벌목 개미과의 일본열마디개미는 국내 토착종이다. 지금까지 야외에서 이 종의 월동기작을 이해하기 위한 내한성 연구는 진행되지 않았다. 저온에서 다양한 온도별 노출시간에 따른 발육태별 저온 저항성을 조사하였다. 성충인 일개미가 다른 발육태와 비교하여 5°C와 10°C의 저온에서 높은 생존율을 보였으며, 급속내한성 유기 조건인 15°C에서 12시간 노출 후 내한성을 획득하였다. 급속내한성 유기는 10°C에서 최대 44%까 지 생존율이 향상되었으며, 체내과냉각점과 체내빙점은 각각 -10.0°C에서 -14.2°C, -11.3°C에서 -15.3°C까지 낮아졌다. 저온처리는 저온 또는 스트레스 관련 유전자인 글리세롤 인산화효소와 열충격 단백질의 발현을 증가시켰다. 이상의 결과는 일본열마디개미의 내한성이 급속내한성 유기에 의해 야기된다는 것을 의미한다.

Glycerol is a polyol that is responsible for the cold hardiness of insects. Glycerol kinase gene, which is an important key enzyme for glycerol biosynthesis, was predicted from whole genome sequencing data from the diamondback moth, Plutella xylostella. Four of P. xylostella glycerol kinase genes (PxGKs) were determined as a functional glycerol kinase through in silico study. Pre-exposure of P. xylostella larvae to 4°C for 7 h significantly enhanced survival (rapid cold hardiness: RCH) under a freezing temperature (-10°C) and increased glycerol titers. To determination of functional GK gene, expressions of all GK genes were measured by RT-PCR analysis. All GK genes were expressed in all larval stage and tissues (gut, hemocyte, and fat body). Expressions of all GK genes were suppressed by its specific dsRNA treatment into 4 th instar larva. Each 150 ng of dsRNA PxGK2 treatment significantly decreased glycerol amount in hemolymph by HPLC analysis. Larval treated by dsRNA PxGK2 also significantly lost the RCH under -10°C exposure. These results indicate that glycerol is a crucial RCH agent and its synthesis is regulated by a specific PxGK2 gene among GK gene isoforms in P. xylostella. In addition, the beet armyworm, Spodpotera exigua, encodes RCHassociated SeGK1, which has been functionally identified by RNA interference.

Cold tolerance of the palm thrips, Thrips palmi Karny, was investigated to predict its survival in field during winter. Supercooling temperatures of T. palmi ranged from -26.4 to -18.4°C. However, exposure to subzero temperatures (from -5°C to -15°C) gave significant mortality to all developmental stages of T. palmi. Thus, T. palmi was determined to be a freeze-susceptible and suffered with cold injury. A brief pre-exposure to a low temperature (4°C) for 7 h significantly increase the cold tolerance of all stages of T. palmi with respect to survival at -10°C and supercooling capacity. A pre-exposure of T. palmi at 4°C significantly increased the survival rate on all developmental stages at -10°C. The rapid cold hardiness (RCH) was dependent on the duration of the pre-exposure period at 4°C in adult stage. Cryoprotectant analysis using an HPLC showed that the pre-exposure treatment increased the adult to synthesize glycerol, trehalose, mannitol, and mannose, at which trehalose represented the highest content. This study suggests that all stages of T. palmi are able to become cold-hardy by RCH, in which several polyols may play crucial roles as cryoprotectant.

The beet armyworm, Spodoptera exigua, is a freeze-susceptible species and overwinters without diapause in temperate zone. Depression of supercooling point (SCP) and rapid cold hardiness (RCH) allow S. exigua to survive at low temperatures. This study reports a polyol which is responsible for the cold hardiness of S. exigua. Pre-exposure of S. exigua larvae to 4°C for 6 h significantly enhanced survival under a freezing temperature (-10°C). This pre-exposure treatment also significantly depressed larval SCPs. Analysis of polyols indicated that glycerol titers significantly increase with increase of pre-exposure time. Glycerol kinase (GK) and glycerol-3-phosphate dehydrogenase (GPDH) are involved in glycolysis pathway of insect. The S. exigua GK (SeGK1) and G3PDH (SeG3PDH1) genes were predicted from 454 pyrosequencing transcripts from fifth instar larvae of the beet armyworm, S. exigua. The SeGK1 and SeG3PDH1 genes both were expressed in all larval stage by RT-PCR analysis. Expression of SeGK1 and SeG3PDH1 genes were suppressed by its specific dsRNASeGK1 or dsRNASeG3PDH1 injection into hemocoel of 5th instar larva. Each 200 ng of dsRNASeGK1 or dsRNASeG3PDH1 injection also significantly decreased glycerol amount in hemolymph. Larval treated by either dsRNASeGK1 or dsRNASeG3PDH1 significantly lost the RCH under -10°C exposure. These results indicate that glycerol is a crucial RCH agent and its synthesis is regulated by SeGK1 and SeG3PDH1 genes in S. exigua.