Cold hardiness-informed budbreak reveals role of freezing temperatures and daily fluctuation in chill accumulation model.

Fundamental questions in bud dormancy remain, including what temperatures fulfill dormancy requirements (i.e., chill accumulation). Recent studies demonstrate freezing temperatures promote chill accumulation and cold hardiness influences time to budbreak - the phenotype used for dormancy evaluations. Here we evaluated bud cold hardiness (CH) and budbreak responses of grapevines (Vitis hybrids) throughout chill accumulation under three treatments: constant (5 °C), fluctuating (-3.5 to 6.5 °C daily), and field conditions (Madison, WI, USA). Chill treatments experiencing lower temperatures promoted greater gains in cold hardiness (CHfield>CHfluctuating>CHconstant). All treatments decreased observed time to budbreak with increased chill accumulation. However, perceived treatment effectiveness changed when time to budbreak was adjusted to remove cold acclimation effects. Among three classic chill models (North Carolina, Utah, and Dynamic), none were able to correctly describe adjusted time to budbreak responses to chill accumulation. Thus, a new model is proposed that expands the range of chill accumulation temperatures to include freezing temperatures and enhances chill accumulation under fluctuating temperature conditions. Most importantly, our analysis demonstrates adjustments for uneven acclimation change the perceived effectiveness of chill treatments. Therefore, future work in bud dormancy would benefit from simultaneously evaluating cold hardiness.

The freeze-avoiding mountain pine beetle (Dendroctonus ponderosae) survives prolonged exposure to stressful cold by mitigating ionoregulatory collapse.

Insect performance is linked to environmental temperature, and surviving through winter represents a key challenge for temperate, alpine and polar species. To overwinter, insects have adapted a range of strategies to become truly cold hardy. However, although the mechanisms underlying the ability to avoid or tolerate freezing have been well studied, little attention has been given to the challenge of maintaining ion homeostasis at frigid temperatures in these species, despite this limiting cold tolerance for insects susceptible to mild chilling. Here, we investigated how prolonged exposure to temperatures just above the supercooling point affects ion balance in freeze-avoidant mountain pine beetle (Dendroctonus ponderosae) larvae in autumn, mid-winter and spring, and related it to organismal recovery times and survival. Hemolymph ion balance was gradually disrupted during the first day of exposure, characterized by hyperkalemia and hyponatremia, after which a plateau was reached and maintained for the rest of the 7-day experiment. The degree of ionoregulatory collapse correlated strongly with recovery times, which followed a similar asymptotical progression. Mortality increased slightly during extensive cold exposures, where hemolymph K+ concentration was highest, and a sigmoidal relationship was found between survival and hyperkalemia. Thus, the cold tolerance of the freeze-avoiding larvae of D. ponderosae appears limited by the ability to prevent ionoregulatory collapse in a manner similar to that of chill-susceptible insects, albeit at much lower temperatures. Based on these results, we propose that a prerequisite for the evolution of insect freeze avoidance may be a convergent or ancestral ability to maintain ion homeostasis during extreme cold stress.

Comparison of the supercooling points of questing Dermacentor variabilis adults in two populations on the Canadian prairies and implications for overwinter survival.

A comparison was made of the supercooling points (SCPs) of questing Dermacentor variabilis adults from two populations located ca. 800 km apart on the Canadian prairies. This is also the first study to examine whether there are seasonal fluctuations in the SCP of questing D. variabilis adults. The SCPs of adult ticks from Lizard Lake Community Pasture, a recently established population in west-central Saskatchewan, varied over spring and summer, with the median SCP warming over time. In addition, the SCPs of ticks from Lizard Lake Community Pasture were significantly higher than those of adult ticks collected from Sandy Hook in Manitoba, a population that has been established for decades. The off-host survival of adults from Sandy Hook between summer and spring has been shown previously to be significantly greater than that of adults from Lizard Lake Community Pasture. The findings of the present study suggest that there may be geographical variation in the SCPs of D. variabilis adults which may be associated with differences in overwinter survival. The relatively low SCPs of questing D. variabilis adults, and the ability of some adults to survive off-host during winter, may be factors contributing to the range expansion of this tick species in Canada.

Time to budbreak is not enough: cold hardiness evaluation is necessary in dormancy and spring phenology studies.

