Title Vertical zonation and heat tolerance of three littorinid gastropods on a rocky shore at Tanjung Chek Jawa, Singapore Author(s) Su-Li Lee and Shirley S. L. Lim Source The Raffles Bulletin Of Zoology, 57(2), 551-560 Published by National University of Singapore

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Archived with permission from the copyright holder. TilE RAFFLES BULLETIN OF ZOOLOGY 2009 57(2): 551-560 Dale of Publicalion: :II Aug.2009 V Natwnal Un1versity of Singapore

VERTICAL ZONATION AND HEAT TOLERANCE OF THREE LITTORINID GASTROPODS ON A ROCKY SHORE AT TANJUNG CHEK JAWA, SINGAPORE

Su-Li Lee Anglo~Chinese Junior College, 25 D01·er Close East, Singapore I 39745, Republic or Singapore

Shirley S. L. Lim Ecology Lal)()ratorr. Natural Sciences & Science Education, National Institute or Education, Nanmng TechnoloRicul Ullll'ersitr. I Nanwmg Walk. Singapore 637616, Repuhlic or Singapore Email: [email protected] (Corresponding alllhor)

ABSTRACT. - Three common rocky shore littorinid taxa, i.e .. Littoraria spp. (a collective term for L Hrigata and L. articulata), ma/accana and £. vidua occupied different tidal heights based on field observations carried out at TanJung [=Cape] Chek Jawa, Pulau [=Island] Ubin, Singapore in 2002. Littoraria spp. were consistently observed at a lower level on the shore than E. ma/accana and £. 1·idua. Manually translocated littorinids returned to their preferred zones in the field, i.e., E. malaccana returned to the region above the MHWS level of 2.7 m while Littoraria spp. remained below the region occupied b) E nwlacwna. Further, E. nwlaccana individuals with their shell nodules removed by filing did not occupy a lower zone than intact conspecifics in the field. Although significant temperature differences (TO) were obsen ed between rock and shell surfaces in the laboratory (TO,,,,.,,.,.,~ TO'"" c ""''"""""

support this hypothesis: the temperature at which 50% mortality (lh LT,11 ) was observed was highest for E. malaccana (50.4°C) compared to E. 1•idua, (48.1 °C) and Littoraria spp. (47.5°C), possibly reflecting their relative tidal positions on the shore. Enzyme stability may also account for high heat tolerance of E. malaccana. Incubation of the enzyme, glutamate oxaloacetate transaminase (GOT) at 55°C showed that £. nw/accana sustained high GOT activity compared to those of Littoraria spp. and£. vidua which decreased sharply at the same temperature, again suggesting inherent physiological adaptation in E. malaccana.

KEY WORDS. - Zonation; heat tolerance; enzyme stability; Littoraria sp.; Echinolittorina malaccana; Echinolittorina vidua.

INTRODUCTION spanning many days (McMahon, 1990). Desiccation may be overcome by behavioural adaptations such as the The intertidal rocky shore is universally divided into withdrawal of the foot and closure of the shell opening zones according to characteristic intertidal flora and fauna with an operculum to reduce evaporation. Attachment to (Raffaelli & Hawkins, 1996): the supralittoral, midlittoral the substratum using a mucous sheet- the mucous holdfast and sublittoral zones. Such zonation is due to the stresses of (Bingham. 1972; Lim, 2008), further ensures that they will varying intensities along the height of the shore. Higher levels not be dislodged easily or maintain minimal contact with the of the shore (e.g .. supralittoral zone) are exposed to the air and heated substratum when out of water. Survival in prolonged sun for a longer period of time when compared with lower aerial exposure also depends on physiological adaptations shore levels (Newell. 1979; Cleland & McMahon. 1989). such as the reduction of metabolic rates, e.g., in Littorina Despite the extreme physical conditions in the supralittoral saxatilis (see McMahon & Russell-Hunter, 1977) and zone, many gastropod species have adapted successfully to elevated thermal tolerance, e.g., in Nodilittorina pyramidalis, life in this harsh zone. Gastropods living above the high tide N. granularis and Littorina hrevicula (see Fraenkel, 1966) mark are especially vulnerable to desiccation and thermal as well as Austrolittorina unifasciata (= N. unifasciata; see stress, as they typically experience prolonged aerial exposure Reid, 2007) (see McMahon, 1990).

551 Lee & Lim: Vertical zonation and heat tolerance of Singapore littorinids

Common littorinids found on the rocky shores of Singapore in the cell (Cooper & Meister, 1985). The metabolic include Littoraria articulata (Philippi. 1846). L. sl riga/a importance of GOT is that it brings about a free exchange (Philippi, 1846) (often as Littorina undulata in previous of amino groups between glutamate and aspartate; both of literature) and Echinolittorina ma/accana (Philippi, 184 7) which are required for separate but essential steps in the (also referred to in the literature variously as Nodilillorina urea cycle for ammonia detoxication and nitrogen excretion. pyramidalis or N. trochoides; see Reid. 2007). A less The free movement of nitrogen between the glutamate and common rough periwinkle, Echinolillorina vidua (Gould, aspartate pools is an important balancing process that is vi tal 1859) (previously Nodilitlorina vidua; see Reid, 2007) is for normal cell metabolism (Cooper & Meister, 1985 ). also found in certain rocky areas in Singapore.

