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Supercooling points and heat coma temperatures in four species of oceanic sea skaters of the genus Halobates (Heteroptera: Gerridae: Halobatinae)
Journal of Asia-Pacific Entomology 16 (2013) 219–222
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Journal of Asia-Pacific Entomology journal homepage: www.elsevier.com/locate/jape
Supercooling points and heat coma temperatures in four species of oceanic sea skaters of the genus Halobates (Heteroptera: Gerridae: Halobatinae) Tetsuo Harada a,⁎, Shiho Takenaka a, Kohki Iyota a, Takashi Shiraki a, Masatoshi Moku b, Chihiro Katagiri c, Vladimír Koštál d a
Laboratory of Environmental Physiology, Graduate School of Integrated Arts and Sciences of Human and Nature, Kochi University, Kochi, Japan Atmosphere and Ocean Research Institute, The University of Tokyo, Kashiwa, Japan c Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan d Institute of Entomology, Biology Center of the Academy of Sciences CR, Ceske Budejovice, Czech Republic b
a r t i c l e
i n f o
Article history: Received 17 December 2011 Revised 22 January 2013 Accepted 24 January 2013 Keywords: Temperature dynamics Distribution Heat tolerance Cold hardiness Cross tolerance
a b s t r a c t The only insects known to live in the open ocean are several species of sea skaters of the genus Halobates (Heteroptera: Gerridae). We have collected four species in the tropical western Pacific Ocean and measured heat coma temperature (HCT) and supercooling point (SCP) in 132 specimens. The SCPs were relatively low (ranging between − 18 °C and −19 °C) and similar in all species. Significant inter-species differences were found in the level of HCT. The lowest HCT 32.9 °C was found in Halobates micans Eschscholtz, 1822, which is distributed in thermally stable tropical waters. The highest HCT levels of 36.6 °C and 36.0 °C were observed in Halobates sericeus Eschscholtz and Halobates germanus White, 1883, respectively, mostly occurring in thermally more fluctuating habitats (subtropical and warm temperate waters, and coastal waters, respectively). A new species, H. sp. (under process of description) showed an intermediate HCT of 34.8 °C. In addition, we found significant negative statistical correlation between HCT and SCP values at the species and individual levels. We discuss our results in the light of putative relationship between thermal physiology of sea skaters and their distribution. © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2013. Published by Elsevier B.V. All rights reserved.
Introduction Despite the enormous biodiversity of insects in terrestrial and freshwater habitats just a few thousand species are marine (Cheng, 1985, 1989, 2003; Cheng and Frank, 1993), and the only insects known to live in the open ocean are sea skaters of the genus Halobates (Heteroptera: Gerridae). Most of the 47 species of Halobates that have been described so far occur in near-shore areas of the tropical seas. They are often associated with mangrove ecosystem and many are endemic to specific islands or island groups (Cheng, 1989). Five species of sea skaters are considered as true open ocean inhabitants (Cheng, 1985): Halobates micans Eschscholtz, 1822, Halobates sericeus Eschscholtz, 1822, Halobates germanus White, 1883, Halobates splendens Witlaczil, 1886, and Halobates sobrinus White, 1883. One additional species is now under the process of description, we will refer to it as H. sp. in this paper (Harada et al., 2010). Relatively good information is currently ⁎ Corresponding author at: Laboratory of Environmental Physiology, Graduate School of Integrated Arts and Sciences of Human and Nature, Kochi University, Kochi 780-8520, Japan. Tel./fax: +81 88 844 8410. E-mail address: [email protected] (T. Harada).
available on the oceanic sea skater distribution, phylogeny, life history and bionomy (Cheng, 1985, 1989; Ikawa et al., 1998, 2004; Damgaard et al., 2000; Andersen and Cheng, 2004; Harada, 2005). Much less is known, however, about specific behavioral and physiological adaptations that have allowed their unique (among insects) evolutionary transition to the oceanic habitat. In her excellent review, Cheng (1989) raised numerous questions which may be condensed into a single challenge: “How does Halobates succeed where others have failed?” In this paper, we present physiological data concerning four different sea skaters from the tropical western Pacific Ocean. We have measured the heat coma temperature (HCT) and ice crystallization temperature (supercooling point, SCP) in the specimens that were collected in 8 locations during November/December 2009. Three well-described species of Halobates included in this study show distinct geographical distributions (Cheng, 1985, 1989). H. micans is the only species found in three oceans (Pacific, Indian and Atlantic). In Pacific, it is mainly associated with the equatorial zone between 20°N and 20°S. According to Cheng (1985, 1989), H. sericeus is absent in the equatorial zone of Pacific but it extends much further north (up to 45°N) and south (up to 30°S) than H. micans. Our recent data, however, show that H. sericeus may occur as far south as
1226-8615/$ – see front matter © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2013. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aspen.2013.01.005
220
T. Harada et al. / Journal of Asia-Pacific Entomology 16 (2013) 219–222
5°N, at least occasionally (Harada et al., 2007). H. germanus is missing in the eastern Pacific, and in the western Pacific, it seems to be more closely associated with the coasts of the islands than the other two species. The main objective of our study was to test the two hypotheses: (1) that the levels of HCT differ among species of Halobates, reflecting the different geographical distributions and probability of encountering temperature fluctuations in their habitats; and (2) that the levels of HCT correlate with the levels of SCP, reflecting some physiological linkage between the two temperature thresholds.