BACKGROUND: Dormancy of buds is an important phase in the life cycle of perennial plants growing in environments where unsuitable growth conditions occur seasonally. In regions where low temperature defines these unsuitable conditions, the attainment of cold hardiness is also required for survival. The end of the dormant period culminates in budbreak and flower emergence, or spring phenology, one of the most appreciated and studied phenological events - a time also understood to be most sensitive to low-temperature damage. Despite this, we have a limited physiological and molecular understanding of dormancy, which has negatively affected our ability to model budbreak. This is also true for cold hardiness. SCOPE: Here we highlight the importance of including cold hardiness in dormancy studies that typically only characterize time to budbreak. We show how different temperature treatments may lead to increases in cold hardiness, and by doing so also (potentially inadvertently) increase time to budbreak. CONCLUSIONS: We present a theory that describes evaluation of cold hardiness as being key to clarifying physiological changes throughout the dormant period, delineating dormancy statuses, and improving both chill and phenology models. Erroneous interpretations of budbreak datasets are possible by not phenotyping cold hardiness. Changes in cold hardiness were very probably present in previous experiments that studied dormancy, especially when those included below-freezing temperature treatments. Separating the effects between chilling accumulation and cold acclimation in future studies will be essential for increasing our understanding of dormancy and spring phenology in plants.

The role of genotypic and climatic variation at the range edge: A case study in winegrapes.

PREMISE: Changes in habitat suitability due to climate change are causing range shifts, with new habitat potentially available at cold range edges. We must predict these range shifts, but forecasters have limited knowledge of how genetic differences in plant physiological tolerances influence range shifts. Here, we focus on a major determinant of species ranges-physiological tolerance to extreme cold-to ask how warming over recent decades and genetic variation shape expansion across complex landscapes. METHODS: We examined how genotypes vary in maximum cold tolerance from 9 years of cold hardiness data across 18 genotypes from 13 sites, using winegrapes (Vitis vinifera subsp. vinifera) as a case study. Combining a Bayesian hierarchical dose-response model with gridded climate data, we then project changes in climatic suitability near winegrapes' current cold range-edge between 1949 and 2016. RESULTS: Plants increased maximum cold hardiness non-linearly with decreasing air temperature (maximum cold hardiness: -23.6°C), but with substantial (by 2°C) variation across genotypes. Our results suggest, since the 1980s, decreasing freeze injury risk has made conditions more favorable for all genotypes at the cold range-edge, but conditions remained more favorable for more cold hardy genotypes and in warmer areas. There was substantial spatial variation in habitat suitability, with the majority of suitably warm habitat located in a narrow north-south oriented strip. CONCLUSIONS: We highlight the importance of genotypic differences in physiological tolerances when assessing range shift potential with climate change. Habitat improvements were unevenly distributed over the spatially complex landscape, though, emphasizing the importance of dispersal in range expansion.

Effect of constant and fluctuating low temperature on the survival of Tuta absoluta pupae.

Temperature is among the key factors impacting the establishment and spread of invasive pests. The tomato leafminer Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) is one of the major pests attacking Solanaceae plants and is known to possess overwintering capacities. However, the cold hardiness of T. absoluta pupae is poorly documented. In this study, we investigated the effect of constant temperature and stepwise cooling on T. absoluta pupae under laboratory conditions. For this purpose, bioassays on pupal development under constant temperature (5°C) for 30, 60 and 90 days, and stepwise changes in temperature (11, 10 and 8°C; in this order every 30 days), were assessed. We found that exposure to 5°C for 30 and 60 days did not affect the post-cooling emergence time of adults compared to the control. Pupae completed their development after 60 days of cold exposure at 5°C, but more adults emerged after 30 than 60 days. Even though alive pupae were observed after 90 days of cold exposure at 5°C, no adults emerged. External colours of pupae depended on the duration of cold periods, and green pupae obtained after 30 and 60 days were found to be positively correlated with the emergence of adults. When pupae were kept at 11°C for 30 days, 47% emerged, and when the temperature was changed to 10, only 12% of pupae emerged for the period 31-60 days. However, the decrease of the temperature to 8°C yielded no emergence for the period 61-90 days. Our study provides useful information to better understand the population dynamics of overwintering T. absoluta, and to underpin the development of monitoring and control strategies for the pest.

