1 Ontogenetic shifts in oxygen uptake in Common ( kalolo) and 2 its role in microhabitat selection 3 4 JENNIE S. SANDBERG, AND WAYNE A. BENNETT 5 6 (WAB) The University of West Florida, Department of Biology; and (DJS) University of Essex, 7 Department of Biological Sciences 8 Running head: Oxygen uptake of Common Mudskipper 9 Manuscript type: Shorter Contribution 10 Key words: amphibious fish, metabolism, air-breathing fish 11 12

1 13 The Common Mudskipper, Periophthalmus kalolo, (family ) is a tropical,

14 amphibious fish capable of utilizing both air and water as a respiratory medium. Although

15 little is known about their early life history, post-metamorphose fish ≤ 2.0 cm in length are

16 thought to be more dependent on tidepools than larger fish. We quantified oxygen uptake in

17 water and air of fish with standard lengths between 1.0 and 10.3 cm to identify size-related

18 shifts in aquatic and aerial oxygen consumption rates. Common 4.0 cm or less

19 in standard length exhibited aquatic mass-adjusted metabolic rates nearly twice those

20 measured for larger fish. Aerial mass-adjusted oxygen uptake in small mudskippers (≤ 2.0

21 cm) was ten-fold higher than values estimated for larger fish. Furthermore, air:water

22 metabolic rate ratios showed that emerged mudskippers ≤ 2.0 cm in length consumed seven

23 and one-half times more oxygen than when submerged. We argue that tidepool dependence

24 of small Common Mudskippers is linked to the markedly high metabolic demand while on

25 land, and not an inability to effectively extract oxygen from air. Elevated aerial metabolic

26 costs likely result from differing demands in air and water for physiological processes such

27 as evaporative water loss and ammonia excretion.

28

2 29 The Common Mudskipper, Periophthalmus kalolo, (family Gobiidae) is an air-breathing

30 fish common to mangal ( biome) habitats throughout the Indo-Pacific (Note:

31 common and scientific names cited throughout the text follow taxonomic clarifications

32 proposed by Graham, 1997). Small post-metamorphose fish less than 2.0 cm standard length

33 prefer shaded tidepools in upper intertidal zones near mangrove stands; whereas larger

34 individuals aggregate in exposed lower intertidal mudflats (Frith, 1977; Tytler & Vaughan,

35 1983) where they may spend as much as 90% of their time out of water (Gordon et al., 1978;

36 Gordon et al., 1985). Both Brillet (1976) and Tytler and Vaughn (1983) report that at low

37 tide, small Common Mudskippers inhabiting mangals in Madagascar and Kuwait Bay,

38 respectively, never stray far from tidepools or at least wet mud, and small fish placed in the

39 open preferentially move away from exposed areas toward wet, shaded zones (Tytler and

40 Vaughn, 1983).

41 Factors influencing size-dependent distribution of Common Mudskippers among mangal

42 microhabitats are not entirely clear. Tamura and co-workers (1976) suggested that recently

43 post-metamorphic mudskippers are more effective at extracting oxygen from water than air,

44 and must therefore remain closely associated with pool environments. However, histological

45 examination has shown that epidermal vascularization in at least one periophthalmid,

46 Shuttles Hoppfish (Periophthalmus modestus), is well-developed only 10 days post-hatch

47 and should be capable of supporting cutaneous aerial respiration (Suzuki and Hagiwara,

48 1995). Likewise, Clayton (1993) has argued that high skin to gill area ratios in smaller

49 mudskippers should support a greater dependence on cutaneous rather than branchial

50 respiration leaving them better suited to aerial habitats than large fish. While numerous

51 studies have assessed aquatic or aerial oxygen uptake in mudskippers larger than 5 cm

3 52 standard length (Ip et al., 1991; Kok et al., 1998; Takeda et al., 1999; Taylor et al., 2005), no

53 study to date has quantified metabolic rates in smaller fish, leaving questions of relative

54 oxygen extraction rate unanswered.

