Concurrent effects of cold and hyperkalaemia cause insect chilling injury

Chilling injury and death are the ultimate consequence of low temperature exposure for chill susceptible insects, and low temperature tolerance is considered one of the most important factors determining insect distribution patterns. The physiological mechanisms that cause chilling injury are unknown, but chronic cold exposure that causes injury is consistently associated with elevated extracellular [K⁺], and cold tolerant insects possess a greater capacity to maintain ion balance at low temperatures. Here, we use the muscle tissue of the migratory locust (Locusta migratoria) to examine whether chill injury occurs during cold exposure or following return to benign temperature and we specifically examine if elevated extracellular [K⁺], low temperature, or a combination thereof causes cell death. We find that in vivo chill injury occurs during the cold exposure (when extracellular [K⁺] is high) and that there is limited capacity for repair immediately following the cold stress. Further, we demonstrate that that high extracellular [K⁺] causes cell death in situ, but only when experienced at low temperatures. These findings strongly suggest that that the ability to maintain ion (particularly K⁺) balance is critical to insect low temperature survival, and highlight novel routes of study in the mechanisms regulating cell death in insects in the cold.     

Cold tolerance of New Zealand alpine insects

New Zealand has extensive alpine and subalpine habitats where, together with some lowland sites, insects are exposed to subzero temperatures. Studies of cold tolerance in New Zealand insects have centred on an alpine weta (Hemideina maori), which is the world's largest freezing tolerant insect, and an alpine cockroach (Celatoblatta quinquemaculata). Both of these insects are moderately freezing tolerant and have ice nucleating agents in their haemolymph and guts. There is some evidence for the survival of intracellular ice formation in the isolated gut tissue of C. quinquemaculata. Trehalose is a suggested cryoprotectant in both H. maori and C. quinquemaculata whilst proline also provides this role in H. maori. Cells and tissues of both insects maintain viability and physiological function during freezing to moderately low temperatures but viability declines at lower temperatures, the most vulnerable tissue presumably setting the limit to the survival of the animal. Antifreeze proteins are found in the gut tissue of C. quinquemaculata and may protect this tissue when freezing occurs in the gut. Several other New Zealand insects are also moderately freezing tolerant and the apparent dominance of this cold tolerance strategy in the New Zealand fauna may reflect the relatively mild climate but unpredictable exposure to subzero temperatures that is typical of many Southern Hemisphere environments.     

Cold treatment enhances low‐temperature flight performance in false codling moth,Thaumatotibia leucotreta (Lepidoptera: Tortricidae)

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Effects of sub-lethal cold stress on the Western Flower Thrips, Frankliniella occidentalis

In order to establish in a new geographical area, introduced insects must be able to survive any period of adverse conditions such as a temperate winter and be capable of subsequent development to adulthood and/or reproduction. However, this aspect of insect overwintering and cold tolerance has been poorly studied. At high latitudes, Frankliniella occidentalis is typically associated with artificially heated glasshouses, but has some ability to tolerate low temperatures and may survive winter field conditions for short periods, or for longer periods of time during mild winters. The effects of overwintering on the viability of survivors are, however, unknown. In this study, acute and chronic cold exposure regimes were imposed on first instar larvae and adult female Western Flower Thrips, after which the longevity, development and reproductive capacity of the survivors were monitored and compared to those of non-stressed individuals. Survival of cold exposure did not affect subsequent survivorship of immature or adult insects, though cold treated larvae took approximately two days longer to reach adulthood than untreated individuals (at 20°C, 18L:6D). Chill treatment of adult females significantly reduced their rate of reproduction (from 1.45 to 0.93 larvae day^-1), reproductive lifespan (from 13.3 to 9.2 days) and as a result, total reproductive output (from 20.4 to 10.8 larvae), compared to control females. Acute exposure resulted in non-significant decreases of the same parameters. The relevance of the above effects to overwintering of F. occidentalis is discussed.     

Influence of soil hydric parameters on the winter cold hardiness of a burrowing beetle, Leptinotarsa decemlineata (Say)

This investigation examined the influence of soil moisture and associated parameters on the cold hardiness of the Colorado potato beetle (Leptinotarsa decemlineata Say), a temperate-zone species that overwinters in terrestrial burrows. The body mass and water content of adult beetles kept in sand at 4 °C varied over a 16-week period of diapause according to the substratum's moisture content. Changes in body water content, in turn, influenced the crystallization temperature (range −3.3 to −18.4 °C; n = 417), indicating that environmental moisture indirectly determined supercooling capacity, a measure of physiological cold hardiness. Beetles held in dry sand readily tolerated a 24-h exposure to temperatures ranging from 0° to −5 °C, but those chilled in sand containing as little as 1.7% water (dry mass) had elevated mortality. Thus, burrowing in dry soils not only promotes supercooling via its effect on water balance, but may also inhibit inoculative freezing. Mortality of beetles exposed to −5 °C for 24 h was lower in substrates composed of sand, clay and/or peat (36–52%) than in pure silica sand (78%) having an identical water content (17.0% dry mass). In addition to moisture, the texture, structure, water potential, and other physico-chemical attributes of soil may strongly influence the cold hardiness and overwintering survival of burrowing insects. Accepted: 10 September 1996     

