of Molluscan Studies, 74, 245e251.