Carabus Granulatus, Pterostichus Oblongopunctatus and Platynus Assimilis

Forestry Studies|Metsanduslikud Uurimused 57, 90–96, 2012 DOI: 10.2478/v10132-012-0007-3

Seasonal cold adaptation dynamics of some carabid beetle species: Carabus granulatus, Pterostichus oblongopunctatus and Platynus assimilis

Angela Ploomi*, Irja Kivimägi, Eha Kruus, Ivar Sibul, Katrin Jõgar, Külli Hiiesaar and Luule Metspalu

Ploomi, A., Kivimägi, I., Kruus, E., Sibul, I., Jõgar, K., Hiiesaar, K., Metspalu, L. 2012. Seasonal cold adaptation dynamics of some carabid beetle species: Carabus granulatus, Pterostichus oblongopunctatus and Platynus assimilis. – Forestry Studies | Metsanduslikud Uurimused 57, 90–96. ISSN 1406-9954.

Abstract. Cold-hardiness can be measured by supercooling points – the temperature at which spontaneous freezing occurs. Seasonal changes in supercooling point were assessed in ﬁeld-collected predacious carabid beetle species: Carabus granulatus L., Pterostichus oblongopunctatus L. and Platynus assimilis Payk. (Coleoptera: Carabidae). Supercooling ability of these beetles changed seasonally. The tested carabid beetles proved to belong to freeze-avoiding cryotype. Key words: Coleoptera, Carabidae, cold-hardiness, supercooling. Authors’ address: Estonian University of Life Sciences, Kreutzwaldi 1, 51014 Tartu, Estonia; *e-mail: [email protected]

Introduction fluids liquid by removing ice nucleators that initiate ice formation, synthesizing Seasonal adaptation of insects depends antifreeze proteins to reduce the nuclea- on many abiotic factors, with tempera- tion potential of seed crystals, and accumu- ture being the most critical factor aside of lating sugars and polyols, such as glycerol humidity and light (Danks, 2006, 2007). or trehalose, which also lower the crystal- Insects survive low temperatures either by lization temperature (defined as its super- keeping their body fluids liquid below their cooling point) and stabilize membranes at ordinary freezing point (freeze avoidance), low temperatures (Vernon & Vannier, 2002; or by surviving the formation of ice in their Lee, 2010). tissues (freeze tolerance) (Bale & Hayward, Freezing-tolerant insects survive ice for- 2010; Lee, 2010). For species inhabiting in mation in body tissues. Ice formation usu- temperate and colder climates, the abil- ally is confined to extracellular fluids, thus ity to supercool is undoubtedly the most avoiding damage to intracellular compo- important component of the overwin- nents. Protective mechanisms for freezing tering strategy. At temperatures below resistance include elevated solute levels, 0 ºC, most insect species remain unfro- presence of nucleating agents, and accu- zen because they supercool. Cold-hardi- mulation of cryoprotectants in body fluids. ness can be measured by indices such as A number of freeze-tolerant insects have supercooling points (SCP), the tempera- supercooling points in the range of –8 ºC to ture at which spontaneous freezing occurs –10 ºC, whereas some freeze-avoiding spe- (Danks, 2004). cies supercool extensively, even to –60 ºC or Freeze-avoiding insects keep their body below. Insects that supercool are freezing-

