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Temperature is one of the influential factors for development and reproduction of ectothermic invertebrates such as insects (Noor-ul-Ane and Jung, 2020), affects seasonal and geographical distribution. Ectothermic insects have colonized regions that are seasonally or perpetually cold, and must cope with temperatures that fall substantially below 0°C (Costanzo and Lee, 2013). Survival during the extreme temperature condition is crucial for sustained existence and proliferation (Teets and Denlinger, 2013). For these cold-hardy organisms, capacity for cold tolerance is tuned to the temperatures and exposure durations that a given species encounters within its habitat (Addo-Bediako et al., 2000; Noor-ul-Ane and Jung, 2021). Survival under low temperature depends on either freeze avoidance through supercooling or freeze tolerance (Sinclair et al., 2003). Supercooling is a metastable state in which body fluids remain liquid below the subzero condition, promoted by increased hemolymph cryprotectants such as glycerol or other antifreeze proteins which protect against chilling injury or remove inoculative freezing ice-nucleating agents (Duman et al., 1991). Freeze tolerance is the strategy allowing survival even with part of the body frozen, but managing tissue and cell from frost damage. Freeze tolerant insects could initiate the freezing at relatively high temperatures through inoculative freezing by ice nucleating proteins (Sinclair et al., 2003; Jakobs et al., 2015; Teets et al., 2020). In temperate regions of the northern hemisphere where the cold comes seasonally for longer periods, the main strategy is freeze avoidance while in the southern, and freeze tolerance is also more prevalent in insects (reported in 85% of species studied; Sinclair et al., 2003).

Ants, bees, termites and wasps are social insects which constitute most of terrestrial habitats (Holldobler and Wilson, 1990). Social insects not only regulate their own body temperature but also for their nest mates, whereas solitary insects only responsible to regulate their own body temperature (Kadochová and Frouz, 2013). Honey bees, plant pollinators, are well known for their contribution in the yield production of the different crops (Jung, 2008; Begna et al., 2020). Winter survival is a challenging aspect of Apis mellifera in temperate regions. A. mellifera survives in winter by forming a cluster and maintaining temperature around 20℃ (Southwick, 1985). Cold temperature may challenge A. mellifera workers during autumn for their foraging and defense purpose. Cold tolerance of sub-species of honey bees has been studied (Li et al., 2012; Qin et al., 2019) but there is no report from Korea. Vespa velutina nigrithorax, an invasive predator of honey bees, has been established in South Korea since 2003 (Park and Jung, 2016). All casts of V. velutina dies in winter except mated queens which overwinters in the soil or tree crevices (Monceau et al., 2014) but there is no report of cold tolerance of V. velutina.

Body size is also another fitness trait in organisms including insects, usually larger individual benefitted more in terms of their survival, fecundity and mating success (Honek, 1993; Andersson, 1994) subsequently contributing for ecosystem functioning (Enquist et al., 2003). Stress resistance is also more evident in larger insects (Hood and Tschinkel, 1990). Larger insects show more desiccation resistance and foraging time (Kaspari, 1993) within and between species (Chown et al., 1999). Starvation is also another stress which helps larger insects for their survival as they have more energy reservoirs or lower metabolic activities (Harshman and Schmid, 1998). Body size also plays an important role in thermoregulation of organisms (Schmidt-Nielsen, 1997) including insects both in cold tolerance and heat tolerance (Baudier et al., 2008; Shepherd et al., 2008). Similarly, body mass is also important for ecosystem functioning, as it controls energy uptake and transformation in ectotherms at organismal level and it also link to stress resistance including thermal tolerance of insects (Oyen et al., 2016; Knapp and Řeřicha, 2020).

Underlying the importance of cold tolerance for these species, we determined Supercooling points of A. mellifera workers and V. velutina queen and also determined influence of body size and body mass on SCP within and across these species and then estimated SCPs compared with SCPs of social hymenopterans.

The SCPs of A. mellifera and V. velutina workers were ranged from -3.5 to -8.2℃ and -4.2 to -8.8℃, respectively, with a median value of -6.2 and -6.5℃, respectively (Fig. 1). The SCP of V. velutina queen was -11.3±0.8℃ sentand significantly higher than values of A. mellifera workers and V. velutina (ANOVA, F=74.5, df=2,37, P>0.001). Histogram shows that SCPs of both A. mellifera, V. velutina queen, and V. velutina workers have unimodal distribution (Fig. 2). SCP of A. mellifera of present study is almost similar to its subspecies of A. mellifera ligustica which has SCP of -5.3℃ calculated by Li et al. (2012) and -6.4℃ in December collected workers in another study of Qin et al. (2019). Even though winter honey bees are fully equipped with fat body and higher density of hemolymph proteins, SCPs of winter honey bees were relatively higher since the honey bees overwinter in the winter cluster within the hive in the protected space (Southwick, 1985). But still honey bees are risky and susceptible for freeze dying if winter cluster being broken or exposed to the cold even relatively shorter time associated with parasitic mites or disease (van Dooremalen et al., 2018).