55 In the present study, we estimate aerial and aquatic oxygen uptake of Common

56 Mudskippers to identify possible size-related metabolic shifts. In addition, we express

57 oxygen uptake in air and water as a ratio to assess relative differences between aerial and

58 aquatic respiration across size classes. Finally, we interpret metabolic rate data relative to

59 environmental and physiological constraints to better understand factors influencing activity

60 and distribution patterns of Common Mudskipper between upper and lower mangal zones.

61

62 MATERIALS AND METHODS

63 Common Mudskippers were collected between June and August 2005 from a mangal

64 habitat on the northwest edge of Hoga Island (05° 27.53′S, 123° 46.33′E) in the Wakatobi

65 National Marine Park, Sulawesi, Indonesia (Fig. 1). Fish larger than 2 cm were collected

66 from open lower mangal mudflat areas, whereas smaller fish were always captured from

67 upper mangal pools near mangrove trees. Diel air temperatures ( 0.5 C) from shaded upper

68 mangal and exposed lower mangal capture sites were measured using iButton® temperature

69 loggers (Thermocron® model DS1921H). Temperatures were recorded at 20 min intervals

70 from 28 June to 24 July 2005.

71 Captured fish were taken to Hoga Marine Research Center and transferred to covered,

72 plastic holding tanks (121  39.6  30.8 cm) containing beach sand covered with 33 ‰

73 seawater. Emergent palm frond and driftwood pieces allowed fish free movement between air

74 and water. Holding tank temperatures were kept between 25 and 28 C with seawater changed

4 75 twice daily and sand changed every 2-3 days to maintain habitat quality. Mudskippers were

76 fasted for 48 h before respirometry trials. Each fish was used in only one aerial or aquatic

77 experiment and all fish were released to their site of capture at the end of the study.

78 Manometric respirometry (Bicudo and Johansen, 1979; Cech, 1990; Taylor et al., 2005)

79 was used to estimate total resting routine metabolic rates in air for 117 Common

80 Mudskippers. The respirometer design was similar to that of Taylor et al. (2005) and

81 consisted of a 20-mL (fish ≤ 3.0 cm standard length), or 250-mL (fish ≥ 3.1 cm) glass

82 respirometer flask connected by a manometer tube to a reference flask of equal volume.

83 Seawater-saturated blotter paper maintained high humidity levels in both flasks, and a strip of

84 10% sodium hydroxide-saturated Whatman paper attached to the respirometer flask stopper

85 removed expired carbon dioxide. An identical control (without fish) was run simultaneously

86 during each trial to correct for non-fish respiration.

87 Prior to each trial, the apparatus was pressure-tested to identify and eliminate system

88 leaks and a randomly selected fish placed into the respirometry flask. Fish were allowed to

89 adjust to the flask environment for 45 min at the experimental set point temperature at which

90 time, the respirometer was sealed and the trial begun. As oxygen was consumed, respirometer

91 flask pressure decreased causing manometer fluid displacement proportional to oxygen

92 uptake. Manometer fluid was returned to its starting level by adding air slowly and in small

93 amounts to the respirometer flask via a 10 cc Hamilton® gas-tight syringe. Measurements of

94 total injected air were recorded every 30 min for two hours with total oxygen consumption

95 (mg h-1) estimated as milligrams of injected air added divided by total trial time.

96 Estimates of total aquatic resting routine oxygen uptake were made for 92 Common

97 Mudskippers via flow-through respirometry techniques (Cech, 1990). For each trial, a fish

5 98 was randomly selected from the holding tank and transferred into 25 mL (fish ≤ 3.00 cm

99 standard length) or 250 mL (fish ≥ 3.01 cm) glass respirometer flask. Filtered seawater was

100 pumped through the respirometer flask at rates between 3 and 20 mL min-1. Flow rates varied

101 with fish mass so that oxygen content of water leaving the flask never fell below 15% of total

