Larval Cold Tolerance Research Articles

Geographic variation in larval cold tolerance and exposure across the invasion front of a widely established forest insect.

Under global climate change, high and low temperature extremes can drive shifts in species distributions. Across the range of a species, thermal tolerance is based on acclimatization, plasticity, and may undergo selection, shaping resilience to temperature stress. In this study, we measured variation in cold temperature tolerance of early instar larvae of an invasive forest insect, Lymantria dispar dispar L. (Lepidoptera: Erebidae), using populations sourced from a range of climates within the current introduced range in the Eastern United States. We tested for population differences in chill coma recovery (CCR) by measuring recovery time following a period of exposure to a nonlethal cold temperature in 2 cold exposure experiments. A 3rd experiment quantified growth responses after CCR to evaluate sublethal effects. Our results indicate that cold tolerance is linked to regional climate, with individuals from populations sourced from colder climates recovering faster from chill coma. While this geographic gradient is seen in many species, detecting this pattern is notable for an introduced species founded from a single point-source introduction. We demonstrate that the cold temperatures used in our experiments occur in nature during cold spells after spring egg hatch, but impacts to growth and survival appear low. We expect that population differences in cold temperature performance manifest more from differences in temperature-dependent growth than acute exposure. Evaluating intraspecific variation in cold tolerance increases our understanding of the role of climatic gradients on the physiology of an invasive species, and contributes to tools for predicting further expansion.

Hundreds of billions of silent outbreaks: A historic outbreak record of the gall midge Schizomyia castanopsisae (Diptera: Cecidomyiidae) on the Izu Islands, Tokyo, Japan, and its potential mechanism

AbstractHerbivore outbreaks cause serious damage to forest trees. In recent decades, outbreaks of the gall midge Schizomyia castanopsisae (Diptera: Cecidomyiidae) inducing inflorescence galls on Castanopsis sieboldii (Fagaceae) occurred in the Izu Islands, Tokyo, Japan. We investigated the gall density on the islands and estimated the larval population on Miyakejima Island. We also surveyed seasonal changes in larval age structure, presence of natural enemies, larval cold tolerance, and thermal effects on the adult emergence. In addition, we conducted molecular phylogenetic analyses of S. castanopsisae populations among different localities. The gall density was particularly high in southern parts (from Miyakejima to Aogashima) of the Izu Islands. The gall midge seemed to have been expanding its range toward northern parts in recent years. On Miyakejima, the larval population was estimated to be approximately 158 and 36 bn in 2017 and 2018, respectively. Parasitoids were not found in the dissection of galls. A cold temperature treatment did not affect the percentage of adult emergence. Developmental zero and thermal constant during the period from larval departure to adult emergence were estimated to be 4.7°C and 588.2°C‐days in males and 7.4°C and 476.2°C‐days in females, and the adults were predicted to emerge in April. Populations in Kyushu were genetically distinct from those in Okinawajima and the Izu Islands, but a common haplotype was found between the latter two localities. These imply that S. castanopsisae on the Izu Islands may be a recent invasion, and the absence of natural enemies causes its outbreaks.

The HSP/co-chaperone network in environmental cold adaptation of Chilo suppressalis

Winter cold is one of the major environmental stresses for ectotherm species. Chilo suppressalis, a notorious lepidopteran pest of rice, has a wide geographic region that includes temperate zones with severe environmental conditions. Although C. suppressalis exhibits remarkable cold tolerance, its cold-adaptation mechanisms remain unclear. Here, we used bioinformatics approaches to evaluate transcript levels of genes comprising the C. suppressalis heat shock protein (HSP)/co-chaperone network in response to cold-induced stress. Using all such genes identified in the C. suppressalis genome, we experimentally examined the corresponding transcript levels under cold-acclimation or intermittent cold-shock stresses in diapause and non-diapausing larvae. In total, we identified 19 HSPs and 8 HSP co-chaperones in the C. suppressalis genome. Nine (hsp90, hsp75, hsp70, hsp40, small hsp, activator of 90 kDa heat shock protein ATPase-like, heat shock factor, heat shock factor binding protein 1-like and HSPB1-associated protein 1) were highly cold-inducible and likely comprise the principal cold-response HSP/co-chaperone network in C. suppressalis. We also found that transcriptional regulation of the HSP/co-chaperone networks response differs between cold-acclimation and short-term cold-shock. Moreover, activation of the HSP/co-chaperone network depends on the diapause state of overwintering larvae and cold acclimation may further increase larval cold tolerance. These results provide key new insights in the cold-adaptation mechanisms in C. suppressalis.