Materials and methods This study represents part of research program conducted during the cruise no. MR-09-04 of the R/V MIRAI ship (8687 t, owned by JAMSTEC, Japan Agency for Marine-Earth Science and Technology) to western Pacific Ocean in 2009 (Fig. 1A). In total, 907 living sea skaters were collected from the surface of ocean between November 12th and December 4th in 8 different locations (see Fig. 1B for details) by trailing the Neuston Net (diameter 1.3 m, length 6.0 m) for 45 min in each location (ship speed of 2.0 knot to the sea water). The temperature of ocean surface was between 28 and 29 °C. Three well established species were distinguished: H. micans, H. sericeus, H. germanus, plus one species, H. sp, which is under the process of description currently (Harada et al., 2010). All specimens were immediately moved to aquaria with sea water, where temperature was maintained at 28± 1 °C. The sea skaters
were supplied with adult flies, Lucillia illustris, as food and were acclimated in the aquaria for 12–180 h prior to the experiments. The heat coma temperature (HCT) was measured inside the transparent aquarium where the temperature was increased stepwise from initial 28 to 29 °C by 1 °C every 1 h (fluctuations were within the limits of ±0.3 °C). HCT was defined as the temperature at which the ventral surface of sea skater's body was caught by water film. The same specimens that were previously used for HCT measurement were further used for the determination of their supercooling point (SCP). Surface of each insect was dried with filter paper, and nickel/bronze thermocouple was attached to the ventral surface of the abdomen. The insect, inside a double-wall Styrofoam insulating box, was cooled at the rate of approximately 1 °C/min in the freezer, where the temperature was set to −35 °C. The lowest temperature reached prior to the occurrence of freeze exotherm (release of latent heat of ice crystallization) was recorded as the SCP. All statistical analyses of data were performed using Prism4 (GraphPad Software, San Diego, CA).
Results We observed that the sea skaters regularly ceased their movements prior to reaching the HCT. Typically, the movements became very slow or ceased completely at temperatures that were 2–3 °C below the HCT (2.75± 2.43 °C, mean± SD, range: 0–8 °C, n = 132). The assessment
Fig. 1. (A) Geographic distribution of three species of sea skaters (redrawn from Cheng, 1985, 1989). Rectangle shows the area where sea skaters were sampled. This area is enlarged in (B) showing eight locations (grey circles) where the samples were collected between November 12th and December 4th, 2009. The numbers flanking the locations give total numbers of specimens belonging to four different species (in the order: Halobates germanus, H. sericeus, H. micans, and H. sp). The vector lines show direction and speed of surface currents as measured along the path of R/V MIRAI cruise. The directions of main oceanic currents are shown by arrows: solid line, Equatorial Countercurrent; upper dashed line, North Equatorial Current; two lower dashed lines, two branches of South Equatorial Current.
T. Harada et al. / Journal of Asia-Pacific Entomology 16 (2013) 219–222
of movements, however, is ambiguous. The periods of inactivity/rest might be confused with paralysis by heat. Therefore, we decided to analyze the HCT data only. Table 1 summarizes the data on HCT and SCP for all 132 measured adult specimens belonging to the four species. Both datasets (HCT and SCP) are presented as means ±SD, as they passed normality tests in all species (at α = 0.05). There were no statistical differences between females and males in any species. Therefore, we pooled datasets for both sexes and used them for analysis of between-species differences by one-way ANOVA, followed by Bonferroni's multiple comparison test (Fig. 2A). No differences in the SCP value were found between the species (ANOVA: df=3, R2 =0.01962, P=0.4705). The means ranged between −17.9 and −18.9 °C in individual species. On the other hand, HCT values significantly differed between species (ANOVA: df =3, F=4.100, P=0.0082). The lowest HCT (32.9±3.2, n=10) was found in H. micans. The highest HCT values were shown in H. sericeus (36.6±2.5, n=27) and H. germanus (36.0±3.2, n=77). The values seen in H. sp were intermediate (34.8±3.7, n=18). Next, we used linear regressions to analyze the relationship between HCT and SCP in each species individually. Because neither the slopes (F = 0.474722, P = 0.7004) nor the Y-intercepts (F = 0.767035, P = 0.5145) of the regression lines differed significantly among the four species, we pooled data for all species together and conducted Pearson's correlation analysis for the whole dataset (Fig. 2B). We found significant negative statistical correlation between HCT and SCP values (Pearson r = −0.3500, R2 = 0.1225, P b 0.0001). Discussion The surface temperature of the tropical western