90 Seasonal cold adaptation dynamics of some carabid beetle species

��

Figure 1. The generalized diagram of insect response to subzero temperatures. Insect body temperatures (heavy line) in relation to the melting point, the supercooling point, and the nucleation of ice in body fluids. The bars on the right convey general ranges of insect response to low temperatures, the top of the bar for the range of freeze tolerance and the bottom of the bar for freeze avoidance correspond to the supercooling point value illustrated in the center of the figure (Lee, 1989; Ellsbury et al., 1998). Joonis 1. Putukas toimuvate temperatuurist põhjustatud muutuste näitlik diagramm (Lee, 1989; Ellsbury et al., 1998). Tugev must joon näitab putuka kehatemperatuuri (body temperature) muutusi temperatuuri langedes; viirutatud ala vastab jää (ice) tekkele ja olemasolule putuka organismis. Kehavedeliku sulamispunkt (melting point of body fluids) on temperatuur, mille juures sulab viimane kehas olev jääkristall. Enne allajahtumispunkti (supercooling point) ehk tegelikku külmumistemperatuuri on kehavedelikud (supercooled fluids) 0 °C madalamas temperatuuris jahtunud olekus. AJP (allajahtumispunkti) juures eraldub kudede spontaansel külmumisel soojust (heat of crystallization). Putukate seisundid temperatuuri langemisel: normaalne aktiivsus (normal activity), külmašokk (cold shock), külmumise vältimine ehk külmatundlikkus (allajahtumine) (freeze avoidance (supercooled)), kül- mumise talumine (freeze tolerance). Külmašokk on jahtumine ilma külmumata, mõne külmatundliku putuka jaoks on pikaajaline madal temperatuur letaalne. Toimuvad muutused on tihedalt seotud aja- (time) ja temperatuurifaktoriga (temperature).

avoiding since they avoid freezing damage ing carabid beetles known of their sen- to body tissues only at a temperature above sitive reaction to environmental changes the supercooling point. Below the super- (Koivula, 2011). Carabid beetles (Coleoptera: cooling point, death results from ice forma- Carabidae) are species rich and abundant tion in body tissues (Danks, 2004). These in arable and forest habitats (Kromp, 1999). relationships are summarized in Figure 1 Carabid beetles are natural enemies attack- adapted from Lee (1989) and Ellsbury et al. ing also both immature and adult pine wee- (1998). vils (Salisbury & Leather, 1998; Dillon & Cold-hardiness is equally important Grifﬁn, 2008). Adult carabid beetles exhibit to pests and beneﬁcial predators, includ- scarcely any activity in winter. Carabid

91 A. Ploomi et al. species that inhabit cultivated fields spend mined. For measuring supercooling points the winter in soil or migrate into the field (SCPs), the beetles were positioned so that boundaries, whereas some species migrate its integument (thoracic tergit) was in con- into the forest (Thiele, 1977). The microenvi- tact with the copper-constantan thermo- ronment of overwintering sites is at least as couples-thermometer (RS-232 Data logger important in allowing winter survival as are thermometer; TES Electrical Electronic, physiological adaptations (Danks, 2004). Taiwan), placed in glass vial closed by cap, The aim of the present research was and then transferred to circulator bath to study seasonal cold adaptation dynam- (Ministat 230w-2, Huber, Germany, –33 °C ics of carabid beetles Carabus granulatus L., to +200 °C). SCP was determined using Pterostichus oblongopunctatus L. and Platynus a 0.5 °C min−1 cooling rate. The SCP was assimilis Payk. These species are farely com- taken as the thermocouple recorded the mon, all univoltine spring breeders (Luff, lowest point before the emission of the 1993) and represent three Carabidae gen- latent heat of crystallization (when body era. As they overwinter in older and par- fluids freeze). From each species five indi- tially decayed tree stumps they can be used viduals were taken for determination of as model species for Carabids of similar cold-hardiness strategies (freeze-tolerant or biology associated with both agricultural freeze-avoidance) by supercooling. When and forest ecosystems. the temperature limit to supercooling is reached, heat of crystallisation appears, species who will survive recooling to the Materials and methods SCP have freeze-tolerant overwintering Insects strategy, whereas those who will not sur- Laboratory experiments were conducted to vive are classified as freeze-avoiding spe- test three carabid species: Carabus granulatus cies (Lee, 1989; Bale, 2002). The temperature (Linneaus 1758), Pterostichus oblongopuncta- curve was registered and saved by using tus (Linnaeus 1758) and Platynus assimilis data logger (Almemo 2890-9, Ahlborn Mess- (Paykull 1790). Adult insects were collected und Regelungstechnik GmbH, Germany). from their hibernating sites – tree stumps at wintertime (January) and from the field Statistical analysis by using dry pitfall traps in spring (May) Supercooling points were tested for nor- and autumn (September). Beetles were col- mality using the Kolmogorov-Smirnov lected from the Tartu County, Estonia (ca. Test. Statistical comparisons of the seasonal 58°26′ N, 27°7′ E) in 2012. Samplings were change of mean supercooling points of performed early in the morning in cool individuals were performed with repeated- weather (temperatures between 0 °C to measures one-way analysis of variance 10 °C) and beetles were placed in the refrig- (ANOVA) followed by Fisher’s LSD test, erator at 4 °C immediately after the arrival using STATISTICA 12 package. The col- to avoid major temperature fluctuations. To lected data were analysed separately in obtain field-collected beetles with empty each species. All means were considered guts, they were held for 24 h prior to the significantly different at the p ≤ 0.05 level. testing without food in plastic boxes filled with moisturized moss (for retaining mois- Results and discussion ture and providing hiding areas). Mean supercooling points are shown in Experimental design Figure 2. Seasonal mean SCPs examined in Together 216 beetles were recorded (9 vari- C. granulatus were determined as follows: ances (3 months × 3 species) × 24 insect rep- –5.4 ºC (in January), –5.2 ºC (in May) and licates), the sex of insects were not deter- –6.3 ºC (in September). The SCP in autumn