Autumn larval cold tolerance does not predict the northern range limit of a widespread butterfly species.

Climate change is driving range shifts, and a lack of cold tolerance is hypothesized to constrain insect range expansion at poleward latitudes. However, few, if any, studies have tested this hypothesis during autumn when organisms are subjected to sporadic low‐temperature exposure but may not have become cold‐tolerant yet. In this study, we integrated organismal thermal tolerance measures into species distribution models for larvae of the Giant Swallowtail butterfly, Papilio cresphontes (Lepidoptera: Papilionidae), living at the northern edge of its actively expanding range. Cold hardiness of field‐collected larvae was determined using three common metrics of cold‐induced physiological thresholds: the supercooling point, critical thermal minimum, and survival following cold exposure. P. cresphontes larvae were determined to be tolerant of chilling but generally die at temperatures below their SCP, suggesting they are chill‐tolerant or modestly freeze‐avoidant. Using this information, we examined the importance of low temperatures at a broad scale, by comparing species distribution models of P. cresphontes based only on environmental data derived from other sources to models that also included the cold tolerance parameters generated experimentally. Our modeling revealed that growing degree‐days and precipitation best predicted the distribution of P. cresphontes, while the cold tolerance variables did not explain much variation in habitat suitability. As such, the modeling results were consistent with our experimental results: Low temperatures in autumn are unlikely to limit the distribution of P. cresphontes. Understanding the factors that limit species distributions is key to predicting how climate change will drive species range shifts.

Cold Acclimation of Trogoderma granarium Everts Is Tightly Linked to Regulation of Enzyme Activity, Energy Content, and Ion Concentration.

In this study, cold hardiness and some physiological characteristics of the Khapra beetle, Trogoderma granarium Everts (Coleoptera: Dermestidae) larvae, were investigated under different thermal regimes, i.e., control, cold-acclimated (CA), fluctuating-acclimated (FA), and rapid cold-hardened (RCH). In all the regimes, the larval survival rate decreased with a decrease in temperature. CA larvae showed the highest cold hardiness following 24 h exposure at -15 and -20°C. Control larvae had the highest glycogen content (34.4 ± 2.3 μg/dry weight). In contrast, CA larvae had the lowest glycogen content (23.0 ± 1.6 μg/dry weight). Change in trehalose content was reversely proportional to changes in glycogen content. The highest myo-inositol and glucose contents were detected in CA larvae (10.7 ± 0.4 μg/dry weight) and control (0.49 ± 0.03 μg/dry weight), respectively. In control and treated larvae, [Na+] decreased, though [K+] increased, with increasing exposure time. The shape of the thermal reaction curve of AMP-depended protein kinase and protein phosphatase 2C followed the same norm, which was different from protein phosphatase 1 and protein phosphatase 2A. Protein phosphatase 2A and 2C showed a complete difference in thermal reaction norms. Indeed, thermal fluctuation caused the highest changes in the activity of the enzymes, whereas the RCH showed the lowest changes in the activity of the enzymes. Our results showed a significant enhancement of larval cold tolerance under CA regime, which is related to the high levels of low molecular weight carbohydrates under this regime. Our results showed that among the different thermal regimes tested, the CA larvae had the lowest supercooling point (about -22°C) and the highest cold hardiness following 24 h exposure at -15 and -20°C.