Two previous littorinid distribution studies have been MATERIAL AND METHODS conducted in Singapore on the south-eastern coast of Singapore; one at a man-made breakwater (see Lam. 1980) Distribution studies and the other at a monsoon drain (see Tan. 1988). To date, there is no published literature with quantitative data on Two distribution studies were carried out at the rocky the vertical distribution of Littoraria spp .. Echinolittorina shore of Tanjung Chek Jawa on Pulau Ubin (approx. I- nzalaccana and E. vidua at natural rocky shores in Singapore. 24'N. l(J3o 59'E). an island off the north-eastern coast of The purpose of the first part of this study was to determine Singapore. The mean high water spring tide level (MHWS) the zonation pattern of these littorinids at the rocky shores at the study site is 2.7 m (Hydrographic Department. 200 I l of Tanjung [=Cape] Chek Jawa. Pulau [=Island] Ubin, These studies were made at (i) a vertical rock and (ii l a Singapore, through a distribution survey. sloping rock (angle of inclination "'35.6"). The vertical rock is located further inland (i.e., more up-shore in a little A translocation experiment involving Littoraria spp. and 'cove-like· recess) as compared to the sloping rock located E. ma/accana was also carried out to verify the zonation at a small spit jutting out sea wards. Hence, the vertical rock pattern observed. Dislodged littorinids are able to crawl surface studied is exposed earlier by the receding tide and upwards to their original zone. e.g., N. exigua (see Ohgaki. inundated later by the incoming tide when compared to the 1988a), Lillorina lillorea (see Petraitis, 1982) and Lillorina sloping rock. Both rocks have sparse algal cover and a fair un(fasciala (see Chapman, 1999). It is hypothesized that number of crevices. displaced Liltoraria spp. and E. malaccana individuals in this study would return to their prefeiTed zones. Reid ( 1986) reported that the two closely related 'pecie.., Littoraria arliculata and L. .1·1rigata possess shells with Shell sculpturing was reported by Yermeij (1973) to reduce similar shape. colour and banding pattern. and without the relative effective areas for absorption. i.e .. area in a plane examination of internal anatomical features. it was difficult that is perpendicular to incident sun's rays. This implies that to ascertain their identities in the field. Thus. these two there is greater heat loss by reflection of solar radiation by littorinid species are collectively referred to as Lilloraria an uneven surface, thereby reducing heat absorption. The spp. in this study as dissection of every individual shell of E. ma/accana is nodulated while that of Littoraria encountered was neither feasible nor possible. Data from the spp. individuals is smooth. Since E. malaccana occupies vertical rock were collected during three sampling sessions: a higher region of the shore, the nodulation may be an June/July. September and December 2002, with a total advantage for survival under prolonged aerial exposure. of I 0 transect belts laid at each session. The distribution The increased surface area of the shell due to the presence surveys were all carried out during three to five days of of nodules could facilitate convection and heat loss from consecutive spring tides. Accessibility to the study site the shell. To test this hypothesis, the nodulated shells of was only possible when the tides were suitably low. Each E. malaccana individuals were filed down and included belt transect consisted of a transparent 10 em-wide plastic in the translocation study to elucidate the possible role of strip with a measuring tape attached. The position of each nodulation in zonation. The effect of nodulation on heat Littoraria spp. (collectively Liuoraria) and Echinoliltorina reflection was tested both in the field (for E. malaccana and ma/accana (E. ma/accana) individual encountered beneath Littoraria spp.) and in the laboratory (for E. malaccana. the strip was recorded to the nearest 0.5 em. Distribution both intact and filed. and Littoraria spp. ). The temperature of Littoraria, E. malaccana and E. 1·idua was similarly difference between rock and shell surfaces was used as the determined at the sloping rock on 28 and 29 August 2002. response variable. A region covered by a total of 18 belt transects. each 15 em in width. was surveyed. The tide level (TL) at the top Two laboratory experiments were conducted to establish of the vertical rock is 0. I m above MHWS. possible physiology-related reasons for the zonation pattern: (I) a heat tolerance test, represented by 1 hour lethal temperatures (l h L T ,0), of the littorinids, and (2) a Translocation experiment heat stability test of the enzyme, glutamate oxaloacetate transaminase (GOT), extracted from these littorinids. The A translocation experiment was conducted in December enzyme GOT. also known as aspartate aminotransferase, 2002. Thirty individuals of Lilloraria and 60 E. malaccana was chosen because it is one of the most active enzymes individuals were collected and transported to the laboratory