A small survey of introduced Zaprionus indianus (Diptera: Drosophilidae) in orchards of the eastern United States.

The African fig fly, Zaprionus indianus (Gupta), is a generalist fruit fly that typically breeds in decaying fruits from over 70 plant species. The species has spread globally from its native range in tropical Africa, becoming an invasive pest on ripening figs in Brazil. First reported in the United States in 2005 in Florida, Z. indianus has since been documented as far north as Canada and is hypothesized to recolonize northwards from southern refugia each year. We sampled drosophilid communities over the growing season at 2 orchards in Virginia from 2020 to 2022 and 11 orchards along the East Coast during the fall of 2022 to quantify the abundance of Z. indianus relative to other drosophilids across locations, seasons, and fruit crops. Massachusetts had the northernmost population, with no Z. indianus detected in Maine and no correlation between latitude and relative abundance. Variation in Z. indianus relative abundance was high between nearby orchards and abundance was higher on peaches relative to apples within orchards. Comparisons of seasonal abundance curves between 2 Virginia orchards showed similar dynamics across years with individuals first detected around July and becoming absent around December, with peaks in late summer and mid-fall. The variation in seasonal and latitudinal abundance shown here highlights a need for broader sampling to accurately characterize the range, spread, and environmental tolerances of Z. indianus in North America.

Cold tolerance and overwintering survival of Aphelinus certus (Hymenoptera: Aphelinidae), a parasitoid of the soybean aphid (Hemiptera: Aphididae) in North America.

Broad-spectrum insecticides are the main control measure of the invasive and economically damaging soybean aphid (Aphis glycines) in North America, although biological control by resident natural enemies can also greatly diminish population levels. One such natural enemy is the accidentally introduced Eurasian parasitoid Aphelinus certus (Hymenoptera: Aphelinidae), though its impact appears to be limited by low rates of parasitism early in the growing season. We tested the hypothesis that A. certus might experience high overwintering mortality. In the laboratory, we used thermocouple thermometry to measure the supercooling points of diapausing parasitoids and assessed parasitoid survival after exposure to ecologically relevant durations of low temperature. We found A. certus to be freeze-intolerant with a median supercooling point of -28°C. When exposed to temperatures of 0°C for up to 7 months, adults emerged only after exposures of at least 60 days and survival decreased with durations beyond 150 days. We also conducted in-field studies at sites from northern Minnesota to southern Iowa to determine if diapausing A. certus could overwinter above and below the snowpack. Survival was negatively correlated with increasing latitude and was greater for parasitoids placed on the ground than 1 meter off the ground, likely due to the warmer and stabler temperatures of the subnivean microclimate. Our results suggest that A. certus is capable of overwintering in the region inhabited by soybean aphid but may experience substantial mortality even under ideal conditions. Climate change is predicted to bring warmer, drier winters to the North American Midwest, with decreased depth and duration of snow cover, which may further reduce overwintering survival.

Variations of freezing tolerance and sugar concentrations of grape buds in response to foliar application of abscisic acid.

The purpose of this study was to explore the mechanism of ABA-induced freezing tolerance increase in grapevines. The specific objectives were to evaluate the impact of ABA treatment on soluble sugar concentration in grape buds and determine the correlations between freezing tolerance and ABA-affected soluble sugar concentration. Vitis spp 'Chambourcin' and Vitis vinifera 'Cabernet franc' were treated with 400 and 600 mg/L ABA in the greenhouse and field. The freezing tolerance and soluble sugar concentration of grape buds were measured monthly during the dormant season in the field and at 2wk, 4wk, and 6wk after ABA application in the greenhouse. It was observed that fructose, glucose, and sucrose are the main soluble sugars that correlate with freezing tolerance of grape buds and the synthesis of these sugars can be enhanced by ABA treatment. This study also found that ABA application can promote raffinose accumulation, however, this sugar may play a more important role in the early acclimation stage. The preliminary results suggest that raffinose accumulated first in buds, then its decrease in mid-winter corresponded with the increase of smaller sugars, such as sucrose, fructose, and glucose, which in turn, corresponded with reaching maximum freezing tolerance. It is concluded that ABA is a cultural practice tool that can be used to enhance freezing tolerance of grapevines.