Fig. 1. 
Supercooling points of A. mellifera workers, V. velutina workers and V. velutina queens.

Fig. 2. 
Frequency distribution the Supercooling points of A. mellifera and V. velutina.

The SCP of V. velutina worker from South Korean population was -6.5℃ which is almost similar to some of other hymenopteran workers of Polistes annularis (-6.8℃) and Bombus terrestris (-7.1℃) (Strassman et al., 1984; Owen et al., 2016). However, SCP of V. velutina queen (-11.3℃), was higher than SCPs of worker V. velutina (F=74.5, df=2,37, P>0.001), also close to SCPs of P. annularis, -12.9℃ and Sirex noctilio, -11.8℃ (Strassman et al., 1984; Li et al., 2019). As queens of V. velutina should overwinter, but not workers, and establish new colonies in the next spring (Monceau et al., 2014), there are physiological differences between workers and queens and that fat body appearance is also a reliable indicator of caste. For P. exclamans, 83% of the future queens survived at the lower conditioning temperature, +5℃ of 15 days exposure while 24% of the workers survived during the same exposure.

SCPs were not influenced by the body size and body mass for both A. mellifera (F=1.5, df=1,19, P=0.2354), and V. velutina worker (F=0.51, df=1,19, P=0.4919), and V. velutina queen (F=0.16, df=1,10, P=0.69554) (Fig. 3). Similarly, body length also did not influence SCPs of both A. mellifera (F=1.04, df=1,19, P=0.3217), V. velutina worker (F=0.23, df=1,11, P=0.6437), and V. velutina queen (F=2.70, df=1,10, P=0.1317) (Fig. 4). There are some observations which showed that body trait such as body size is linked with SCP (Zachariassen, 1985). Larger body size increased supercooling point in someinsects (Sinclair and Chown, 2005). While, no differences were also reported (Knapp and Uhnavá, 2014; Yunik and Chilton, 2021). Yunik and Chilton (2021) reported that the different size and weight of male and female ticks showed no difference of SCPs. We found difference in SCPs between different casts of overwintering queen and non-overwintering worker of Vespa velutina but no difference in SCPs of individuals in the same cast (worker or queen) with different body size. Also overwintering honey bee worker and non-overwintering vespa worker which differ in body size, showed similar SCPs, suggesting workers of V. velutina and A. mellifera may have similar cold tolerance. The study suggested that larger insects do not necessarily have higher SCPs.

Fig. 3. 
Supercooling points of A. mellifera and V. velutina of different body weights. ◊: V. velutina queens (Y=1.0685x+10.687, R²=0.0160), ▲: V. velutina workers (Y=2.5866x+5.0168, R²=0.0439) and ■: A. mellifera workers (Y= -16.601x+7.9167, R²=0.0733).

Fig. 4. 
Supercooling points of A. mellifera and V. velutina of different body lengths. ◊: V. velutina queens (Y=0.2483x+4.3517, R²=0.2123), ▲: V. velutina workers (Y=0.1941x+1.0513, R²=0.0201), and ■: A. mellifera workers (Y= -1.0337x+7.9938, R²=0.0517).

There are numerous factors influencing SCPs; cryoprotectant level (Somme, 1982), developmental stage (Bouchard et al., 2006), feeding (Cannon, 1986) apart from body size. The workers of ant, Coptotermes herculeanus, have the lowest SCP (-40℃) followed by a mason bee, Osmia cornuta, (-31℃) (Table 1). The highest SCP of value -4.3 and -6.1℃, belongs to Myrmica angulinodis and A. mellifera, respectively. The queen of paper wasp, “Polistes anularis” showed SCP of value -6.8℃. While, the queens of Vespula pensylvanica showed lower SCP values (-23.3℃). In conclusion, present study shows that SCP of V. velutina queen is lower than V. velutina workers and A. mellifera workers. Body length and body mass did not have any influence on SCP of within and across species of A. mellifera, V. velutina queen, and V. velutina workers.