Geographic variation in winter hardiness of a common agricultural pest,Leptinotarsa decemlineata,the Colorado potato beetle

Successful latitudinal expansions into temperate climates depend largely upon the evolution of novel adaptive traits or the presence of pre-adaptive or exapted mechanisms for survival in seasonal climates. Geographic comparisons of ancestral (pre-expansion) and derived (post-expansion) populations provide a useful framework for understanding the evolutionary conditions that facilitate geographic expansions. Using a common agricultural pest, the Colorado Potato Beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae) as a model, we conducted a regional comparison of cold hardiness and overwintering success among ancestral (southern Mexico) and derived (Vermont and Kansas, USA) L. decemlineata populations. In order to determine if ancestral and derived beetle populations vary physiologically for cold hardiness, we compared supercooling points (SCPs) of three geographic populations of L. decemlineata. We also tested if ancestral and derived beetle populations differed in their overwintering behavior and success by performing an overwintering field experiment. Ancestral and derived populations did not express different physiological responses (i.e. SCPs) to freezing temperatures. However, ancestral and derived populations responded differently to the onset of winter conditions and displayed dissimilar overwintering behaviors. The majority of ancestral beetles failed to initiate diapause and dug upward within experimental mesocosms. Differences in overwintering behavior also resulted in significant variation in overwintering success as derived populations displayed higher overwintering survivorship when compared with ancestral populations. Given our results, it is evident that research exploring the interaction of the ecological factors and evolutionary processes is necessary to fully realize the dynamics of biological invasions.     

Studies on the low temperature induced biogenesis of glycerol by adult Protophormia terranovae

Physiological and biochemical changes accompanying cold stress in the diapausing adult arctic blowfly, Protophormia terranovae, have been observed. In the laboratory, this insect survives prolonged periods at temperatures in the range of ?1°C to +4°C. Concentrations of free glycerol in excess of 10% of fresh body weight have been measured and the rate of its synthesis is greater at +1°C to +4°C than at ?1°C to 0°C. Under these conditions Protophormia also undergoes significant weight loss (up to 58% over 39 days) presumably in part due to dehydration. Its respiration rate decreases as expected when first shifted from 20°C to 0°C but the rate declines an additional 70% after exposure to 0°C for 24 hr. This lowest rate, which is then maintained, when considered with the initial faster one suggests positive thermal modulation is coupled to inverse thermal compensation during cold stress. This was not observed with nondiapausing Protophormia.Increments in free glycerol are accompanied by decreases in the insect's total glycogen reserves but upon rewarming, they return to pre-cold stress levels. While pre-stress glycogen stores are insufficient to provide for most of the free glycerol which accumulates, ingested carbohydrate present in the crop provides sufficient quantities. Studies with [¹⁴C] glucose indicate it is also metabolically active at low temperature.Neutral glyceride glycerol cannot contribute to net synthesis of free glycerol in significant amounts since the steady state concentrations present in pre-cold stressed insects decrease only slightly during cold stress. Furthermore, the specific radioactivity of acyl glyceride glycerol labelled in vivo with 2-[³H] glycerol before cold stress, remains unchanged during hibernation indicating that acyl glycerides are not turning over glycerol units produced by catabolism of hexose. The results of these studies argue that carbohydrate and not lipid glycerol is the source of the free glycerol which accumulates in Protophormia at low temperatures. The relationship of the above results to possible mechanisms which should permit glycerol accumulation under aerobic conditions are discussed.     

Change in the temperature preferences of <Emphasis Type="Italic">Beauveria bassiana sensu lato</Emphasis> isolates in the latitude gradient of Siberia and Kazakhstan

The radial growth of twenty isolates of the entomopathogenic fungus Beauveria bassiana sensu lato from different natural zones of Western Siberia and Kazakhstan (from 65 to 43°N) was tested under different temperatures (5–35°C). It was shown that the thermotolerance of the fungal isolates increased significantly from the north to south. The cold activity of the cultures did not significantly correlate with the latitude of origin and the sum positive temperatures of the regions. A distinct group of the steppe thermotolerance isolates was shown by the analysis of genomic polymorphism using seven intermicrosatellite DNA markers (ISSR). The steppe isolates had high levels of virulence to the wax moth Galleria mellonella and the Colorado potato beetle Leptinotarsa decemlineata at high temperatures (>30°C) compared to that of the forest-steppe isolates. The obtained data indicate that the use of isolates from the steppe zone will be most promising for the insect pest control under the conditions of continental and arid climate.     