Cold tolerance of third-instar Drosophila suzukii larvae

Drosophila suzukii is an emerging global pest of soft fruit; although it likely overwinters as an adult, larval cold tolerance is important both for determining performance during spring and autumn, and for the development of temperature-based control methods aimed at larvae. We examined the low temperature biology of third instar feeding and wandering larvae in and out of food. We induced phenotypic plasticity of thermal biology by rearing under short days and fluctuating temperatures (5.5–19°C). Rearing under fluctuating temperatures led to much slower development (42.1days egg-adult) compared to control conditions (constant 21.5°C; 15.7days), and yielded larger adults of both sexes. D. suzukii larvae were chill-susceptible, being killed by low temperatures not associated with freezing, and freezing survival was not improved when ice formation was inoculated externally via food or silver iodide. Feeding larvae were more cold tolerant than wandering larvae, especially after rearing under fluctuating temperatures, and rearing under fluctuating temperatures improved survival of prolonged cold (0°C) to beyond 72h in both larval stages. There was no evidence that acute cold tolerance could be improved by rapid cold-hardening. We conclude that D. suzukii has the capacity to develop at low temperatures under fluctuating temperatures, but that they have limited cold tolerance. However, phenotypic plasticity of prolonged cold tolerance must be taken into account when developing low temperature treatments for sanitation of this species.

The protective effect of rapid cold-hardening develops more quickly in frozen versus supercooled larvae of the Antarctic midge, Belgica antarctica

During the austral summer, larvae of the terrestrial midge Belgica antarctica (Diptera: Chironomidae) experience highly variable and often unpredictable thermal conditions. In addition to remaining freeze tolerant year-round, larvae are capable of swiftly increasing their cold tolerance through the rapid cold-hardening (RCH) response. The present study compared the induction of RCH in frozen versus supercooled larvae. At the same induction temperature, RCH occurred more rapidly and conferred a greater level of cryoprotection in frozen versus supercooled larvae. Furthermore, RCH in frozen larvae could be induced at temperatures as low as -12°C, which is the lowest temperature reported to induce RCH. Remarkably, as little as 15 min at -5°C significantly enhanced larval cold tolerance. Not only is protection from RCH acquired swiftly, but it is also quickly lost after thawing for 2 h at 2°C. Because the primary difference between frozen and supercooled larvae is cellular dehydration caused by freeze concentration of body fluids, we also compared the effects of acclimation in dehydrated versus frozen larvae. Because slow dehydration without chilling significantly increased larval survival to a subsequent cold exposure, we hypothesize that cellular dehydration caused by freeze concentration promotes the rapid acquisition of cold tolerance in frozen larvae.

Temporal and Species Variation in Cold Hardiness Among Invasive Rhizophagous Weevils (Coleoptera: Curculionidae) in a Northern Hardwood Forest

Abstract A complex of invasive rhizophagous weevils has established in North American northern hardwood forests. Little is known regarding the overwintering biology of these weevils and thus how cold hardiness and weather affect population dynamics. Field data from winter 2006–2007 showed a decline in larval abundance but an increase in larval weight of the surviving individuals. During winter 2008–2009, we examined several aspects of overwintering biology of Phyllobius oblongus (L.), Polydrusus sericeus (Schaller), and Barypeithes pellucidus (Boheman). Larvae were collected in the Upper Peninsula of Michigan and transported in bulk field soil to the University of Notre Dame, South Bend, IN, for laboratory assays. Supercooling points (SCPs) of P. oblongus and B. pellucidus larvae not in contact with ice were highest in October and lowest in March, but SCPs of larvae that were in contact with ice did not differ among sampling dates. Larval cold tolerance increased over the winter, with 11% of P. oblongus and 40% of P. sericeus surviving 24 h at -12°C. Few B. pellucidus or P. oblongus survived 30 d at temperatures of -3.3°C or lower. Body water content increased from January to March in P. sericeus and B. pellucidus. Larval hemolymph from all species showed some thermal hysteresis and hexagonal crystal formation, indicative of low levels of antifreeze proteins or glycolipids. These subterranean-dwelling larvae are buffered from ambient winter temperatures, but our data also suggest low levels of freeze avoidance. We discuss how these overwintering strategies may affect adult population dynamics.