Pacific Ocean is maintained relatively constant between 27 and 29 °C throughout the year (Ingmanson and Wallace, 1989). The pacific distribution of H. micans nearly overlaps with the tropical zone, which means that the species lives in a thermally highly stable habitat. Almost peri-equatorial distribution area of H. sericeus is situated in colder and more seasonal waters (Ingmanson and Wallace, 1989). The third species, H. germanus is often found in coastal waters. The temperature of coastal waters may easily increase above 30 °C (Tropical weather, 2012) during hot summer months. The thermal habitats of the three species thus significantly differ in their mean temperature and seasonality. In this study, we found small but statistically significant differences in the heat coma temperature of the four Halobates species. While H. micans, occupying the most stable habitat thermally, showed relatively low HCT of 32.9 °C, the two species inhabiting more fluctuating habitats, H. sericeus and H. germanus, showed high HCTs of 36.6 °C and 36.0 °C, respectively. The HCTs in all three species are relatively close to typical surface temperatures of the tropical ocean. Moreover, we observed first signs of heat paralysis (slowing down or cessation of movements) typically occurring at temperatures 2–3 °C below HCT, which shifts the limit of supra-optimal temperatures even closer to typical ambient temperatures. Also the chill coma temperature lies just a few degrees of Celsius
Table 1 Heat coma temperatures (HCT) and supercooling points (SCP) in the adults of sea skaters of four different species. Species
Sex
n
HCT mean ± SD
t-testa
SCP mean ± SD
t-testa
H. germanus
F M F M F M F M
48 29 13 14 8 2 15 3
36.0 ± 3.3 36.0 ± 3.1 36.1 ± 3.0 37.1 ± 1.7 33.4 ± 3.3 31.0 ± 1.4 35.2 ± 3.6 33.0 ± 4.4
t = 0.01057 P = 0.9916 t = 1.135 P = 0.2671 t = 0.9630 P = 0.3638 t = 0.9414 P = 0.3605
−18.6 ± 2.2 −18.3 ± 1.9 −18.7 ± 1.6 −19.0 ± 1.8 −18.0 ± 1.5 −17.7 ± 1.3 −18.9 ± 1.8 −18.7 ± 0.7
t = 0.5920 P = 0.5556 t = 0.3255 P = 0.7475 t = 0.2864 P = 0.7819 t = 0.2562 P = 0.8011
H. sericeus H. micans H. sp. a
Unpaired, two-tailed t-tests were used to compare means in females and males.
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Fig. 2. (A) Analysis of heat coma temperatures (HCT) and supercooling points (SCP) in four sea skater species. Each column represents mean ± SD, source data are presented in Table 1 in detail. Means flanked by different letters are significantly different (ANOVA followed by Bonnferoni's MCT). (B) Analysis of the correlation between HCT and SCP in all four species. The formula of regression line is presented. See text for more details.
bellow the ambient in the oceanic sea skaters (Harada, unpublished observations). Collectively, oceanic sea skaters seem to be relatively stenothermic. The exact full range of permissive temperatures (from chill coma to heat coma) is not currently known, however, for any Halobates species. The temperature of ice crystallization (SCP) does not necessarily represent the lower lethal temperature. Most insects suffer from chillinjuries and high mortality at temperatures far above the SCP (for discussion, see Bale, 1987, 1993; Renault et al., 2002; Koštál et al., 2004). Nevertheless, the meta-analysis of data collected for over 350 terrestrial insect species revealed that some correlation between the SCP and cold tolerance exists. The more severe (colder) was the habitat (scored on an 11-point scale from tropics to polar regions), the lower was the SCP of its inhabitants (Turnock and Fields, 2005). We found that all four sea skater species showed very similar SCP levels ranging between − 18 °C and − 19 °C. This value is unusually low for an active insect and resembles rather the values characteristic for winter-acclimated terrestrial insects in temperate regions (Somme, 1982). For comparison, the SCP ranges between −5 to −7 °C in the adults of closely related subtropical freshwater halobatine species, Metrocoris histrio, irrespective of season (summer, fall, winter) (Harada unpublished observations). Another freshwater strider, Aquarius paludum (Heteroptera, Gerridae) also shows relatively high SCP, ranging between −8 °C and −16 °C depending on season (summer and winter, respectively) (Harada et al., 2000; Harada, 2003). As the SCPs are exceptionally low and similar in all four oceanic halobatids, there may be a link between the exceptional low value of SCPs feature and a high osmotic pressure of haemolymph due to the surrounding sea water salinity, though there has been no evidence. Even with the extremely low and limited SCPs range, there is statistically significant correlation between SCP and HCT. At the level of individual insects (irrespective of species), the higher was the level of HCT, the lower was the level of SCP. Similar relationship may be observed also at the species level: while H. sericeus had the highest HCT and the lowest