Effects of cold stress on metabolic regulation in the overwintering larvae of the Chinese white pine beetle,Dendroctonus armandi

The Chinese white pine beetle, Dendroctonus armandi Tsai & Li (Coleoptera: Curculionidae, Scolytinae), is considered the most destructive forest pest in the Qinling and Bashan Mountains of China. In recent years, winter temperature has dropped in these regions, and extremely low temperatures are hard to survive for insects. Cold hardiness becomes a crucial strategy because temperature change often leads to fluctuations in insect abundance, and the metabolism rate is a key index of resistance to cold in overwintering insects. Therefore, we investigated the relationship between the change in respiratory rate and the activity of metabolism-related mitochondrial enzymes in D. armandi larvae under cold conditions. We found that the respiratory rate decreased, and it was matched with the activity of glutamate dehydrogenase, aconitase, and lipase during overwintering. Among the various test times under cold conditions, the respiratory rate also decreased with decreasing temperature and increased under very low temperatures. At all cold stress periods, glutamate dehydrogenase and lipase showed increased activity at higher temperatures and decreased activity under lower temperatures, but the activity of NAD-malic enzyme, NADP-malic enzyme, mitochondrial isocitrate dehydrogenase, and aconitase were contrary. Under all low temperatures, the activity of enzymes – except for NADP-malic enzyme, glutamate dehydrogenase, and lipase – increased in short-term cold stress and decreased in long-term cold stress at 4, 0, −4, −6, −8, and −10 °C. However, at −2 °C, the activity of enzymes showed a decreasing trend in short-term treatments and an increasing trend in long-term treatments, except for mitochondrial isocitrate dehydrogenase. The results not only improve our understanding of the metabolic mechanism of cold adaptation in D. armandi, but also provide an important experimental basis for further study and biological pest control.     

Effects of thermal stress on lipid peroxidation and antioxidant enzyme activities of the predatory mite,Neoseiulus cucumeris (Acari: Phytoseiidae)

Changes in temperature are known to cause a variety of physiological stress responses in insects and mites. Thermal stress responses are usually associated with the increased generation of reactive oxygen species (ROS), resulting in oxidative damage. In this study, we examined the time-related effect (durations for 1, 2, 3, and 5 h) of thermal stress conditions—i.e., relatively low (0, 5, 10, and 15 °C) or high (35, 38, 41, and 44 °C) temperatures—on the activities of antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), peroxidase (POX), glutathione S-transferases (GSTs), and total antioxidant capacity (T-AOC) of the predatory mite Neoseiulus cucumeris. Also the lipid peroxidation (LPO) levels of the predatory mite were measured under thermal stress conditions. The results confirmed that thermal stress results in a condition of so-called oxidative stress and the four antioxidant enzymes play an important role in combating the accumulation of ROS in N. cucumeris. CAT and POX activity changed significantly when the mites were exposed to cold and heat shock, respectively. The elevated levels of SOD and GSTs activity, expressed in a time-dependent manner, may have an important role in the process of antioxidant response to thermal stress. However, the levels of LPO in N. cucumeris were high, serving as an important signal that these antioxidant enzyme-based defense mechanisms were not always adequate to counteract the surplus ROS. Thus, we hypothesize that thermal stress, especially extreme temperatures, may contribute much to the generation of ROS in N. cucumeris, and eventually to its death.     

Effects of acclimation and diapause on the cold tolerance of Trogoderma granarium