102 inflow oxygen concentration (Cech, 1990). Fish were allowed to adjust to the flask

103 environment for 45 min, after which three respirometer outflow water samples were collected

104 in biological oxygen demand (BOD) bottles. Bottles were filled slowly from the bottom and

105 allowed to overfill at least two times their volume to minimize the chance of atmospheric

106 oxygen uptake (Cox, 1990). Oxygen concentration of each BOD sample was determined

107 using standard Winkler titration techniques (Cox, 1990). A control flask, identical in all

108 respects but containing no fish, was included in each trial to allow correction for oxygen

109 consumption from non-fish sources. Total oxygen uptake (mg h-1) was determined as the

110 mean difference in oxygen content (mg L-1) between water leaving the control flask and

111 respirometry flask, multiplied by the respirometer flow rate (L h-1).

112 Aerial and aquatic respirometers were held at a constant temperature by submerging each

113 system into a temperature controlled, recirculating water bath maintained at 26.5 ± 0.09 C.

114 The experimental temperature was based on the average temperatures encountered in Hoga’s

115 mangal habitat. Fishes were carefully observed during experiments, and trials in which

116 individuals exhibited more than intermittent movements were omitted. At the end of each

117 trial, mudskippers were weighed (wet mass ± 0.001 g), measured (standard length ± 0.01

118 mm), and returned to a second holding tank.

119 Mass-adjusted oxygen consumption rates were calculated as the total oxygen consumed

120 (mg h-1) divided by wet mass raised to the power of 0.80. The mass adjustment exponent of

6 121 0.80 is commonly used in fish respirometry to correct for allometric relationships between

122 mass and metabolic rate (Winberg, 1956) and replaces the practice of dividing total metabolic

123 rate by mass alone (see discussion by Cech, 1990). All values were corrected to standard

124 temperature and pressure. Fish in each media were grouped into 5 size classes (≤ 2.00, 2.01-

125 4.00, 4.01-6.00, 6.01-8.00, ≥ 8.01 cm in standard length) and final oxygen consumption

126 measures for each size class reported as the arithmetic mean of the collective mass-adjusted

127 oxygen consumption rates of individual fish. Mass-adjusted oxygen consumption values in

128 air and water were plotted on fish mass to identify differences across size classes or between

129 respiratory media. Mass-adjusted oxygen consumption values between size classes within

130 media were explored using one-way analysis of variance (ANOVA). Where significant

131 changes were found, a Student-Newman-Keuls (SNK) multiple range test (MRT) was used to

132 identify statistical relationships between size groups. Statistical decisions for within media

133 ANOVA and SNK MRT comparisons were based on an alpha level of 0.05. Comparisons of

134 mass-adjusted metabolic rates within size classes between media were made using Student’s

135 independent t-test and all statistical decisions based on a Bonferroni corrected alpha level of

136 0.01.

137

138 RESULTS

139 Common Mudskippers used in our experiments covered a wide size range (Table 1), with

140 lengths varying by an order of magnitude (1.00 to 10.3 cm), and wet mass changing by three

141 orders of magnitude (0.03 to 17.3 g). The largest fish captured had a standard length of 12.9

142 cm and wet mass of 28.9 g but could not be used in experimental trials due to respirometry

143 flask size constraints.

7 144 Air temperature profiles for upper and lower mangal habitats between 28 June and 24

145 July 2005 (Fig. 2) showed that when emerged, mudskippers from exposed lower mangal

146 regions experienced temperatures ranging from 24.5 to 36.5 C (Δ = 12 C), whereas air

147 temperatures from shaded upper mangal zones exhibited more moderate temperature changes

148 of 24 to 31 C (Δ = 7 C). Lower mudflat regions exhibited maximum diel temperature shifts

149 of 11.5 C. Shaded upper mangal areas, on the other hand, exhibited temperatures ranging no

150 more than 6.5 C within a 24-h period. The average daytime high temperature difference

151 between low and high mangal zones was 4.6 C (34.3 and 29.7 C, respectively), while the

152 average night temperature difference was only 0.1 C (25.3 and 25.2 C, respectively).