552 THE RAFFLES BULLETIN OF ZOOLOGY 2009 in a container (with seawater from the study site) for colour­ would not be confounded by other factors, e.g., shade or coding with nail polish. The nodules of 30 E. rnalaccana reflection of heat from adjacent surfaces. adult individuals were tiled off (henceforth termed "'tiled E. malaccana") using an ordinary machinist's file. All The temperature difference (TD) between rock surface and experimental snails were returned to the study site within that of the shell surface (i.e., TD = Tro,k surrace - Tshcn surraccl two days to minimize stress and ensure their survival during for E. rnalaccana and Littoraria were tested separately for the translocation experiment. Based on the distribution significance using /-tests. TDL"'""'"" and TDE rnalauaaa were patterns observed from previous sampling periods, the then compared using the Mann-Whitney test statistic, as MHWS level (2.7 m) was found to approximately separate there was non-normality in the data set. In the laboratory, the regions occupied by Lilloraria and E. rnalacc·ana. 25 E. malaccana, 25 filed E. malaccana and 25 Littoraria Thus, the marked littorinids were returned to the 2.7 m were used in an experiment involving heat treatment. Five TL with the MHWS level clearly marked at the study E. malaccana, five tiled E. malaccana and five Littoraria site. Crowe & Underwood (1999) suggested that a snail's were placed on a piece of relatively smooth rock that behaviour changed by merely dislodging it from its original most resembled that at the field site. The rock was then position. This may affect the results of the translocation placed approximately 20 em beneath a lamp and left to study. However. Chapman ( 1999) found that although warm up for 5 minutes. Pairs of temperature readings translocation affected the distance moved by the snails, it were then measured: the temperature of the shell surface did not result in any change in the direction at which the and the average temperature of the substrate surface at the snails dispersed. perimeter of the snail. The procedure was repeated until all 75 were used. A one-way ANOV A was used to test During the next four consecutive days ( 16 to 20 December for significant differences among TDLawana• TDE malacrana and 2002). the distance between the locations of every marked TD,~c. 1 E ""''"""""' In order to maintain approximately equal individual from the MHWS level was measured to the variances, two obvious outliers were removed prior to nearest 0.1 em. The mean tidal level occupied by each ANOVA analyses. The distance of the infrared thermometer species on each of the four days was calculated. Crowe & from the surface was kept as constant as possible by Underwood ( 1999) cited that upon disturbance. littorinids maintaining the laser spot at approximately the same size are likely to require two days before resuming their natural during each measurement. ways. Thus. four days would be sufficient for the duration of the translocation study. Data collected were analysed with one-way Analysi' of Variance (A;\/OV A) tests to ascertain Lethal temperatures study whether the preferred zones occupied by the two species differed significantly. The Tukey's multiple comparison test Lilloraria, E. malaccana and E. vidua individuals with shell was applied when ANOVA results were significant. lengths between 7-11 mm were collected from the sloping rock at Tanjung Chek Jawa in November 2002. Snails were used within 24 hours of collection. The experiment Effect of shell nodulation of E. malaccana involved 12 temperature treatments between 44 and 52°C on heat reflection (see below). For each temperature treatment, 30 littorinid individuals (i.e., 10 Lilloraria, 10 E. malaccana and 10 E. Collection of data was carried out on two occasions: at 1•idua) were placed in an aluminium can containing I OOml Tanjung Chek Jawa and in the laboratory. Temperature of artificial seawater. Heat treatment of the snails submerged measured by a Raynger® STn1 infrared thermometer in seawater served to eliminate the effect of desiccation (see (Raytek®) was taken as an indication of the amount of Evans, 1948). A plastic cover was placed over the can to heat energy given off by the li ttorinid. The thermometer prevent water loss due to evaporation during heating. The detects emitted energy (e.g., radiated heat energy from seawater was brought to the required temperature in a water within the snail/gastropod) and reflected heat energy (from bath from room temperature (22-23°C). The temperature an external source e.g., the sun). At Tanjung Chek Jawa, the of the seawater was measured with "K" type thermocouple average temperature of the shell of the rough periwinkle, probe connected to an AI I 04 humidity temperature meter E. malaccana, was measured using the average-function of (Azex Instruments (S) Pte. Ltd.). The probe was inserted the infrared thermometer. The average temperature of the through a hole in the plastic cover. Once the required rock surface. i.e .. at the perimeter of the . was then temperature was attained, the time was noted and the measured. Likewise, the temperatures of the shell surface seawater was maintained at a relatively constant temperature of the 'imooth periwinkle. Lilloraria, and the surrounding for an hour (the temperature was monitored and recorded surface were measured under direct sunlight. A total of 55 at 15 minute intervals). This length of time was used by pairs of temperature readings were recorded for individuals Fraenkel ( 1966) in a heat stress experiment conducted of various sizes in each species. All individuals, whose shell on 12 species of intertidal prosobranchs in south-western temperatures were measured, were selected such that they Japan. The time taken for the seawater to reach the required were found on relatively smooth surfaces and not in any temperature ranged between 20 and 30 minutes. Monitoring sort of pit or crevice. This was to ensure that measurements of seawater temperatures indicated that the seawater could