Cold tolerance and diapause within and across trophic levels: Endoparasitic wasps and their fly host have similar phenotypes.

Low temperatures associated with winter can limit the survival of organisms, especially ectotherms whose body temperature is similar to their environment. However, there is a gap in understanding how overwintering may vary among groups of species that interact closely, such as multiple parasitoid species that attack the same host insect. Here, we investigate cold tolerance and diapause phenotypes in three endoparasitoid wasps of the apple maggot fly Rhagoletis pomonella (Diptera: Tephritidae): Utetes canaliculatus, Diachasma alloeum, and Diachasmimorpha mellea (Hymenoptera: Braconidae). Using a combination of respirometry and eclosion tracking, we found that all three wasp species exhibited the same three diapause duration phenotypes as the fly host. Weak (short duration) diapause was rare, with <5 % of all three wasp species prematurely terminating diapause at 21 °C. Most D.mellea (93 %) entered a more intense (longer duration) diapause that did not terminate within 100 d at this warm temperature. The majority of U.canaliculatus (92 %) and D. alloeum (72 %) averted diapause (non-diapause) at 21 °C. There was limited interspecific variation in acute cold tolerance among the three wasp species: wasps and flies had similarly high survival (>87 %) following exposure to extreme low temperatures (-20 °C) as long as their body fluids did not freeze. The three wasp species also displayed little interspecific variation in survival following prolonged exposure to mild chilling of 8 or more weeks at 4 °C. Our study thus documents a remarkable conservation of cold tolerance and diapause phenotypes within and across trophic levels.

Phenological physiology: seasonal patterns of plant stress tolerance in a changing climate.

The physiological challenges posed by climate change for seasonal, perennial plants include increased risk of heat waves, postbudbreak freezing ('false springs'), and droughts. Although considerable physiological work has shown that the traits conferring tolerance to these stressors - thermotolerance, cold hardiness, and water deficit stress, respectively - are not static in time, they are frequently treated as such. In this review, I synthesize the recent literature on predictable seasonal - and therefore, phenological - patterns of acclimation and deacclimation to heat, cold, and water-deficit stress in perennials, focusing on woody plants native to temperate climates. I highlight promising, high-throughput techniques for quantifying thermotolerance, cold hardiness, and drought tolerance. For each of these forms of stress tolerance, I summarize the current balance of evidence regarding temporal patterns over the course of a year and suggest a characteristic temporal scale in these responses to environmental stress. In doing so, I offer a synthetic framework of 'phenological physiology', in which understanding and leveraging seasonally recurring (phenological) patterns of physiological stress acclimation can facilitate climate change adaptation and mitigation.

Internal freezing and heat loss of apple (Malus domestica Borkh.) and sweet cherry (Prunus avium L.) reproductive buds are decreased with cellulose nanocrystal dispersions.

Cold damage has caused more economic losses to fruit crop growers in the U.S. than any other weather hazard, making it a perennial concern for producers. Cellulose nanocrystals (CNCs) represent a new generation of renewable bio-nanomaterials, with many unique physical and chemical properties, including their low thermal conductivity. Our team has developed a process for creating CNC dispersions that can be sprayed onto woody perennial crops, forming a thin insulating film around buds which has been shown to increase cold tolerance. Using digital scanning calorimetry (DSC) on dormant apple (Malus domestica Borkh.) reproductive buds, we investigated the thermodynamic properties of plant materials treated with CNC dispersion at lower temperatures. Scanning electron microscopy (SEM) was used to evaluate the thickness of the CNC films and their deposition on the sweet cherry bud surface. Apple buds treated with 3% CNC exhibited lethal freezing at temperatures 3.2°C and 5.5°C lower than the untreated control when sampled 1 and 3 days after application, respectively. Additionally, the latent heat capacity (J/g) of the 3% CNC-treated buds was 46% higher compared with untreated buds 1 day after application, and this difference increased 3 days after application to 168% higher. The emissivity of cherry buds treated with 3% CNC was reduced by an average of 16% compared with the untreated buds. SEM was able to detect the dried films on the surface of the buds 3 days after application. Film thickness measured with SEM increased with material concentration. The emissivity, HTE, and LTE results show that CNC-treated reproductive buds released thermal energy at a slower rate than the untreated buds and, consequently, exhibited internal ice nucleation events at temperatures as much as 5.5°C lower. The increased enthalpy during the LTE in the CNC-treated apple buds shows more energy released at lethal internal freezing, indicating that CNC coatings are increasing the amount of supercooled water. The effects of CNC shown during the DSC tests were increased by CNC concentration and time post-application. These results suggest that CNC dispersions dry into nanofilms on the bud surface, which affects their thermodynamic processes at low temperatures.