Integrating biophysical models and evolutionary theory to predict climatic impacts on species’ ranges: the dengue mosquitoAedes aegyptiin Australia

Summary 1. Climate change will alter the distribution and abundance of many species, including those of concern to human health. Accurate predictions of these impacts must be based on an understanding of the mechanistic links between climate and organisms, and a consideration of evolutionary responses. 2. Here we use biophysical models of energy and mass transfer to predict climatic impacts on the potential range of the dengue fever vector, Aedes aegypti, in Australia. We develop a first-principles approach to calculate water depth and daily temperature cycles in containers differing in size, catchment and degree of shading to assess habitat suitability for the aquatic life cycle phase. We also develop a method to predict the potential climatic impacts on the evolutionary response of traits limiting distribution. 3. Our predictions show strong correspondence with the current and historical distribution and abundance of Ae. aegypti in Australia, suggesting that inland and northern limits are set by water availability and egg desiccation resistance, and southern limits by adult and larval cold tolerance. 4. While we predict that climate change will directly increase habitat suitability throughout much of Australia, the potential indirect impact of changed water storage practices by humans in response to drought may have a greater effect. 5. In northern Australia, we show that evolutionary changes in egg desiccation resistance could potentially increase the chances of establishment in a major centre (Darwin) under climate change. 6. Our study demonstrates how biophysical models of climate‐animal interactions can be applied to make decisions about managing biotic responses to climate change. Mechanistic models of the kind we apply here can provide more robust and general predictions than correlative analyses. They can also explicitly incorporate evolutionary responses, the outcomes of which may significantly alter management decisions.

Detecting freeze injury and seasonal cold-hardening of cells and tissues in the gall fly larvae, Eurosta solidaginis (Diptera: Tephritidae) using fluorescent vital dyes

This study identified a hierarchy in levels of cold tolerance for diverse tissues from larvae of Eurosta solidaginis. Following freezing at −80 °C, larval survival and the viability of specific tissues were assessed using membrane-permeant DNA stain (SYBY-14) and propidium iodide. Integumentary muscle, hemocytes, tracheae, and the crystal-containing portion of the Malpighian tubules were most susceptible to freezing injury. A second group consisting of fat body, salivary glands, and the proximal region of the Malpighian tubules were intermediate in their susceptibility, while the foregut, midgut, and hindgut were the most resistant to freezing injury. Seasonal increases in larval cold tolerance were closely matched by changes in the cold tolerance of individual tissues. Compared to larvae collected in September, the survival rates for each of the six tissues tested from October-collected larvae increased by 20–30%. The survival rate in all tissues was notably higher than that of whole animals, indicating that larval death could not be explained by the mortality in any of the tissues we tested. This method will be useful for assessing the nature of chilling/freezing injury, the role cryoprotectants, and cellular changes promoting cold tolerance.

Geographical and diapause-related cold tolerance in the blow fly, Calliphora vicina

Three geographical strains of the blow fly, Calliphora vicina, were tested for cold tolerance at 0°, −4° and −8°C. Survival to eclosion after 1 to 18 days of cold exposure was greater for diapause-destined larvae than for nondiapause-destined larvae of the two northern strains (Nallikari, Finland 65°N and Edinburgh, Scotland 55°N) but not for the southernmost strain (Barga, Italy 44°N) where no clear differences were apparent. Diapause-destined larvae of the Edinburgh strain were more cold tolerant than those from Nallikari, at both −4° and −8°C, a difference possibly attributable to the long-lasting snow cover in the more northern locality, which might insulate the overwintering soil microclimate. At 0°C, however, Nallikari larvae were more cold tolerant than Edinburgh or Barga. This was also the case for nondiapause-destined larvae, indicating that cold tolerance may occur, in part, independently of the diapause programme. In all three strains diapausing larvae were more cold tolerant than same-age (nondiapausing) pupae. For Nallikari, but not Barga, wandering larvae from short-day exposed flies, therefore initially programmed for diapause, but diverted from the diapause pathway by larval breeding at 19°C, were significantly more cold tolerant than nondiapause larvae from long-day parents, indicating some maternal regulation of larval cold tolerance. There was, however, no evidence for an additional cold hardiness in larvae acclimatised to cold by a gradual reduction of temperature.