92 Seasonal cold adaptation dynamics of some carabid beetle species

��

Figure 2. Supercooling seasonal dynamics of the carabid beetles C. granulatus, P. oblongopunctatus and P. assimilis. Columns with different letters are signiﬁcantly different (ANOVA Fisher’s LSD test, p ≤ 0.05). The differences were calculated between one species. Box-and-whisker plots: boxes show standard errors, whiskers are standard deviation bars, small box in the centre is mean. Joonis 2. Sõmerjooksiku (C. granulatus), metsa-süsijooksiku (P. oblongopunctatus) ja süsi-ketasjooksiku (P. assimilis) külmakindluse sesoonne dünaamika. Allajahtumispunkte (supercooling point ºC) on võrreldud liigi piires. Statistiliselt usaldusväärsed erinevused (p ≤ 0.05) on tähistatud erinevate tähtedega (ANOVA Fisher’s LSD test).

differed signiﬁcantly from that in January dynamic strategy the greatest changes in (F2,69 = 14.17, p ≤ 0.05) and in May (p ≤ 0.05), cold-tolerance take place in autumn – the but there were no difference between win- ability to supercool attains its winter max- ter and spring SCPs (p = 0.43). The SCPs imum as early as autumn, in September- of P. oblongopunctatus ranged from –6.3 ºC October. In the course of winter (from (May) to –7.2 ºC (September). Similarly to November to April) it does not change C. granulatus, supercooling of P. oblongo- noticeably. Supercooling capacity changes punctatus was lower in autumn compared during the year and this is increasing when to winter (F2,69 = 25.49, p ≤ 0.05) and spring insect prepares for resting stage. Recorded (p ≤ 0.05), but also no differences between SCPs were higher in January, compared to winter and spring supercoolings were September. It is now known that many spe- found (p = 0.44). The SCPs of C. granulatus cies complete diapause by mid-winter, but and P. oblongopunctatus reached their min- development is still suppressed by contin- imum during autumn, and then raised by uing low temperature. In such species, and mid-winter to values as high as in spring. those without a winter diapause, polyol This pattern, which matches the autumn- concentrations and low SCP are usually dynamic strategy characterized by Merivee maintained until spring, indicating that (1978), could be an physiological adaptive rising temperature triggers the reversible response in association with behavioral synthesis or degradation of the carbohy- patterns of these species in Northern tem- drates and consequent loss of cold-hardi- perate condition. According to the autumn- ness (Leather et al., 1993).