553 Lee & Lim: Vertical zonation and heat tolerance of Singapore littorinids

not be assumed to be at the temperature shown by the water calculated. Glutamate oxaloacetate transaminase (GOT) bath as the temperature probe that was inserted into the activity (1-Hnol NADH/min/g wet weight) was calculated experimental container registered some fluctuations. Hence, according to the following formula: the temperature of each heat treatment was taken to be the average temperature of the temperatures recorded during the (~A/min) (total volume) (volume of buffer hour. The resultant 12 temperature treatments were 44.1 oe, + wet weight of tissue) x 50 44.rC 45.6oe, 47.ooe, 47.4°C, 48.0°C, 48.4°C, 48.7°C, GOT 49.SOC, 50.3°C, 50.8cC and 5l.8°C, The temperature range 6.22 x sample volume x wet weight of tissue selected was based on temperatures observed in the field. There were six replicates for each of the four temperature After heat treatment, the littorinids were then transferred to treatments for all three littorinid taxa. GOT activity was the centre of a holding tank containing artificial seawater converted to a percentage value based on the results and an algae-covered rock. The tank was left in a normal of previous temperatures. as an indication of change in external environment (i.e., out of the air-conditioned activity due to increase in temperature. Data were arcsine­ laboratory). At the end of 24 hours in the holding tank, transformed and analysed with one-way A;--JOV A. This the number of live individuals was recorded. An individual was performed only on data for 50°C and ssoe, as most was considered to be alive if it displayed foot attachment, of the data for 25°C and 45°C were greater than 100'7r and had scaled up the sides of the tank, moved onto the rock or could not be arcsine-transformed. All statistical analyses responded to gentle prodding of the foot. The percentage in this study were carried out using MIN IT AB (Release mortalities obtained were converted into probit values and 12.21. 1998). graphed against temperature. The !-hour lethal temperature

(I h L T 50 ) of each group was taken to be the temperature at which there was 50% mortality. As it was not feasible to RESULTS have a large sample size, statistical analyses of the I h L T 50s was not carried out. Distribution studies

At the vertical rock study site. Lilloraria and E. malaccww Heat stability of GOT were found in two distinct regions of the high intertidal zone in June/July (Fig. Ia): Littoraria occupied the lower region Six samples of tissues (each consisting approximately 0.5 g and E. malaccana. the higher region. The boundary between fresh weight of pooled flesh from a few individuals) of each the two regions was located approximately at 2.6 m (0.1 m of the three littorinid taxa were frozen using liquid nitrogen below MHWS). The mean tide level (mean TL) occupied and stored at -80°C The frozen samples were pounded to by £. malaccana was 2.8 ± 0.2 m (range = 2.4 m to 3.6 m) powder in liquid nitrogen, reweighed and homogenised while that of Littoraria was 2.0 ± 0.2 m (range = 1.8 m to with thrice their weight of cold O.OSM Tris-HCl buffer 3.1 m) (Fig. la). In September 2002. the mean TL occupied (from Sigma), pH 7.0. Homogenisation was done using a by E. nwlaccana increased to 3.0 m ± 0.3 m (range = 2.1 Polytron PT 1300D homogeniser for a duration of 3 x 20 s m to 3.5 m). while that of Littoraria also increased to 2.3 with 20 s intervals. The homogenates were sonicated with ± 0.2 m (range= 1.9 m to 3.2 m) (Fig. I b). The band of E. a Heat Systems sonicator at 40% output power for one malaccana had a 0.6 m overlap with of the Liltoraria band. minute. The mixtures were centrifuged at 9000 rpm, 4 oc In December 2002, the overlapping region decreased to 0.3 for 10 min in a Beckman J2-MC refrigerated centrifuge. The min width (Fig. lc). £. malaccana was found between TL supernatants were pipetted into clean test tubes, which were of 2.5 m to 3.6 m (mean TL = 3.2 ± 0.3 m) and Littoraria kept in ice. Enzyme analyses were conducted within a few was found between TL of 2.0 m to 3.5 m (mean TL = 2.4 of hours of sample preparation using the GOT optimized ± 0.2 m) (Fig. I c). At the sloping rock. £. malaccana also aspartate-aminotransferase EC 2.6.1.1 UV -test kit (Sigma). occupied the highest region of the rock (range = 2.6m to The sample used for enzyme analyses was a SOx dilution 2.8 m) while E. l'idua individuals occupied a region that of the extract (0.03 ml of the supernatant mixed with I .4 7 overlapped with that of Lilloraria (Fig. 2). The distribution ml of Tris-HCl buffer, pH 7 .0). Prior to enzyme analyses, of E. l'idua extended slightly higher than the Littoraria zone Reagent A (260 mmol!L aspartate, I 04 mmol/L phosphate but was distinctly below that of E. ma/accana. buffer pH 7.4, 0.234 mmol/L NADH, 780 UIL MDH and 1560 U/L LDH) and the samples were incubated separately at 25°C, 45°C, 50°C and 55°C for 30 minutes. After the Translocation experiment 30 min incubation, 0.1 ml of sample was added to 1.3 ml of Reagent A and mixed by inversion. After a minute of The mean TL occupied by Littoraria on the four consecutive incubation in a temperature controlled cuvette compartment days was slightly lower than the MHWS level of 2.7 rn while of a UV -160 I spectrophotometer with a CPS temperature E. nzalaccana and filed E. malaccana were found at least controller (Shimadzu), 0.125 ml of Reagent B ( 156 mmol!L 0.3 m above the MHWS level (Table 1 ). One-way ANOV A 2-oxoglutarate) was added and mixed. The absorbance of results showed that on all four days there was a significant the mixture was recorded eight times at 25 s intervals and difference (P < 0.05) among the mean TLs occupied by the mean absorbance change per minute (~A/min) was the three species of littorinids (Table 2) Tukey's multiple