Screening of cold hardiness-related indexes and establishment of a comprehensive evaluation method for grapevines (V. vinifera).

The goals of this work were to screen physiological and biochemical indexes to assess a set of V. vinifera germplasm resources, to compare evaluation methods for cold hardiness, and to establish a comprehensive method that can be used for more accurate screening for cold hardiness in V. vinifera. Four single methods were used to evaluate the cold hardiness of 20 germplasms resources and 18 physiological and biochemical indexes related to cold hardiness were determined. The LT50 values determined by electrical conductivity (EL), 2,3,5-triphenyltetrazolium chloride staining (TTC), differential thermal analysis (DTA), and recovery growth (RG) methods showed extremely significant positive correlation. Bound water content (BW), proline content (Pro), total soluble sugar content (TSS), malondialdehyde content (MDA), catalase content (CAT), and ascorbic acid content (ASA) exhibited significant correlation with LT50 values measured by different evaluation methods. The comprehensive cold hardiness index calculated by principal component analysis (PCA) combined with subordinate function (SF) was negatively correlated with LT50 values measured by different evaluation methods. Meili and Ecolly exhibited the highest cold hardiness, indicating their potential for use as parents for cold hardiness breeding. EL, DTA, TTC, and RG methods successfully distinguished cold hardiness among different V. vinifera germplasm lines. Measurements of BW, Pro, TSS, MDA, CAT, and ASA in dormant shoots also can be used as main physiological and biochemical indexes related to cold hardiness of V. vinifera. Comprehensive evaluation by PCA combined with SF can accurately screen cold hardiness in V. vinifera. This study provides a reference and accurate identification method for the selection of cold hardiness parents and the evaluation of cold hardiness of germplasm of V. vinifera.

Pre-stress salicylic-acid treatment as an intervention strategy for freeze-protection in spinach: Foliar versus sub-irrigation application and duration of efficacy.

Exogenous application of salicylic acid (SA) to plant tissues has been shown to confer tolerance against various abiotic stresses. Recently, SA application through sub-irrigation was shown to improve plant freezing tolerance (FT). For SA treatment to be employable as an effective intervention strategy for frost protection under field conditions, it is important to study its effect on FT when applied as a foliar spray to whole plants. It is also important to determine for how long the FT-improvement by SA lasts. Present study was conducted to compare SA-induced FT of spinach (Spinacia oleracea L. 'Reflect') seedlings following SA-application by foliar spray vs. sub-irrigation. Durability of FT-promotive effect of SA was evaluated using three freeze-tests over a 4-d period, i.e., at 10-d, 12-d, and 14-d after the SA application. Freezing stress was applied using a temperature-controlled freeze-thaw protocol, and FT was assessed by visual observations (leaf flaccidness vs. turgidity) as well as ion-leakage assay. Data indicated that both foliar spray and sub-irrigation methods improved FT of the seedlings against a relatively moderate (-5.5 °C) as well as severe stress (-6.5 °C). Moreover, improved FT against moderate stress was sustained over a 4-d period, whereas such benefit waned somewhat against the severe stress. SA-treated leaves' growth performance was similar to the non-treated control based on dry weight, fresh weight, leaf area, and dry weight/leaf area parameters. Our results suggest that SA application as a foliar spray can potentially be used to protect field-grown transplants against episodic frosts.

Acquisition of Freezing Tolerance in Vaccinium macrocarpon Ait. Is a Multi-Factor Process Involving the Presence of an Ice Barrier at the Bud Base.