Khapra beetle, Trogoderma granarium Everts (Coleoptera: Dermestidae), is a pest of stored grain in Africa, Asia, and Europe. It is a quarantine insect for much of the rest of the world. Control of T. granarium can be achieved with methyl bromide, but this fumigant is an ozone‐depleting substance and is being phased out worldwide. Thus, there is an urgent need to find new methods of control, including the use of low temperatures. Here, we assess the effects of diapause and cold acclimation on the cold tolerance of T. granarium. The percentage of larvae in diapause increased with larval density, reaching 57.3% when reared at a density of 73 larvae g^?1 diet. The cold tolerance of T. granarium was assessed by the supercooling points (SCPs) of various life stages. The SCP of non‐acclimated insects ranged from ?26.2 ± 0.2 °C (mean ± SEM) for eggs to ?14.4 ± 0.4 °C for larvae. The lowest SCP for larvae, ?24.3 ± 0.3 °C, was obtained for diapausing‐acclimated larvae. Based on mean LT₅₀ values, the most cold‐tolerant stage at ?10 °C was the diapausing‐acclimated larvae (87 days) followed by non‐diapausing‐acclimated larvae (51 days), diapausing non‐acclimated larvae (19 days), adults (4 days), non‐diapausing non‐acclimated larvae (2 days), pupae (0.4 days), and eggs (0.2 days). The estimated times to obtain 99.9968% mortality (Probit 9) for diapausing‐acclimated larvae are 999, 442, 347, 84, and 15 days at 0, ?5, ?10, ?15, and ?20 °C, respectively. Probit 9 is an estimated value used by quarantine experts to estimate conditions that are required to kill all insects. In light of the long exposure time needed to control T. granarium even at ?20 °C, cooling to below ?27 °C (i.e., below the SCP of eggs) will quickly kill all life stages and may be the best way to control this insect with low temperatures.     

Characterization of two copper/zinc superoxide dismutases (Cu/Zn-SODs) from the desert beetle Microdera punctipennis and their activities in protecting E. coli cells against cold

Superoxide dismutases (SODs) are crucial in scavenging reactive oxygen species (ROS); however, studies regarding SOD functions in insects under cold conditions are rare. In this paper, two novel Cu/Zn-SOD genes in the desert beetle Microdera punctipennis, an extracellular copper/zinc SOD (MpecCu/Zn-SOD) and an intracellular copper/zinc SOD (MpicCu/Zn-SOD), were identified and characterized. The results of quantitative real-time PCR showed that MpecCu/Zn-SOD expression was significantly up-regulated by 4 °C exposure for 0.5 h, but MpicCu/Zn-SOD was not. Superoxide anion radical (O₂•^-) content in beetles under 4 °C exposure for 0.5 h showed an initial sharp increase and fluctuated during the cold treatment period, which was consistent with the relative mRNA level of MpecCu/Zn-SOD. The total SOD activity in the beetle was negatively correlated to the O₂•^- content with a correlation coefficient of −0.437. An E. coli system was employed to study the function of each MpCu/Zn-SOD gene. The fusion proteins Trx-His-MpCu/Zn-SODs were over expressed in E. coli BL21 using pET32a vector, and identified by SDS-PAGE and Western blotting. The transformed bacteria BL21(pET32a-MpecCu/Zn-SOD) and BL21(pET32a-MpicCu/Zn-SOD) showed increased cold tolerance to −4 °C as well as increased SOD activity compared to the control BL21(pET32a). The relative conductivity and malondialdehyde content in the two MpCu/Zn-SODs transformed bacteria under −4 °C were significantly lower than the control BL21(pET32a). Furthermore, BL21(pET32a-MpecCu/Zn-SOD) had significantly higher SOD activity and cold tolerance than BL21(pET32a-MpicCu/Zn-SOD) under −4 °C treatment, and had lower conductivity than BL21(pET32a-MpicCu/Zn-SOD). In conclusion, low temperature led to the accumulation of O₂•^- in M. punctipennis, which stimulated the expression of extracellular MpCu/Zn-SOD gene and the increase of total SOD activity. E. coli overexpressing Trx-His-MpCu/Zn-SODs increased resistance to cold treatment-induced oxidative stress. Our findings will be helpful in further study of Cu/Zn-SOD genes in insect cold-tolerance.     

Cold tolerance of the maize orange leafhopper,Cicadulina bipunctata

Cicadulina bipunctata was originally distributed in tropical and subtropical regions of the Old World. This leafhopper recently expanded its distribution area to southern parts of temperate Japan. In this study, factors affecting the overwintering ability of C. bipunctata were examined. A series of laboratory experiments revealed that cold acclimation at 15 °C for 7 days enhanced the cold tolerance of C. bipunctata to the same level as an overwintering population, adult females were more tolerant of cold temperature than adult males, and survival of acclimated adult females was highly dependent on temperature from −5 to 5 °C and exposure duration to the temperature. The temperature of crystallization of adult females was approximately −19 °C but temperatures in southern temperate Japan rarely dropped below −10 °C in the winter, indicating that overwintering C. bipunctata adults in temperate Japan are not killed by freezing injury but by indirect chilling injury caused by long-term exposure to moderately low temperatures. An overwintering generation of C. bipunctata had extremely low overwinter survival (<1%) in temperate Japan; however, based on winter temperature ranges, there are additional areas amenable to expansion of C. bipunctata in temperate Japan.