554 THE RAFFLES BULLETIN OF ZOOLOGY 2009

3 7 ' LL (b) EM LL (c) LL 3 6 "

3 5 ' ! 3 4 ;

3 3 ,

3 2 i

3 1

E j 0 ' _J 2 9 - Ll.J > Ll.J 2 8 j _J Ll.J 0 i= 2 6 j 2 5 j

2 4 ,

2 3 ;

2 2 1

I 2 1 1 0 300 L___j 2 o 1 Number/mL I 1.9 j

Fig. I Kite diagrams showing the distributions of Lilloraria spp. (LL) and Echinuiillorina malaccana (EM) on a vertical rock at Tanjung Chek Jawa in (a) June/July, (bi September, and (c) December 2002. tMHWS is represented by dashed line).

2.8

2 7

2.6

25

2.4

E 2 3 _J w 2.2 w> _J 2.1 w 0 2.0 f-

1.9

1.8

1.7

1 6 0 60

1 5 EM EV LL

hg 2 Kite dtagrams showing the distributions of l.1111mma spp. (LL), Eclwwlillorina nwlaccana (EM) and E. l'idua (EV) on a sloping rock (angle of elevat1on of approximately .15.6 J at TanJung Chek Jawa. iMHWS is represented hy dashed line).

555 Lee & Lim: Vertical zonation and heat tolerance of Singapore littorinids

Table I. Mean tide levels occupied by marked Littomria spp. (Lirtoraria), Echmolirtorina nwlaccana (F. nwlaccana) and tiled F. maluccanu (filed E. malaccana) on four consecutive days (16- 20 Dcc.2002) at TanJung Chek Jawa. Singapore.

Mean tide level ± S.D. (m) Littoraria E. malaccana Filed£. malaccana

Day 1 2.69 ± 0.20 3.11 ±0.23 3.03 ± 0.23 Day 2 2.54 ± 0 24 3.11 ± 0.20 3.04 ± 0.23 Day 3 2.59 ± 0.25 3.14 ± 0.19 3.11 ± 0.26 Day 4 2.67 ± 0.21 3.16 ± 0.21 3.09 ± 0.21

Table 2. Results of one way ANOYA and Tukey's multiple comparison for tide levels that were occupied by marked Lit/!lraria spp. (Littoraria), Echinolittorina maloceana (E. malaccana) and filed E. nwlaccana (filed E. malaccana) on four cnnsecutJ\e days (I 6 - 20 December 2002) at Tanjung Chek Jawa, Singapore. (*: significant).

AN OVA Tukey's test F dfl, df2 p

Day I 29.99 2. 87 0.00 * Littoraria < filed E. malaccana = E. nwluccww Day 2 54.23 2. 87 0.00 * Littoraria < filed E. malaccana = E. nw/accana Day 3 52.4 I 2, 87 0.00 * Littomria < filed E. nwlucuuw = E. ma/accuna Day 4 33.84 2. 87 0.00 * Littoraria < lilcd E. malaccana = t:. nwlaccuna

comparison tests showed that there was no significant 50.4 oc (Fig. 3 ). Echinnlittorina vidua had an intermediate difference between the TLs occupied by filed E. malaccana I h LT"1 of approximately 48.1 oc (Fig. 3). and E. malaccana on the four days (P > 0.05) (Table 2). Both E. ma/accana and filed E. malaccana were found at a significantly higher TL (P < 0.05) than Littoraria (i.e., Heat stability of GOT Littoraria < filed E. ma/accana "' E. malaccana) (Table 2). Incubation of GOT at 45°C resulted in increased GOT activity for all three species of littorinids with greatest increase in GOT activity seen in E. malaccana (236.48 ± Effect of shell nodulation of EM on heat reflection 6.11 o/c, Table 4) The maximum GOT activity for E. vidua occurred after incubation at 50°C while those of Littoraria Results from /-tests showed that TDLoromno and TD£ nudcuumo and E. ma/accana were at 55°C (Fig. 4). A~OVA re~ults recorded at Tanjung Chek Jawa were both significantly showed that after incubation at 5oce, the mean percentage greater than ooc (1 = 3.36 and t = 7.61 respectively. P < GOT activity remaining for E. malaccana was significantly 0.05 for both). The mean TD between rock surface and shell greater than that of E. vidua but not significantly different surface for E. ma/accana and Littoraria were 0.55 ± 0.54 oc from that of Littoraria (P < 0.05, E. l'idua < Littoraria < and 0.15 ± 0.34 oc respectively. TDE ""''"''"""was significantly E. malaccana). After incubation at 55°C, mean percentage greater than TD1."'""'"" (W = 3942.5. P < 0.05). GOT activity remaining for E. ma/accana was significantly greater than those from E. vidua and Uttomria. and that of The ANOV A results of the laboratory heat experiment Littoraria significantly greater than that of E. \·idua (P < showed that mean TD varied significantly with the 0.05, E. \'idua < Littoraria < E. malaccana). treatment group of littorinids in question (Table 3. P < 0.05). Tukey's multiple comparison test showed that there was no significant difference between TDL"""""" and TD 11 ~cu DISCUSSION E "'""""'""but TDE "'"'""""" was significantly greater than both