SCP, H. micans had the lowest HCT and the highest SCP among the four species. Because the temperature of SCP correlates with the temperature of melting (m.p.), which in turn is colligatively influenced by osmolality of body fluids (Zachariassen, 1985), the osmolality might be a good candidate as a linking physiological trait which is responsible for the relatively low SCP levels and for the correlation between HCT and SCP. Relatively high body fluid osmolality should be expected in insects inhabiting marine waters with the mean salinity of 3.5% (approximately 1000 mosmol/kg). Unfortunately, no rigorous data are available for oceanic sea skaters. Unpublished observations by Edney and Cheng (mentioned in Cheng, 1989) refer to the value of 523.6 mosmol/kg. Such
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value is indeed at the upper limit of a range seen in various terrestrial and freshwater insects (250–550 mosmol/kg) (Hadley, 1994), which may explain, at least partially, why the SCP values are so low. The question is how the correlation between SCP and HCT is induced. Heat shock protein (HSP) is well known to be in transcript higher than that under normal conditions in response to the exposure to heat in insects, especially in Drosophila (Velazquez et al., 1983; Goto et al., 1998). There is heat shock protein defense from the damage of proteins due to heat, for example (Neven, 2000). The HSP70 protein may ‘chaperone’ the protein to the lysosome for degradation (Chiang et al., 1994) or may help the protein re-fold after return to favorable temperatures (Parsell and Lindquist, 1994). The over expression of HSP may be speculated to promote the expression of other substances to increase the osmotic pressures in the case of oceanic sea skaters. Heat shock proteins promote the synthesis of sorbitol in white flies, polyol in whiteflies and aphids, and the trehalose synthesis in yeast (Salvucci, 2000; Salvucci et al., 2000). As we consider our results to be introductory, we do not see the conclusions as authoritative. Nevertheless, we suggest that our data may be further extended for the research on the relationship between the thermal characteristics of sea skater habitat and the complex of linked physiological parameters, which may serve to better understand the distribution and biology of these unique insects. Acknowledgments We would like to thank Dr. Yuji Kashino (Head Scientist of the cruise MR-09-04) and Mr. Yasushi Ishioka (Captain of R/V MIRAI) for their support during our study. Prof. Yoshiro Watanabe (Ocean Research Institute, University of Tokyo, Japan) kindly rented the Neuston Net to us. Dr. Tomáš Ditrich (Pedagogical Faculty, University of South Bohemia, České Budějovice, Czech Republic) helped with the data processing and analysis. References Andersen, N.M., Cheng, L., 2004. The marine insect Halobates (Heteroptera: Gerridae): biology, adaptations distribution, and phylogeny. Oceanogr. Mar. Biol. Annu. Rev 42, 119–180. Bale, J.S., 1987. Insect cold hardiness: freezing and supercooling — an ecophysiological perspective. J. Insect Physiol. 33, 899–908. Bale, J.S., 1993. Classes of insect cold hardiness. Funct. Ecol. 7, 751–753. Cheng, L., 1985. Biology of Halobates (Heteroptera: Gerridae). Annu. Rev. Entomol. 30, 111–135. Cheng, L., 1989. Factors limiting the distribution of Halobates species. Reproduction, genetics and distribution of marine organisms. In: Ryland, J.S., Tyler, P.A. (Eds.), Proceedings of 23rd European Marine Biology Symposium. Olsen & Olsen, Fredensbor, pp. 357–362.
Cheng, L., 2003. Marine insects. In: Resh, V.H., Carde, R.T. (Eds.), Encyclopedia of Insects. Academic Press, San Diego, pp. 679–682. Cheng, L., Frank, J.H., 1993. Marine insects and their reproduction. Oceanogr. Mar. Biol. Annu. Rev 31, 479–506. Chiang, H., Terlecky, S.R., Plant, C.P., Dice, J.F., 1994. A role for a 70-kilodaton heat shock protein in lysosomal degradation of intracellular proteins. Science 246, 382–385. Damgaard, J., Andersen, N.M., Cheng, L., 2000. Phylogeny of sea skaters, Halobates Eschscholtz (Hemiptera, Gerridae), based on mtDNA sequence and morphology. Zool. J. Linne. Soc. 130, 511–526. Goto, S.G., Yoshida, K.M., Kimura, M.T., 1998. Accumulation of Hsp70 mRNA under environmental stresses in diapausing and nondiapausing adults of Drosophila triauraria. J. Insect Physiol. 44, 1009–1015. Hadley, E.B., 1994. Water Relations of Terrestrial Arthropods. Academic Press, London. Harada, T., 2003. Hardiness to low temperature and drought in a water strider, Aquarius paludum in comparison with other insect groups. (Res. Trends, Trivandrum, India) Trends Entomol. 3, 29–41. Harada, T., 2005. Geographical distribution of three oceanic Halobates spp. and an account of the behaviour of H. sericeus (Heteroptera: Gerridae). Eur. J. Entomol. 