93 A. Ploomi et al.

The SCPs were lowest in the beginning ice formation occurs (supercooling point) of the overwintering (in September) in all by producing antifreeze agents, minimiz- examined insects: in C. granulatus –6.3 ± ing internal nucleation sites, and avoiding 0.73 ºC; in P. oblongopunctatus –7.2 ± 0.41 ºC contact with external ice (Sømme, 1999). and in P. assimilis –9.3 ± 1.58 ºC. In P. assimi- Unseasonal cold in summer could cause lis, beetles collected in May produced high- some mortality in freeze avoiding insects, est SCP (–6.8 ºC), three months earlier in as there is not enough time for such insects January the SCP was –8.7 ºC. There were to clear their guts and undergo the bio- significant differences between all the sea- chemical changes associated with the long- sonal SCPs of P. assimilis beetles (F2,69 = 27.17, term acclimation for winter conditions p ≤ 0.05). Earlier research by Kivimägi et al. (which is the process that may take days or (2009) has measured the January SCP at – weeks) (Sinclair et al., 2003). All the studied 5.5 °C on P. assimilis, which indicates that insect species overwinter in protected place the SCP and thus the cold-hardiness is reg- – in tree stumps. After moving to overwin- ulated by environmental conditions and tering sites beetles stop feeding, enabling depends on the physiological state of the them to increase their supercooling capac- insect (Marshall & Sinclair, 2012). For exam- ity. The greatest risk of mortality from low ple carabid beetle Pterostichus brevicornis temperatures comes from the combination (Kirby 1837) was studied in Alaska, where of no snow and low temperatures. Snow is it overwinters in decaying tree stumps and an important insulator against low temper- felled timber, although the habitat is at atures and a cover of snow or vegetation times partly covered with snow. These bee- also helps to protect overwintering indi- tles may experience temperatures as low as viduals against lethal temperatures, there- –60 ºC, with prolonged exposure to –60 ºC fore snow cover could have significant mat- over several weeks. Seasonally collected ter in insects overwintering (Danks, 2004; beetles showed SCPs from –6 ºC in sum- Bale & Hayward, 2010). According to the mer (when freezing was fatal) to –11 ºC, Estonian Meteorological and Hydrological when beetles became freeze-tolerant (Baust Institute’s (EMHI) measurements, the mean & Miller, 1970). The most striking feature air temperature in insects overwintering of freezing intolerant insects is the sea- time in January 2012 was –5.8 ºC and snow sonal increase in supercooling from sum- cover 23 cm. Consequently, overwintering mer through autumn to winter. Hiiesaar et mortality due to low temperature may be al. (2012) found that supercooling ability of rare, for example, only in extremely severe horseradish flea beetles Phyllotreta armora- snow-less winters. For a better interpreta- ciae (Koch 1803) is depending on their feed- tion of supercooling of studied carabid bee- ing status. In nature, most overwintering tles, more research is required. insects of the temperate or colder climates evacuate the digestive tract as a part of Conclusions a multifactorial process that maximises supercooling in winter. Cold adaptation dynamics of carabid bee- The tested carabid beetle species didn’t tle species C. granulatus, P. oblongopunctatus survive the recooling until SCP, therefore and P. assimilis varied seasonally. Carabid they belong to freeze-avoiding insect group beetle species C. granulatus, P. oblongopunc- (Lee, 1989; Bale, 2002). The large majority tatus and P. assimilis belong to freeze-avoid- of terrestrial arthropods that have been ing cryotype. They are likely to survive investigated are freeze-avoiding. Freeze- unseasonal cold and overwintering could avoiding species tend to (1) choose ther- be harmful only in very cold snow-less mally buffered microsites for overwinter- winters. ing and (2) lower the temperature at which