TDLIIrurana and TDtilcJ E. mo/(l({(//1f1 (TDLI//OI(Ir/11 ~ TDhlcd E mufwnmu < During all three sampling periods, Littoraria (i.e. L. TDE malcwc·ana) (Table 3). articulata and L. strigata) was generally found below the MHWS level of 2.7 m. In consultation with the Singapore Tide Tables and Port Information (Hydrographic Lethal temperature study Department. 2001 ), it was estimated that the MHWS level could be left dry for at least eight days during a two-week

Littoraria recorded the lowest I h L T 50 of approximately tidal cycle before the next high tide wetted the region. 47.5°C, and E. malaccana, the highest. at approximately Littoraria individuals may not be able to cope with the physical stresses beyond the MHWS level during the

556 THE RAFFLES BULLETIN OF ZOOLOGY 2009

Tahle 3. Result;, of one-way ANOV A and Tukey' s multiple comparison of the variable. temperature difference (TO) between rock surface and shell surface of Littoraria spp. (Littoraria). Echinulittorina ma/accana (E. malaccana) and filed E. malaccana in the laboratory. (*: sigmficant).

AN OVA Variable Mean ± S.D. (°C) Thkey's test F p

TDc"'"'""" 0.128 ± 0.177 ± 2, 72 TO,."'"'""""" 0.300 0.204 5.85 0.004 * TDLIII

TDhkd/ mullli

aerial exposure. At the sloping rock, E. l'idua was found Lillorina granularis) and Litroraria (as L. undulata), Lam to occupy a region distinctly below that of E. malaccana ( 1980) found that E. malaccana was most tolerant to and it overlapped with the upper region in which Litroraria desiccation at 38 ± I °C, a trait that partly accounts for its were found. successful survival above the MHWS level.

Echinolillorina ma/accana was generally found above The distributions of Littoraria and E. malaccana were and at the MHWS level. Lam ( 1980) also reported that E. generally distinct from each other, with the former found malaccana (as Tectarius malaccensis) inhabited the splash at a lower level than the latter. This zonation observed zone above MHWS on a man-made breakwater. A similar at Tanjung Chek J awa was verified by the translocation distribution pattern was observed by Ohgaki ( 1988b) study, whereby displaced individuals of Littoraria and E. for another rough periwinkle, Nodilittorina exigua, in malaccana returned to their preferred regions on the shore. Shirahama, Japan. Ohgaki ( 1988b) suggested that N. exigua Such movements were also observed in Austrolittorina remained above and around the MHWS level to escape unifasciata (as L. unifasciata) and Nodilittorina pyramidalis harsh conditions (e.g., strong wave action) that exist lower in New South Wales, Australia by Chapman (1999). In down the shore. In addition, these nodulated littorinids addition, physical removal of shell nodules in E. malaccana are adapted for survival under prolonged aerial exposure. did not result in a change of preference for a tide level, i.e., Among E. ma/accana (as T malaccensis), E. vidua (as the filed E. malaccana did not move to a level significantly lower or higher than that of their unfiled counterparts. However, results from the laboratory experiment showed

10 0 that filed Echinolittorina shells and smooth Littoraria shells • 0 0 were both warmer than nodulated shells, as evidenced by the mean TDr "'"'""""" being significantly greater than both g 0 TD,,bl E mnlau ana and TD Lawnna' This suggests that although shell ornamentation helps to reduce shell temperature, it is probably not a major factor in determining the choice of R 0 tidal level at which the species remained.

Rough, nodulated shells reflect more heat energy 7 J (Nybakken, 2001 ). The higher TD of the rough periwinkle. E. malaccana. observed in this study may be due to this (/) I ' 1- I reason, as the infrared thermometer senses reflected heat Ci.i 0 6.0 energy (refer to Raynger® STTM manual). The significant a: I Q._ I • difference between TDL"'"'"'"' and TDE malanana at Tanjung I ' ' 0 Chek Jawa suggested that the nodulation on E. malaccana 50 o/ o 0 helped in reducing the amount of heat transmitted into the

I ' body of the littorinid. This is because the more heat energy I 0 I is reflected, less is absorbed and transmitted (Jones, 2000). 4.0 Another factor that may account for the difference in TDs I ' 0 0 • I is colour, which affects absorption and reflection of heat.

3.0 According to Vermeij (1973), a light-coloured surface absorbs less radiation from visible light. Many high shore gastropods (e.g., Nerita peleronta, Tectarius muricatus and

2.0 +---~--~--~---~-----~ Littorina unifasciata antipodium) are light coloured while 46.0 47 0 48.0 49.0 50.0 51.0 52.0 gastropods at lower shores have darker shells (Nyabakken,

TEMPERATURE (oC) 2001; Vermeij, 1973). The nodules on E. malaccana are of a much lighter colour (pale yellow) than the rest of the Fig. ·' Prohit re

557 Lee & Lim: Vertical zonation and heat tolerance of Singapore littorinids

Table 4. Mean percentage glutamate oxaloacetatc transaminase (GOT) activity remaining for Littoraria sp. (Littoraria). Echinolittorina vidtw and £. malaccana after incubation at 45°C, 500C and 55°C.