153 Plots of mass-adjusted oxygen consumption on wet mass for Common Mudskippers

154 demonstrated similar trends of oxygen uptake in air and water (Fig. 3), with fish 4.00 cm or

155 less in standard length exhibiting higher mass-adjusted rates than larger fish. Mass-adjusted

156 aerial metabolic rates of fish 2.00 cm or less in standard length were significantly elevated,

157 consuming on average ten times more oxygen than larger size classes; however, aerial

158 oxygen consumption values of size classes 2.01 cm and above did not differ significantly

159 from each other (ANOVA: F4,112 = 40.29, P < 0.0001; SNK, α = 0.05). Metabolic rates of

160 submerged fish also fell into two statistically distinct subsets (Table 1). Mudskippers with

161 standard lengths of 4.00 cm or less had significantly higher metabolic rates than fish within

162 the three remaining size classes; however, mean oxygen uptake values of fish within subsets

163 did not differ significantly from each other (ANOVA: F4,87 = 6.89, P < 0.0001; SNK, α =

164 0.05). When oxygen consumption values at each size class were compared, only the smallest

165 mudskipper size class ( 2.00 cm) differed significantly between air and water (t-test: df =

166 34, t = 3.44, P < 0.0001). Mudskippers 2 cm in standard length or smaller consumed over

8 167 seven times more oxygen in air than water resulting in an air:water oxygen uptake ratio of

168 7.5:1. All other size classes had similar rates of oxygen uptake in air and water and air:water

169 oxygen uptake ratios fell to approximately 1:1 (Table 1).

170

171 DISCUSSION

172 Tamura and co-workers (1976) assumed correctly that mudskipper distribution within

173 mangals was related to differing metabolic rates in air and water; however, we would argue

174 that it is high, and not low, aerial oxygen extraction rates that bind smaller fish to tidepool

175 microhabitats. Mass-adjusted metabolic rates for Common Mudskippers less than 2 cm in

176 standard length were ten times higher than seen in larger fish — an observation consistent

177 with existing morphological (Clayton, 1993) and histological (Suzuki and Hagiwara, 1995)

178 evidence in periophthalmids. The high cost of being on land likely restricts small Common

179 Mudskippers to short emergence times, and requires them to remain near tidepools. Only

180 later, as fish reach sizes of approximately 2 cm, and aerial metabolic rates decrease, do fish

181 begin to move into more exposed mangal areas. Tytler and Vaughn (1983) found that small

182 Common Mudskippers from Kuwait Bay remained in shallow pools or wet mud until

183 reaching sizes between 1.5 and 2.5 cm in standard length after which, they could be found

184 moving into drier microhabitats. Likewise, Brillet (1976) noted that small Common

185 Mudskippers in Madagascar were never found more than a few centimeters away from

186 mangrove tidepools and it was not until reaching standard lengths of 2 cm or more that fish

187 ventured into drier exposed areas. Common Mudskippers in our study showed a similar

188 distribution at low tide, with fish less than 2 cm in length remaining close to pools in upper

189 mangal regions near , and larger fish most numerous in lower mangal mudflats.

9 190 High mass-adjusted metabolic rates noted for small mudskippers in both air and water

191 (Fig. 3) might be explained by demands of rapid growth or higher activity levels, but it is

192 unclear why the increase becomes more pronounced when fish are emerged. High surface

193 area to volume ratios of small mudskippers are believed to support greater activity levels

194 (Graham 1997). Hillman and Withers (1987), for example, found that aerial metabolic rates

195 of Barred Mudskipper, Periophthalmus argentilineatus, between 0.7 and 3.2 g, more than

196 tripled in active fish. Small fish in our aerial trials, however, were not noticeably more active

197 than larger fish, and size would not account for the more than seven fold increase in mass-

198 adjusted oxygen demand by small fish in air. We propose that the marked differences in air