102, 299–302. Harada, T., Inoue, T., Ono, I., et al., 2000. Endocrine, ecophysiological and ecological aspects of seasonal adaptations in a water strider, Aquarius paludum (a mini review). Entomol. Sci. 3, 157–165. Harada, T., Nakajyo, M., Inoue, T., 2007. Geographical Distribution and Heat-tolerance in the Oceanic Sea Skaters of Halobates and Oceanic Dynamics (Heteroptera: Gerridae). : The Cruise Report of MR-06-05-Leg, 3. JAMSTEC, Yokosuka. Harada, T., Sekimoto, T., Iyota, K., et al., 2010. Comparison of the population density of oceanic sea skater of Halobates (Heteroptera: Gerridae) among several areas in the tropical Pacific Ocean and the tropical Indian Ocean. Formos. Entomol. 30, 307–316. Ikawa, T., Okubo, A., Okabe, H., et al., 1998. Oceanic diffusion and the pelagic insects Halobates spp. (Gerridae: Hemiptera). Mar. Biol. 131, 195–201. Ikawa, T., Okabe, H., Hoshizaki, S., et al., 2004. Distribution of the oceanic insects Halobates (Hemiptera: Gerridae) off the south coast of Japan. Entomol. Sci. 7, 351–357. Ingmanson, D.E., Wallace, E.J., 1989. Oceanography. Wadsworth Publications, Belmont, CA. Koštál, V., Vambera, J., Bastl, J., 2004. On the nature of pre-freeze mortality in insects: water balance, ion homeostasis and energy charge in the adults of Pyrrhocoris apterus. J. Exp. Biol. 207, 1509–1521. Neven, L.G., 2000. Physiological responses of insects to heat. Postharvest Biol. Technol. 21, 103–111. Parsell, D.A., Lindquist, S., 1994. In: Morimoto, R.I., Tissieres, A., Georgopoulos, C. (Eds.), The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, pp. 457–494. Renault, D., Salin, C., Vannier, G., et al., 2002. Survival at low temperatures in insects: what is the ecological significance of the supercooling point. Cryo-Letters 23, 217–228. Salvucci, M.E., 2000. Sorbitol accumulation in white flies: evidence for a role in protecting proteins during heat stress. J. Therm. Biol. 25, 353–361. Salvucci, M.E., Stecher, D.S., Henneberry, T.J., 2000. Heat shock proteins in whiteflies, an insect that accumulates sorbitol in response to heat stress. J. Therm. Biol. 25, 363–371. Somme, L., 1982. Supercooling and winter survival in terrestrial arthropods. Comp. Biochem. Physiol. 73, 519–543. Tropical weather, 2012. Tropical weather & hurricanes. http://www.wundergroupnd. com/tropical/?index-region=wp. Turnock, W.J., Fields, P.G., 2005. Winter climates and cold hardiness in terrestrial insects. Eur. J. Entomol. 102, 561–576. Velazquez, J.M., Sonoda, S., Bugaisky, G., Lindquist, S., 1983. Is the major Drosophila heat shock protein present in cells that have not been heat shocked? J. Cell Biol. 96, 286–290. Zachariassen, K.E., 1985. Physiology of cold tolerance in insects. Physiol. Rev. 65, 799–832.
Contents lists available at SciVerse ScienceDirect
Journal of Asia-Pacific Entomology journal homepage: www.elsevier.com/locate/jape
Supercooling points and heat coma temperatures in four species of oceanic sea skaters of the genus Halobates (Heteroptera: Gerridae: Halobatinae) Tetsuo Harada a,⁎, Shiho Takenaka a, Kohki Iyota a, Takashi Shiraki a, Masatoshi Moku b, Chihiro Katagiri c, Vladimír Koštál d a
Laboratory of Environmental Physiology, Graduate School of Integrated Arts and Sciences of Human and Nature, Kochi University, Kochi, Japan Atmosphere and Ocean Research Institute, The University of Tokyo, Kashiwa, Japan c Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan d Institute of Entomology, Biology Center of the Academy of Sciences CR, Ceske Budejovice, Czech Republic b
a r t i c l e
i n f o
Article history: Received 17 December 2011 Revised 22 January 2013 Accepted 24 January 2013 Keywords: Temperature dynamics Distribution Heat tolerance Cold hardiness Cross tolerance
a b s t r a c t The only insects known to live in the open ocean are several species of sea skaters of the genus Halobates (Heteroptera: Gerridae). We have collected four species in the tropical western Pacific Ocean and measured heat coma temperature (HCT) and supercooling point (SCP) in 132 specimens. The SCPs were relatively low (ranging between − 18 °C and −19 °C) and similar in all species. Significant inter-species differences were found in the level of HCT. The lowest HCT 32.9 °C was found in Halobates micans Eschscholtz, 1822, which is distributed in thermally stable tropical waters. The highest HCT levels of 36.6 °C and 36.0 °C were observed in Halobates sericeus Eschscholtz and Halobates germanus White, 1883, respectively, mostly occurring in thermally more fluctuating habitats (subtropical and warm temperate waters, and coastal waters, respectively). A new species, H. sp. (under process of description) showed an intermediate HCT of 34.8 °C. In addition, we found significant negative statistical correlation between HCT and SCP values at the species and individual levels. We discuss our results in the light of putative relationship between thermal physiology of sea skaters and their distribution. © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2013. Published by Elsevier B.V. All rights reserved.