Mean percentage GOT activity ± S.D. (%) Species 25°C 45°C 50°C ssoc

Littoraria 100.00 ± 0.00 176.32 ± 69.78 112.27 ± 56.57 67.87 ± 7.95 E vidua 100.00 ± 0.00 191.99 ± 10.39 98.41 ±9.39 47.27 ± 8.73 E ma/accana 100.00 ± 0.00 236.48 ± 6.11 101.94 ± 22.97 97.59 ± 3.169

heat reflection by the nodules. Filed shells were much darker an almost terrestrial life above and about MHWS such that in colour than intact conspecifics. McQuaid and Schermann it is unable to cope with prolonged immersion. Interspecific ( 1988) reported that the pale-shelled L. a. africana had competition between Lil!oraria and E. malaccana could a lower body temperature than the dark brown morph L. also influence the zonation pattern. However. more studies a. knysnaensis. They also found that there was a higher would have to be conducted to elucidate the interaction mortality rate among individuals of the two sub-species that between the two genera at the study site. were painted black. The I h L T",s of the three taxa of littorinids in this stud> The preference for a higher or lower shore level may increased with their relative position on the shore. Lillnnrriu

likely be due to physiological adaptations. McMahon found lowest on the shore had the lowest l h L T, 11 of (200 I) hypothesized that mid-shore gastropods had 47.5°C while the inhabitant of the highest level on the

modified respiratory systems for aerial and aquatic gas shore. E. malaccana, had the highest I h L T 50 of :i0.4T. exchange. which disabled them from surviving in aquatic The I h L T "' of £. l'idua ( 48.1 oc) reflected ib position environments. Likewise. E. malaccana may have adapted to on the shore relative to £. ma/accana and Lilloraria, i.e .. between the two genera. The increase in thermal tolerance with increase in position on the shore is consistent with findings of other researchers. Southward ( 1958) found that the high-shore top-shells Monodonta linea/a and Gihhula 60 umbilica/is tolerated higher temperatures that G. cineraria and Cal/ipstoma ;:i;:yphinum, which inhabited low water. Fraenkel (1966) observed that N. pvramidalis at the upper part of the intertidal zone was most heat resistant. while Chlorostoma argyrostoma /ischkei collected from the 50 water's edge was least resistant. In addition. Wolcott ( 1973) I I reported that two littoral fringe limpets (Acmaea digitalis I I and A. scabra) maintained higher tissue temperatures that I I two other species (A. pella and A. testudinalis scutum) I I I present in the eulittoral zone. 40 I I I I I I The lh LT,0 value for N. pvramidalis in Japan \\as I I determined by Fraenkel ( 1966) to he ..J.8.5T. which was I lower than the lh LT,0 of 50.4°C obtained in this .-;rudy. His samples were heated in air instead of seawater. which 30 ///+ l\ may have resulted in some degree of desiccation (sec E\ ans. 1948) thus affecting the thermal tolerance of the littorinid. Wolcott (1973) reported that removal of body water by ///// 1 desiccation reduced the thermal tolerance of the limpet A. digitalis by about 3°C. 20

r·/,/ In addition, the different I h LT,0 may be due to the geographical location in which the species inhabits. Thermal tolerance is reportedly negatively correlated to

10+---~----~--~~--~----~--~----~--~ latitude (Fraenkel, 1968; McMahon, 2001) This may he a 20 25 30 35 40 45 50 55 60 result of the higher level of solar radiation all year round 0 TEMPERATURE ( C) in the tropics when compared with that of temperate areas Fig. 4 Mean glutamate oxaloacetate transaminase !GOT) activities liJmnl (Moore. 1972). Southward ( 1958) reported that Lillorina NADH/min/g wet weight) of Littoraria sp., Echlllolutonna malaccana and neritoides found in North Africa was more heat tolerant than E 1·idua from TanJung Chek Jawa at four temperatures: 25 oc. 45 oc. L. saxatilis found all around Britain. Of the 60 gastropods 50 oc and 55 oc. (Vertical bars represent standard errors). Llltoraria sp. e---e: Echino/utorina nwlaccwza, o ...... -0: E. l'idua G---G. collected from various localities (e.g .. Australia, England.