199 and water metabolic rates result from different physiological demands imposed on fish by

200 each medium. For example, ammonia build-up while on land is known to provoke higher

201 metabolic rates in at least three mudskipper (Gordon, 1969; Gordon et al., 1978; Ip et

202 al., 2004). Likewise, rapid heat exchange is a serious problem for emerged mudskippers

203 (Tytler and Vaughn, 1983) that may lead to elevated metabolic rates. Taylor et al. (2005)

204 found that Common Mudskipper oxygen consumption increased by 44% as ambient

205 temperatures rose from 26 to 32 C. Upward shifts in metabolic rates would be especially

206 taxing for small amphibious fish owing to their high surface area to volume ratios and

207 elevated specific metabolic rates. For these fish, frequent submersion may be the best way to

208 ameliorate problems of ammonia or heat buildup.

209 While the high cost of being amphibious dictates, or at least reinforces, a close

210 association with pool environments, small Common Mudskippers no doubt realize additional

211 benefits from inhabiting upper mangal regions. Clayton (1993) suggested that temperature

212 relationships are the primary factor in dictating behavior and physiological processes in

10 213 mudskipper fishes. Dry season temperatures on Hoga can be extreme with daytime air

214 temperatures increasing rapidly by as much as 12 C in exposed areas (Fig. 2). High

215 temperatures not only elevate metabolic rate but also promote high rates of evaporative water

216 loss in mudskippers (Tytler and Vaughn, 1983). Small mudskippers on Hoga avoided

217 problems associated with extreme temperatures when emerged by remaining in shaded upper

218 mangals where daytime temperatures were on average 5 C lower than those experienced by

219 fish on exposed mudflats. Their proximity to pools may have the added advantage of

220 allowing small fish to quickly move between air and water thus effectively reducing both

221 aerial and aquatic predatory pressure (Brillet, 1976). In addition, spatial separation of small

222 and large Common Mudskipper within the mangal may reduce direct competition and

223 intraspecific cannibalism helping these fish more effectively exploit sparse mangal resources.

224

225 ACKNOWLEDGEMENTS

226 We thank the 2005 Indonesia Research Team for their contributions. Operation Wallacea

227 and University of West Florida, College of Arts and Sciences provided research funding. All

228 were collected under Operation Wallacea collection permit #OP 647-05, and treated

229 in accordance with the guidelines established by Operation Wallacea and the Care

230 and Use Committee at the University of West Florida, protocol #2005-006.

11 231 LITERATURE CITED

232 BICUDO, J.E.P.W. AND K. JOHANSEN. 1979. Respiratory gas exchange in the airbreathing fish, 233 Synbranchus marmoratus. Environmental Biology of Fishes 4:55-64. 234

235 BRILLET, C. 1976. Structure of the burrow, reproduction and behavior of the young of the 236 amphibious fish Periophthalmus sobrinus. La Terre et la vie; revue d’ecologie appliquée 237 30:465-483. 238

239 CECH, J.J. 1990. Repirometry, p 335-361. In: Methods for Fish Biology. C.B. Schreck, P.B. 240 Moyle (eds.). American Fisheries Society, Bethesda, Maryland. 241

242 CLAYTON, D.A. 1993. Mudskippers. Oceanography and Marine Biology an Annual Review 243 31:507-577. 244

245 COX, G.W. 1990. Laboratory Manual of General Ecology. W.C. Brown Publishers, Dubuque, 246 Iowa. 247

248 FRITH, D.W. 1977. A preliminary list of macrofauna from a mangrove forest and adjacent 249 biotopes at Surin Island, western peninsular Thailand. Phuket Marine Biological Center 250 Research Bulletin 17:1-14. 251

252 GORDON, M.S., I. BOETIUS, D.E. EVANS, R. MCCARTHY, L.C. OGLESBY. 1969. Aspects of the 253 physiology of terrestrial life in amphibious fishes. I. The mudskipper Periophthalmus 254 sobrinus. Journal of Experimental Biology 50:141-149. 255