Introduction Despite the enormous biodiversity of insects in terrestrial and freshwater habitats just a few thousand species are marine (Cheng, 1985, 1989, 2003; Cheng and Frank, 1993), and the only insects known to live in the open ocean are sea skaters of the genus Halobates (Heteroptera: Gerridae). Most of the 47 species of Halobates that have been described so far occur in near-shore areas of the tropical seas. They are often associated with mangrove ecosystem and many are endemic to specific islands or island groups (Cheng, 1989). Five species of sea skaters are considered as true open ocean inhabitants (Cheng, 1985): Halobates micans Eschscholtz, 1822, Halobates sericeus Eschscholtz, 1822, Halobates germanus White, 1883, Halobates splendens Witlaczil, 1886, and Halobates sobrinus White, 1883. One additional species is now under the process of description, we will refer to it as H. sp. in this paper (Harada et al., 2010). Relatively good information is currently ⁎ Corresponding author at: Laboratory of Environmental Physiology, Graduate School of Integrated Arts and Sciences of Human and Nature, Kochi University, Kochi 780-8520, Japan. Tel./fax: +81 88 844 8410. E-mail address: [email protected] (T. Harada).
available on the oceanic sea skater distribution, phylogeny, life history and bionomy (Cheng, 1985, 1989; Ikawa et al., 1998, 2004; Damgaard et al., 2000; Andersen and Cheng, 2004; Harada, 2005). Much less is known, however, about specific behavioral and physiological adaptations that have allowed their unique (among insects) evolutionary transition to the oceanic habitat. In her excellent review, Cheng (1989) raised numerous questions which may be condensed into a single challenge: “How does Halobates succeed where others have failed?” In this paper, we present physiological data concerning four different sea skaters from the tropical western Pacific Ocean. We have measured the heat coma temperature (HCT) and ice crystallization temperature (supercooling point, SCP) in the specimens that were collected in 8 locations during November/December 2009. Three well-described species of Halobates included in this study show distinct geographical distributions (Cheng, 1985, 1989). H. micans is the only species found in three oceans (Pacific, Indian and Atlantic). In Pacific, it is mainly associated with the equatorial zone between 20°N and 20°S. According to Cheng (1985, 1989), H. sericeus is absent in the equatorial zone of Pacific but it extends much further north (up to 45°N) and south (up to 30°S) than H. micans. Our recent data, however, show that H. sericeus may occur as far south as
1226-8615/$ – see front matter © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2013. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aspen.2013.01.005
220
T. Harada et al. / Journal of Asia-Pacific Entomology 16 (2013) 219–222
5°N, at least occasionally (Harada et al., 2007). H. germanus is missing in the eastern Pacific, and in the western Pacific, it seems to be more closely associated with the coasts of the islands than the other two species. The main objective of our study was to test the two hypotheses: (1) that the levels of HCT differ among species of Halobates, reflecting the different geographical distributions and probability of encountering temperature fluctuations in their habitats; and (2) that the levels of HCT correlate with the levels of SCP, reflecting some physiological linkage between the two temperature thresholds.
Materials and methods This study represents part of research program conducted during the cruise no. MR-09-04 of the R/V MIRAI ship (8687 t, owned by JAMSTEC, Japan Agency for Marine-Earth Science and Technology) to western Pacific Ocean in 2009 (Fig. 1A). In total, 907 living sea skaters were collected from the surface of ocean between November 12th and December 4th in 8 different locations (see Fig. 1B for details) by trailing the Neuston Net (diameter 1.3 m, length 6.0 m) for 45 min in each location (ship speed of 2.0 knot to the sea water). The temperature of ocean surface was between 28 and 29 °C. Three well established species were distinguished: H. micans, H. sericeus, H. germanus, plus one species, H. sp, which is under the process of description currently (Harada et al., 2010). All specimens were immediately moved to aquaria with sea water, where temperature was maintained at 28± 1 °C. The sea skaters
were supplied with adult flies, Lucillia illustris, as food and were acclimated in the aquaria for 12–180 h prior to the experiments. The heat coma temperature (HCT) was measured inside the transparent aquarium where the temperature was increased stepwise from initial 28 to 29 °C by 1 °C every 1 h (fluctuations were within the limits of ±0.3 °C). HCT was defined as the temperature at which the ventral surface of sea skater's body was caught by water film. The same specimens that were previously used for HCT measurement were further used for the determination of their supercooling point (SCP). Surface of each insect was dried with filter paper, and nickel/bronze thermocouple was attached to the ventral surface of the abdomen. The insect, inside a double-wall Styrofoam insulating box, was cooled at the rate of approximately 1 °C/min in the freezer, where the temperature was set to −35 °C. The lowest temperature reached prior to the occurrence of freeze exotherm (release of latent heat of ice crystallization) was recorded as the SCP. All statistical analyses of data were performed using Prism4 (GraphPad Software, San Diego, CA).
Results We observed that the sea skaters regularly ceased their movements prior to reaching the HCT. Typically, the movements became very slow or ceased completely at temperatures that were 2–3 °C below the HCT (2.75± 2.43 °C, mean± SD, range: 0–8 °C, n = 132). The assessment
Fig. 1. (A) Geographic distribution of three species of sea skaters (redrawn from Cheng, 1985, 1989). Rectangle shows the area where sea skaters were sampled. This area is enlarged in (B) showing eight locations (grey circles) where the samples were collected between November 12th and December 4th, 2009. The numbers flanking the locations give total numbers of specimens belonging to four different species (in the order: Halobates germanus, H. sericeus, H. micans, and H. sp). The vector lines show direction and speed of surface currents as measured along the path of R/V MIRAI cruise. The directions of main oceanic currents are shown by arrows: solid line, Equatorial Countercurrent; upper dashed line, North Equatorial Current; two lower dashed lines, two branches of South Equatorial Current.