94 Seasonal cold adaptation dynamics of some carabid beetle species

Acknowledgements. The research was sup- Kromp, B. 1999. Carabid beetles in sustainable agricul- ported by the Estonian Science Foundation ture: a review on pest control efficacy, cultivation impacts and enhancement. – Agriculture, Ecosys- (grant 9449), Estonian target financing tems and Environment, 74(1–3), 187–228. project number SF170057s09 and State Forest Kuusik, A., Metspalu, L., Hiiesaar, K.1995. Insektit- Management forest protection project 8-2/ siidide toimemehhanismide uurimine putukatel. T12115MIMK (2012–2015). (Studies on insecticide modes of action). Estonian Agricultural University, Tartu, 71–80. (In Esto- nian). Leather, S.R., Walters, K.F.A., Bale, J.S. 1993. The References Ecology of Insect Overwintering. Cambridge, UK. 300 pp. Bale, J.S. 2002. Insects and low temperatures: from Lee, R.E. 1989. Insect cold-hardiness: to freeze or not molecular biology to distributions and abundance. to freeze. – BioScience, 39 (5), 303–313. – Philosophical Transactions of the Royal Society Lee, R.E. 2010. A primer on insect cold-tolerance. of London (B), 357, 849–862. – Lee, R.E., Denlinger, D.L. (eds.). Insects at low Bale, J.S., Hayward, S.A.L. 2010. Insect overwintering temperature. New York, USA, 3-24. in a changing climate. – The Journal of Experimen- Luff, M.L. 1993. The Carabidae (Coleoptera) larvae tal Biology, 213, 980–994. of Fennoscandia and Denmark. – Fauna Entomo- Baust, J., Miller, L.K. 1970. Variations in glycerol and its logica Scandinavica, 27, 1–186. influence on cold hardiness in the Alaskan carabid Marshall, K.E., Sinclair, B.J. 2012. The impacts of re- beetle Pterostichus brevicornis. – Journal of Insect peated cold exposure on insects. – The Journal of Physiology, 16, 979–990. Experimental Biology, 215, 1607–1613. Danks, H.V. 2004. Seasonal adaptations in arctic Merivee, E. 1978. Cold-hardiness in insects. Tallinn, Es- insects. – Integrative and Comparative Biology, tonia, 186 pp. (In Estonian, Russian and English). 44, 85–94. Merivee, E., Hansen, T., Kuusik, A. 1968. Mis saab Danks, H.V. 2006. Insect adaptations to cold and putukatest talvel? (What will become of the insects changing environments. – Canadian Entomolo- in the winter?). – Eesti Loodus, 43, 723–728. (In gist, 38, 1–23. Estonian). Danks, H.V. 2007. The elements of seasonal adaptations Paljak, T. 2007. Õhutemperatuuri järskude muutuste in insects. – Canadian Entomologist, 139, 1–44. statistiline ja sünoptiline analüüs külmal poolaas- Dillon, A., Griffin, C. 2008. Controlling the large pine tal ajavahemikul 1951-2006. (Rapid changes of air weevil, Hylobius abietis, using natural enemies. temperature and their statistical and synoptical COFORD Connects: Silviculture and Forest Man- analysis in cold period 1951-2006). Master thesis. agement, 15, 1–8. Institute of Geography, University of Tartu. 52 pp. Ellsbury, M.M., Pikul, J.L.Jr., Woodson, W.D. 1998. (In Estonian). A review of insect survival in frozen soils with Salisbury, A.N., Leather. S.N. 1998. Migration of larvae particular reference to soil-dwelling stages of corn of the large Pine Weevil, Hylobius abietis L. (Col., rootworms. – Cold Regions Science and Technol- Curculionidae): Possible predation a lesser risk ogy, 27, 49–56. than death by starvation? – Journal of Applied Hiiesaar, K., Kaasik, R., Williams, I.H., Švilponis, E., Entomology, 122 (1–5), 295–299. Jõgar, K., Metspalu, L., Mänd, M., Ploomi, A., Sinclair, B.J., Addo-Bediako, A., Chown, S.L. 2003. Cli- Luik, A. 2012. Cold hardiness of horserad- matic variability and the evolution of insect freeze ish flea beetle (Phyllotreta armoraciae (Koch)). tolerance. – Biological Reviews, 78, 181–195. –Žemdirbystė=Agriculture, 99 (2), 203–208. Sømme, L. 1999. The physiology of cold hardiness Kivimägi, I., Ploomi, A., Metspalu, L., Švilponis, E., in terrestrial arthropods. – European Journal of Jõgar, K., Hiiesaar, K., Luik, A., Sibul, I., Kuusik, Entomology, 96, 1–10. A. 2009. Physiology of a carabid beetle Platynus Thiele, H.U. 1977. Carabid beetles in their environment. assimilis. – Agronomy Research, 7 (spec. iss. I), A study on habitat selection by adaptations in 328–334. physiology and behaviour. Berlin. 330 pp. Koivula, M.J. 2011. Useful model organisms, indicators, Vernon, P., Vannier, G. 2002. Evolution of freezing or both? Ground beetles (Coleoptera, Carabidae) susceptibility and freezing tolerance in terrestrial reflecting environmental conditions. – ZooKeys, arthropods. – Comptes Rendus Biologies, 325, 100, 287–317. 1185–1190.