558 THE RAFFLES BULLETIN OF ZOOLOGY 2009

Hong Kong, Jamaica, Texas), McMahon (2001) found that Chapman, M. G .. 1999. Assessment of variability in responses Nodilirrorina ::ic::ac from Jamaica (18°N) had the highest of intertidal periwinkles to experimental transplantations. mean heat coma temperature (HCT) of 46. 9°C. Lirrorina Journal of Experimenral Marine Biology and Ecolog_v, 236: mariae found in Robin Hoods Bay, England (54°N) had 171-190. the lowest mean HCT of 26.4 oc. Similarly, N. pwamidalis Cleland. J.D. & R. F. McMahon, 1989. Upper thermal limit of nine at Wakayama, Japan (34°N) had a slightly lower thermal intertidal gastropod species from a Hong Kong rocky shore in tolerance than E. malaccana found Tanjung Chek Jawa. relation to vertical distribution and desiccation associated with 0 evaporative cooling. Proceedings of rhe Second Imernarional Singapore (approx. I N). Tropical E. malaccana possibly Marine Biological Workshop: The Marine Flora and Fauna adapted to higher temperatures near the equator. This of Hong Kong and Southern China, Hong Kong. Hong Kong species is found on rocks that may be heated to temperatures Universiry Press, Hong Kong, pp. 1141-1152. of 45°C to sooc (Lim & Fahmy. 2008). McMahon (200 I) Cooper. A. J. L. & A. Meister. 1985. Metabolic significance reported a HCT of 46.3°C for E. malaccana in Hong Kong of transamination. In: Christen. P. & D. E. Metzler (eds.). (22°~) (c.f. lh LT, 5(l4°C recorded in this study). This 11 Transaminases. Wiley. New York. Pp. 533-563. ts further evidence for the resilience of this species under Crow c. T. P. & A. J. Underwood, 1999. Differences in dispersal high temperatures. of an intertidal gastropod in two habitats: the need for and design of repeated experimental transplantation. Journal of Enzyme stability. such as that observed for GOT in E. E1perimenral Marine Biology and Ecology, 237: 31-60. malaccana. may account for the relatively high heat Evans, R. G., 1948. The lethal temperatures of some common tolerance of this littorinid in the current study. Glutamate British littoral molluscs. Journal of Animal Ecology. 17: oxaloacetate transaminase is required for the regulation of 165-173. carbon and nitrogen flow in various biochemical pathways Fraenkel, G., 1966. The heat resistance of intertidal snails at (Cooper & Meister. 1985). An enzyme's function is highly Shiraharna, Wakayama-ken, Japan. Publications of rhe Sero dependent on its structure. The stability of this structure is Marine Biological Laboratory, 14: 185-195. temperature-sensitive (Somero, 1978), denaturing at high Fracnkcl, G., 1968. The heat resistance of intertidal snails at temperatures. Among Lirroraria sp., E. malaccana and Bimini, Bahamas; Ocean Springs, Mississipi; and Woods E. vidua, the percentage of GOT activity remaining after Hole. Massachusetts. Physiological Zoology, 41: 1-13. incubation at 55 oc was highest for E. malaccana. This Gould, A. A., 1859. Descriptions of new species of shells brought indicated that GOT from E. malaccana was most stable, home by the North Pacific Exploring Expedition. Proceedings even at a temperature above its lh LT,0 (50.4°C). Somero of rhe Bosron Society of Nawral His wry, 7: 138-142. ( 1978) reported that enzymes denature at temperatures that Hydrographic Department, 2001. Singapore Tide Tables and Port are usually higher than the upper lethal temperature of the Inj(Jrnwtion. 2002. Hydrographic Department, Maritime and species. After incubation at this temperature, the activity Port Authority of Singapore. 239 pp. of GOT from Lirroraria and E. vidua fell sharply, a likely Hull. S. L., J. Grahame & P. J. Mill, 1999. Heat stability result of denaturation of the enzymes. Hull et al. ( 1999) and activity levels of aspartate aminotransferase and also found that enzymes of high shore animals (e.g., high alanine aminotransferase in British . Journal of shore L. saxarilis and L. arcana) were more stable. i.e., had Experimental Marine Biology and Ecology, 237: 255-270. higher activity rates at 56°C, than those of low shore groups Jones. H.R.N. 2000. Radiarion hear tranofet: Oxford University (e.g .. L. ohrusara. L. neg/ecru and L. lirrorea). This trend of Press, New York. 83 pp. increased stability with increased height on the shore was Lam, Y. K., 1980. Some studies on the Easr Coast breakwarer also evident within a single species, L saxarilis. found at j(wna, wirh parricular reference to the vertical distribution high and mid-shore levels. of rhe periwinkles ( Litrorinidae) and their tolerances ro oil pollurion. Unpublished Honours thesis, University of Singapore, Singapore. Pp 97 + 9 appendices. ACKNOWLEDGEMENTS Lim. S. S. L., 2008. Body posturing in Nodilillorina pyramidalis and Ausrrolirrorina unifasciara (: : The authors thank the Nature Conservation Branch Littorinidae ): a behavioural response to reduce heat stress. (National Parks Board) for the permit to conduct research In: Davie, P J. F. & J. A. Phillips (eds.), Proceedings of and to collect specimens at Pulau Ubin Nature Park. The rhe Thirreemh lnrernarional Marine Biological Workshop, staff of the National Parks Board Office at Pulau Ubin is The Marine Fauna and Flora of Moreron Bay, Queensland. acknowledged for their assistance in field logistics. Financial Memoirs of' rhe Queensland Museum - Narure, 54: 339-347. support for this study was provided by a grant (RP 19/97 Lim, S. S. L. & M. Fahmy, 2008. Is 'standing' posture an effective LSL) from the National Institute of Education, Nanyang behavioural adaptation to reduce heat stress in Echinolillorina Technological University to the second author (SSLL). malaccana'' Absrracts of rhe IX Inrenzarional Symposium on Linorinid Biolog_\' and Evolurion, Uni1·ersity of Vigo, Spain, p. 17. LITERATURE CITED McMahon, R. F., 1990. 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