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of movements, however, is ambiguous. The periods of inactivity/rest might be confused with paralysis by heat. Therefore, we decided to analyze the HCT data only. Table 1 summarizes the data on HCT and SCP for all 132 measured adult specimens belonging to the four species. Both datasets (HCT and SCP) are presented as means ±SD, as they passed normality tests in all species (at α = 0.05). There were no statistical differences between females and males in any species. Therefore, we pooled datasets for both sexes and used them for analysis of between-species differences by one-way ANOVA, followed by Bonferroni's multiple comparison test (Fig. 2A). No differences in the SCP value were found between the species (ANOVA: df=3, R2 =0.01962, P=0.4705). The means ranged between −17.9 and −18.9 °C in individual species. On the other hand, HCT values significantly differed between species (ANOVA: df =3, F=4.100, P=0.0082). The lowest HCT (32.9±3.2, n=10) was found in H. micans. The highest HCT values were shown in H. sericeus (36.6±2.5, n=27) and H. germanus (36.0±3.2, n=77). The values seen in H. sp were intermediate (34.8±3.7, n=18). Next, we used linear regressions to analyze the relationship between HCT and SCP in each species individually. Because neither the slopes (F = 0.474722, P = 0.7004) nor the Y-intercepts (F = 0.767035, P = 0.5145) of the regression lines differed significantly among the four species, we pooled data for all species together and conducted Pearson's correlation analysis for the whole dataset (Fig. 2B). We found significant negative statistical correlation between HCT and SCP values (Pearson r = −0.3500, R2 = 0.1225, P b 0.0001). Discussion The surface temperature of the tropical western Pacific Ocean is maintained relatively constant between 27 and 29 °C throughout the year (Ingmanson and Wallace, 1989). The pacific distribution of H. micans nearly overlaps with the tropical zone, which means that the species lives in a thermally highly stable habitat. Almost peri-equatorial distribution area of H. sericeus is situated in colder and more seasonal waters (Ingmanson and Wallace, 1989). The third species, H. germanus is often found in coastal waters. The temperature of coastal waters may easily increase above 30 °C (Tropical weather, 2012) during hot summer months. The thermal habitats of the three species thus significantly differ in their mean temperature and seasonality. In this study, we found small but statistically significant differences in the heat coma temperature of the four Halobates species. While H. micans, occupying the most stable habitat thermally, showed relatively low HCT of 32.9 °C, the two species inhabiting more fluctuating habitats, H. sericeus and H. germanus, showed high HCTs of 36.6 °C and 36.0 °C, respectively. The HCTs in all three species are relatively close to typical surface temperatures of the tropical ocean. Moreover, we observed first signs of heat paralysis (slowing down or cessation of movements) typically occurring at temperatures 2–3 °C below HCT, which shifts the limit of supra-optimal temperatures even closer to typical ambient temperatures. Also the chill coma temperature lies just a few degrees of Celsius
Table 1 Heat coma temperatures (HCT) and supercooling points (SCP) in the adults of sea skaters of four different species. Species
Sex
n
HCT mean ± SD
t-testa
SCP mean ± SD
t-testa
H. germanus
F M F M F M F M
48 29 13 14 8 2 15 3
36.0 ± 3.3 36.0 ± 3.1 36.1 ± 3.0 37.1 ± 1.7 33.4 ± 3.3 31.0 ± 1.4 35.2 ± 3.6 33.0 ± 4.4
t = 0.01057 P = 0.9916 t = 1.135 P = 0.2671 t = 0.9630 P = 0.3638 t = 0.9414 P = 0.3605
−18.6 ± 2.2 −18.3 ± 1.9 −18.7 ± 1.6 −19.0 ± 1.8 −18.0 ± 1.5 −17.7 ± 1.3 −18.9 ± 1.8 −18.7 ± 0.7
t = 0.5920 P = 0.5556 t = 0.3255 P = 0.7475 t = 0.2864 P = 0.7819 t = 0.2562 P = 0.8011
H. sericeus H. micans H. sp. a
Unpaired, two-tailed t-tests were used to compare means in females and males.