256 GORDON, M.S., W. WILSON, A. YIP. 1978. Aspects of the physiology of terrestrial life in 257 amphibious fishes. III. The Chinese Mudskipper Periophthalmus cantonensis. Journal of 258 Experimental Biology 72:57-75. 259

260 GORDON, M.S., D.J. GABALDON, A.Y.-W. YIP. 1985. Exploratory observations on microhabitat 261 selection within the intertidal zone by the Chinese mudskipper fish Periophthalmus 262 cantonensis. Marine Biology 85:209-215. 263 264 GRAHAM, J.B. 1997. Air-Breathing Fishes. Academic Press, San Diego, California. 265

12 266 HILLMAN, S.S., AND P.C. WITHERS. 1987. Oxygen consumption during aerial activity in aquatic 267 and amphibious fish. Copeia 1987: 232-234. 268

269 IP, Y. K., S. F. CHEW AND W. P. LOW. 1991. Effects of hypoxia on the mudskipper, 270 Periophthalmus chrysospilos (Bleeker, 1853). Journal of Fish Biology 38:621- 623. 271 272 IP, Y. K., D.J. RANDALL, T.K.T KOK, C. BARZAGHI, P.A. WRIGHT, J.S. BALLANTYNE, J.M. 273 WILSON AND S.F. CHEW. 2004. The giant mudskipper Periophthalmodon schlosseri + 274 facilitates active NH4 excretion by increasing acid excretion and decreasing NH3 275 permeability in the skin. Journal of Experimental Biology 207:787-801. 276

277 KOK, W.K., C.B. LIM, T.J. LAM, Y.K. IP. 1998. The mudskipper Periophthalmodon schlosseri 278 respires more efficiently on land than in water and vice versa for Boleophthalmus 279 boddaerti. Journal of Experimental Zoology 280:86-90. 280

281 SUZUKI, N. AND K. HAGIWARA. 1995. Epidermal ultrastructure of pelagic larvae of the 282 mudskipper, Periophthalmus modestus (Gobiidae). Bulletin of Nansei Regional Fisheries 283 Research Laboratory 28:33-41. 284

285 TAKEDA, T. A. ISHIMATSU, S. OIKAWA, T. KANDA, Y. HISHIDA, K.H. KHOO. 1999. Mudskipper 286 Periophthalmodon schlosseri can repay oxygen debts in air but not in water. Journal of 287 Experimental Biology 284:265-270. 288

289 TAMURA, S.O., H. MORII, M. YUZURIHA. 1976. Respiration of the amphibious fishes 290 Periophthalmus cantonensis and Boleophthalmus chinensis in water and on land. Journal 291 of Experimental Biology 65:97-107. 292

293 TAYLOR, J.R., M.M. COOK, A.L. KIRKPATRICK, S.N. GALLEHER, J. EME, W.A. BENNETT. 2005. 294 Thermal tactics of air-breathing and non air-breathing Gobiids inhabiting mangrove 295 tidepools on Palau Hoga, Indonesia. Copeia 2005:885-892. 296

297 TYTLER, P., T. VAUGHN. 1983. Thermal ecology of the mudskippers, Periophthalmus koelreuteri 298 (Pallas) and Boleophthalmus boddarti (Pallas) of Kuwait Bay. Journal of Fish Biology 299 23:327-337. 300

301 WINBERG, G.G. 1956. Rate of metabolism and food requirements of fishes. Fisheries Research 302 Board of Canada Translation Series 194, Ottawa. 303

13 304 (JSS, WAB) DEPARTMENT OF BIOLOGY, THE UNIVERSITY OF WEST FLORIDA, 11000 UNIVERSITY

305 PARKWAY, PENSACOLA, FLORIDA 32514; AND (DJS) DEPARTMENT OF BIOLOGICAL SCIENCES,

306 UNIVERSITY OF ESSEX, WIVENHOE PARK, COLCHESTER, ESSEX, UNITED KINGDOM CO4 3SQ.

307 reprint requests to WAB.