95 A. Ploomi et al.

Mõne jooksiklase liigi külmakindluse sesoonne dünaamika: sõmerjooksik (Carabus granulatus), metsa-süsijooksik (Pterostichus oblongopunctatus) ja süsi-ketasjooksik (Platynus assimilis) Angela Ploomi, Irja Kivimägi, Eha Kruus, Ivar Sibul, Katrin Jõgar, Külli Hiiesaar ja Luule Metspalu

Kokkuvõte

Putukate sesoonne kohastumine sõltub et al., 1995; Danks, 2007). Jooksiklased lõpe- paljudest teguritest, mille hulgas tempera- tavad peale talvituskohtadesse jõudmist tuur on üheks kriitilisemaks faktoriks. Kül- toitumise, mis võimaldab neil oma allajah- makindlus on väga oluline enamike paras- tumisvõimet tõsta. Antud eksperimendi vöötme putukate talviseks ellujäämiseks eesmärgiks oli määrata kolme jooksiklase ning on inimese seisukohalt ühtviisi täh- liigi – sõmerjooksiku (C. granulatus), metsa- tis nii kasulikele kui kahjulikele organismi- süsijooksiku (P. oblongopunctatus) ja süsi- dele (Danks, 2007). Jooksiklasi (Coleoptera: ketasjooksiku (P. assimilis) külmakindluse Carabidae) esineb liigirikkalt ja arvukalt sesoonne dünaamika ja külmakindluse põllumajandusmaadel ja metsades, kus strateegia. nad mitmetoiduliste röövmardikatena on Sõmerjooksiku keskmine AJP oli –5.4 ºC taimekahjurite tähtsad looduslikud vaenla- (jaanuaris), –5.2 ºC (mais) ja –6.3 ºC (sep- sed, näiteks söövad jooksiklased nii männi- tembris). Metsa-süsijooksiku AJP-d variee- kärsakate vastseid kui valmikuid (Salisbury rusid –6.3 ºC (mais) kuni –7.2 ºC (septemb- & Leather, 1998; Kromp, 1999; Dillon & ris). Mõlema uuritud liigi AJP-d olid sügi- Grifﬁn, 2008). Meie kliimavööndis on putu- sel madalamad kui talvel ja kevadel, samas katele võimalike kriitiliste temperatuuride talve ja kevade näitajate vahel erinevus puu- esinemise aeg küllaltki pikk: novembrist dus. Süsi-ketasjooksiku AJP oli kõige kõr- märtsini (Paljak, 2007), seetõttu on populat- gem maikuus (–6.8 ºC), kolm kuud varem siooni arvukuse hindamiseks oluline teada jaanuari oli AJP –8.7 ºC. Erinevatel kuudel nende külmakindlust. Putukate külma- määratud süsi-ketasjooksiku AJP-d erine- kindlust iseloomustab allajahtumisvõime, sid statistiliselt üksteisest. Uuritud jooksik- mille all mõeldakse putuka kudede jahtu- laste liigid hukkuvad kui peale AJP käigus mist 0 ºC madalamas temperatuuris, ilma et eralduvat soojenemist temperatuuri uuesti sellega kaasneks kudede külmumine (jää- kuni AJP temperatuurini jahutada, seega kristallide moodustumine). Putuka allajah- on tegemist külmumist vältivate ehk kül- tumisvõime mõõduks on tema allajahtu- matundlike liikidega (Lee, 1989; Bale, 2002). mispunkt (AJP), s.o. temperatuur milles Tõenäoliselt on katsetatud jooksiklaste lii- ta tegelikult külmub ning vabaneb kristal- kidele ohtlik allajahtumispunktist madala- lisatsioonisoojus, mis on termoelektriliselt mate temperatuuridega lumevaene talv. registreeritav (Merivee et al., 1968; Kuusik Received March 3, 2014, revised May 22, 2014, accepted May 23, 2014