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Fig. 2. (A) Analysis of heat coma temperatures (HCT) and supercooling points (SCP) in four sea skater species. Each column represents mean ± SD, source data are presented in Table 1 in detail. Means flanked by different letters are significantly different (ANOVA followed by Bonnferoni's MCT). (B) Analysis of the correlation between HCT and SCP in all four species. The formula of regression line is presented. See text for more details.
bellow the ambient in the oceanic sea skaters (Harada, unpublished observations). Collectively, oceanic sea skaters seem to be relatively stenothermic. The exact full range of permissive temperatures (from chill coma to heat coma) is not currently known, however, for any Halobates species. The temperature of ice crystallization (SCP) does not necessarily represent the lower lethal temperature. Most insects suffer from chillinjuries and high mortality at temperatures far above the SCP (for discussion, see Bale, 1987, 1993; Renault et al., 2002; Koštál et al., 2004). Nevertheless, the meta-analysis of data collected for over 350 terrestrial insect species revealed that some correlation between the SCP and cold tolerance exists. The more severe (colder) was the habitat (scored on an 11-point scale from tropics to polar regions), the lower was the SCP of its inhabitants (Turnock and Fields, 2005). We found that all four sea skater species showed very similar SCP levels ranging between − 18 °C and − 19 °C. This value is unusually low for an active insect and resembles rather the values characteristic for winter-acclimated terrestrial insects in temperate regions (Somme, 1982). For comparison, the SCP ranges between −5 to −7 °C in the adults of closely related subtropical freshwater halobatine species, Metrocoris histrio, irrespective of season (summer, fall, winter) (Harada unpublished observations). Another freshwater strider, Aquarius paludum (Heteroptera, Gerridae) also shows relatively high SCP, ranging between −8 °C and −16 °C depending on season (summer and winter, respectively) (Harada et al., 2000; Harada, 2003). As the SCPs are exceptionally low and similar in all four oceanic halobatids, there may be a link between the exceptional low value of SCPs feature and a high osmotic pressure of haemolymph due to the surrounding sea water salinity, though there has been no evidence. Even with the extremely low and limited SCPs range, there is statistically significant correlation between SCP and HCT. At the level of individual insects (irrespective of species), the higher was the level of HCT, the lower was the level of SCP. Similar relationship may be observed also at the species level: while H. sericeus had the highest HCT and the lowest SCP, H. micans had the lowest HCT and the highest SCP among the four species. Because the temperature of SCP correlates with the temperature of melting (m.p.), which in turn is colligatively influenced by osmolality of body fluids (Zachariassen, 1985), the osmolality might be a good candidate as a linking physiological trait which is responsible for the relatively low SCP levels and for the correlation between HCT and SCP. Relatively high body fluid osmolality should be expected in insects inhabiting marine waters with the mean salinity of 3.5% (approximately 1000 mosmol/kg). Unfortunately, no rigorous data are available for oceanic sea skaters. Unpublished observations by Edney and Cheng (mentioned in Cheng, 1989) refer to the value of 523.6 mosmol/kg. Such
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value is indeed at the upper limit of a range seen in various terrestrial and freshwater insects (250–550 mosmol/kg) (Hadley, 1994), which may explain, at least partially, why the SCP values are so low. The question is how the correlation between SCP and HCT is induced. Heat shock protein (HSP) is well known to be in transcript higher than that under normal conditions in response to the exposure to heat in insects, especially in Drosophila (Velazquez et al., 1983; Goto et al., 1998). There is heat shock protein defense from the damage of proteins due to heat, for example (Neven, 2000). The HSP70 protein may ‘chaperone’ the protein to the lysosome for degradation (Chiang et al., 1994) or may help the protein re-fold after return to favorable temperatures (Parsell and Lindquist, 1994). The over expression of HSP may be speculated to promote the expression of other substances to increase the osmotic pressures in the case of oceanic sea skaters. Heat shock proteins promote the synthesis of sorbitol in white flies, polyol in whiteflies and aphids, and the trehalose synthesis in yeast (Salvucci, 2000; Salvucci et al., 2000). As we consider our results to be introductory, we do not see the conclusions as authoritative. Nevertheless, we suggest that our data may be further extended for the research on the relationship between the thermal characteristics of sea skater habitat and the complex of linked physiological parameters, which may serve to better understand the distribution and biology of these unique insects. Acknowledgments We would like to thank Dr. Yuji Kashino (Head Scientist of the cruise MR-09-04) and Mr. Yasushi Ishioka (Captain of R/V MIRAI) for their support during our study. Prof. Yoshiro Watanabe (Ocean Research Institute, University of Tokyo, Japan) kindly rented the Neuston Net to us. Dr. Tomáš Ditrich (Pedagogical Faculty, University of South Bohemia, České Budějovice, Czech Republic) helped with the data processing and analysis. References Andersen, N.M., Cheng, L., 2004. The marine insect Halobates (Heteroptera: Gerridae): biology, adaptations distribution, and phylogeny. Oceanogr. Mar. Biol. Annu. Rev 42, 119–180. Bale, J.S., 1987. Insect cold hardiness: freezing and supercooling — an ecophysiological perspective. J. Insect Physiol. 33, 899–908. Bale, J.S., 1993. Classes of insect cold hardiness. Funct. Ecol. 7, 751–753. Cheng, L., 1985. Biology of Halobates (Heteroptera: Gerridae). Annu. Rev. Entomol. 30, 111–135. Cheng, L., 1989. Factors limiting the distribution of Halobates species. Reproduction, genetics and distribution of marine organisms. In: Ryland, J.S., Tyler, P.A. (Eds.), Proceedings of 23rd European Marine Biology Symposium. Olsen & Olsen, Fredensbor, pp. 357–362.
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