308

14 309 FIGURE LEGENDS

310 Fig 1. Mangal mudskipper collection site on Hoga Island, Republic of Indonesia, Southeast

311 Sulawesi in the Banda Sea

312

313 Fig 2. Daily air temperature collected at 20 min intervals between 28 June and 22 July 2005 from

314 shaded upper (A) and exposed lower (B) mangal zones on Hoga Island, Southeast Sulawesi,

315 Indonesia.

316

317 Fig 3. Graph of mass-adjusted oxygen consumption values on standard length (lower abscissa)

318 and mass (upper abscissa) for Common Mudskippers (Periophthalmus kalolo) in (A) air and (B)

319 water.

320

321

15 322

323 TABLE 1. ESTIMATES OF MEAN (± STANDARD ERROR) MASS-ADJUSTED OXYGEN

-0.8 -1 -1 324 CONSUMPTION (MG G H ) AND TOTAL OXYGEN CONSUMPTION (MG H ) RATES IN AIR

325 AND WATER FOR COMMON MUDSKIPPERS (PERIOPHTHALMUS KALOLO). Standard lengths

326 (SL), and wet mass for all size classes in air and water as well as air:water oxygen uptake

327 ratios are also provided. Mean mass-adjusted oxygen consumption estimates with like

328 superscripts are statistically indistinguishable.

329

Total O2 Mass-adjusted Size class SL (cm) Mass (g) uptake O2 uptake

(cm) n Mean ± SE Mean ± SE Mean ± SE Mean ± SE Air:Water

Air

 2.00 29 1.34 ± 0.033 0.07 ± 0.007 0.33 ± 0.039 3.80 ± 0.464a

2.01-4.00 35 3.05 ± 0.106 0.42 ± 0.050 0.21 ± 0.021 0.51 ± 0.058bc

4.01-6.00 28 5.04 ± 0.106 1.78 ± 0.138 0.40 ± 0.029 0.27 ± 0.023c

6.01-8.00 14 6.78 ± 0.151 4.31 ± 0.300 0.85 ± 0.067 0.27 ± 0.024c

≥ 8.01 11 9.37 ± 0.147 11.69 ± 0.786 2.02 ± 0.197 0.29 ± 0.028c

Water

 2.00 7 1.29 ± 0.112 0.06 ± 0.010 0.05 ± 0.011 0.51 ± 0.109b 7.5:1.0

b 2.01-4.00 28 3.18 ± 0.102 0.56 ± 0.057 0.24 ± 0.027 0.40 ± 0.036 1.3:1.0

c 4.01-6.00 28 4.92 ± 0.118 1.69 ± 0.137 0.41 ± 0.032 0.28 ± 0.020 1.0:1.0

c 6.01-8.00 17 7.03 ± 0.149 5.13 ± 0.264 0.90 ± 0.050 0.25 ± 0.015 1.1:1.0

c ≥ 8.01 12 9.01 ± 0.215 11.09 ± 1.076 1.78 ± 0.180 0.26 ± 0.018 1.1:1.0

16 Kalimantan Sumatra Sulawesi Papua

Java

F1 20 400 N Collection Lower Sites mangal Km Upper mangal

Hoga Island 0 ½ 1

Km

330

17 A

36

34

324

302

028 Temperature (°C) Temperature 826

24

22 06/28 07/02 07/06 07/10 07/14 07/18 07/22

B

36

34

32

30

28 Temperature (°C) Temperature 26

24

22 06/28 07/02 07/06 07/10 07/14 07/18 07/22 Date

331

332

18 Mass (g)

0 2 4 6 8 10 12 14 16 7 )1 - 0.8h 6 (mg g 5

2 adjusted4 VO 3

2

Mass- 1

0

0 1 2 3 4 5 6 7 8 9 10 11 Length (cm)

333

19