Bud freezing survival strategies have in common the presence of an ice barrier that impedes the propagation of lethally damaging ice from the stem into the internal structures of buds. Despite ice barriers' essential role in buds freezing stress survival, the nature of ice barriers in woody plants is not well understood. High-definition thermal recordings of Vaccinium macrocarpon Ait. buds explored the presence of an ice barrier at the bud base in September, January, and May. Light and confocal microscopy were used to evaluate the ice barrier region anatomy and cell wall composition related to their freezing tolerance. Buds had a temporal ice barrier at the bud base in September and January, although buds were only freezing tolerant in January. Lack of functionality of vascular tissues may contribute to the impedance of ice propagation. Pith tissue at the bud base had comparatively high levels of de-methyl-esterified homogalacturonan (HG), which may also block ice propagation. By May, the ice barrier was absent, xylogenesis had resumed, and de-methyl-esterified HG reached its lowest levels, translating into a loss of freezing tolerance. The structural components of the barrier had a constitutive nature, resulting in an asynchronous development of freezing tolerance between anatomical and metabolic adaptations.

Woody species do not differ in dormancy progression: Differences in time to budbreak due to forcing and cold hardiness.

Budbreak is one of the most observed and studied phenological phases in perennial plants, but predictions remain a challenge, largely due to our poor understanding of dormancy. Two dimensions of exposure to temperature are generally used to model budbreak: accumulation of time spent at low temperatures (chilling) and accumulation of heat units (forcing). These two effects have a well-established negative correlation; with more chilling, less forcing is required for budbreak. Furthermore, temperate plant species are assumed to vary in chilling requirements for dormancy completion allowing proper budbreak. Here, dormancy is investigated from the cold hardiness standpoint across many species, demonstrating that it should be accounted for to study dormancy and accurately predict budbreak. Most cold hardiness is lost prior to budbreak, but rates of cold hardiness loss (deacclimation) vary among species, leading to different times to budbreak. Within a species, deacclimation rate increases with accumulation of chill. When inherent differences between species in deacclimation rate are accounted for by normalizing rates throughout winter by the maximum rate observed, a standardized deacclimation potential is produced. Deacclimation potential is a quantitative measurement of dormancy progression based on responsiveness to forcing as chill accumulates, which increases similarly for all species, contradicting estimations of dormancy transition based on budbreak assays. This finding indicates that comparisons of physiologic and genetic control of dormancy require an understanding of cold hardiness dynamics. Thus, an updated framework for studying dormancy and its effects on spring phenology is suggested where cold hardiness in lieu of (or in addition to) budbreak is used.

Identification of Intrinsically Disordered Proteins and Regions in a Non-Model Insect Species Ostrinia nubilalis (Hbn.).

Research in previous decades has shown that intrinsically disordered proteins (IDPs) and regions in proteins (IDRs) are as ubiquitous as highly ordered proteins. Despite this, research on IDPs and IDRs still has many gaps left to fill. Here, we present an approach that combines wet lab methods with bioinformatics tools to identify and analyze intrinsically disordered proteins in a non-model insect species that is cold-hardy. Due to their known resilience to the effects of extreme temperatures, these proteins likely play important roles in this insect's adaptive mechanisms to sub-zero temperatures. The approach involves IDP enrichment by sample heating and double-digestion of proteins, followed by peptide and protein identification. Next, proteins are bioinformatically analyzed for disorder content, presence of long disordered regions, amino acid composition, and processes they are involved in. Finally, IDP detection is validated with an in-house 2D PAGE. In total, 608 unique proteins were identified, with 39 being mostly disordered, 100 partially disordered, 95 nearly ordered, and 374 ordered. One-third contain at least one long disordered segment. Functional information was available for only 90 proteins with intrinsic disorders out of 312 characterized proteins. Around half of the 90 proteins are cytoskeletal elements or involved in translational processes.

Characterization of Cold and Heat Tolerance of Bactrocera tau (Walker).

Bactrocera tau (Walker) (Diptera: Tephritidae) is a serious, economically important invasive pest that has spread and been established in many regions worldwide. Temperature is a crucial abiotic factor governing insect activity, fitness, and geographical distribution. Yet, surprisingly, the tolerance of B. tau to extreme cold and heat stress remains unclear. Here, we measured the supercooling point (SCP) of different life stages of B. tau. Further, several life stages of B. tau (egg, 1st, 2nd, and 3rd instar larvae, 1-day-old pupae, and 3-day-old adult) were subjected to six low temperatures (-9, -7, -5, -3, -1, and 0 °C) and six high temperatures (39, 40, 41, 42, 43, and 44 °C) for various durations (0.5, 1.0, 2.0, and 4.0 h), and three-way survival-time-temperature relationships were investigated. We found that the SCPs differed significantly among different life stages of B. tau, being the lowest for SCP of eggs, at -25.82 ± 0.51 °C. There was no significant effect of sex on the mean SCPs of B. tau adults, except for 45- to 50-day-old flies. In addition, an interaction effect was uncovered between tested temperatures and exposure duration upon B. tau mortality at different life stages. Eggs exhibited the strongest cold tolerance, yet the weakest heat tolerance. The 3rd instar larvae were the most heat- and cold tolerant among larval stages, followed by the 2nd and 1st instar larvae. The upper limit of the chill injury zone (ULCIZ) for 3-day-old adult and 1-day-old pupae was -2.51 °C and -2.50 °C, respectively, while their corresponding lower limit of thermal injury zone (LLTIZ) was 39.39 °C and 38.29 °C. This paper presents valuable data to provide an integrated knowledge for understanding the cold and heat tolerance potential of B. tau and ensure the proper implementation of post-harvest phytosanitary protocols for this pest's disinfestation.

Exploring Cold Hardiness within a Butterfly Clade: Supercooling Ability and Polyol Profiles in European Satyrinae.

The cold hardiness of overwintering stages affects the distribution of temperate and cold-zone insects. Studies on Erebia, a species-rich cold-zone butterfly genus, detected unexpected diversity of cold hardiness traits. We expanded our investigation to eight Satyrinae species of seven genera. We assessed Autumn and Winter supercooling points (SCPs) and concentrations of putatively cryoprotective sugars and polyols via gas chromatography-mass spectrometry. Aphantopus hyperantus and Hipparchia semele survived freezing of body fluids; Coenonympha arcania, C. gardetta, and Melanargia galathea died prior to freezing; Maniola jurtina, Chazara briseis, and Minois dryas displayed a mixed response. SCP varied from -22 to -9 °C among species. Total sugar and polyol concentrations (TSPC) varied sixfold (2 to 12 µg × mg-1) and eightfold including the Erebia spp. results. SCP and TSPC did not correlate. Alpine Erebia spp. contained high trehalose, threitol, and erythritol; C. briseis and C. gardetta contained high ribitol and trehalose; lowland species contained high saccharose, maltose, fructose, and sorbitol. SCP, TSPC, and glycerol concentrations were affected by phylogeny. Species of mountains or steppes tend to be freeze-avoidant, overwinter as young larvae, and contain high concentrations of trehalose, while those of mesic environments tend to be freeze-tolerant, overwinter as later instars, and rely on compounds such as maltose, saccharose, and fructose.

Metabolomic and transcriptomic responses of ticks during recovery from cold shock reveal mechanisms of survival.

Ticks are blood-feeding ectoparasites but spend most of their life off-host where they may have to tolerate low winter temperatures. Rapid cold hardening (RCH) is a process commonly used by arthropods, including ticks, to improve survival of acute low temperature exposure. However, little is known about the underlying mechanisms in ticks associated with RCH, cold shock and recovery from these stresses. In the present study, we investigated the extent to which RCH influences gene expression and metabolism during recovery from cold stress in Dermacentor variabilis, the American dog tick, using a combined transcriptomics and metabolomics approach. Following recovery from RCH, 1860 genes were differentially expressed in ticks, whereas only 99 genes responded during recovery to direct cold shock. Recovery from RCH resulted in an upregulation of various pathways associated with ion binding, transport, metabolism and cellular structures seen in the response of other arthropods to cold. The accumulation of various metabolites, including several amino acids and betaine, corresponded to transcriptional shifts in the pathways associated with these molecules, suggesting congruent metabolome and transcriptome changes. Ticks, D. variabilis and Amblyomma maculatum, receiving exogenous betaine and valine demonstrated enhanced cold tolerance, suggesting cryoprotective effects of these metabolites. Overall, many of the responses during recovery from cold shock in ticks were similar to those observed in other arthropods, but several adjustments may be distinct from the responses in other currently examined taxa.