In many species, polymorphic genomic inversions underlie complex phenotypic polymorphisms and facilitate local adaptation in the face of gene flow. Multiple polymorphic inversions can co-occur in a genome, but the prevalence, evolutionary significance, and limits to complexity of genomic inversion landscapes remain poorly understood. Here, we examine genome-wide genetic variation in one of Europe's most destructive forest pests, the spruce bark beetle Ips typographus, scan for polymorphic inversions, and test whether inversions are associated with key traits in this species. We analyzed 240 individuals from 18 populations across the species' European range and, using a whole-genome resequencing approach, identified 27 polymorphic inversions covering ∼28% of the genome. The inversions vary in size and in levels of intra-inversion recombination, are highly polymorphic across the species range, and often overlap, forming a complex genomic architecture. We found no support for mechanisms such as directional selection, overdominance, and associative overdominance that are often invoked to explain the presence of large inversion polymorphisms in the genome. This suggests that inversions are either neutral or maintained by the combined action of multiple evolutionary forces. We also found that inversions are enriched in odorant receptor genes encoding elements of recognition pathways for host plants, mates, and symbiotic fungi. Our results indicate that the genome of this major forest pest of growing social, political, and economic importance harbors one of the most complex inversion landscapes described to date and raise questions about the limits of intraspecific genomic architecture complexity.

• Keywords
• Ips typographus, forest pest, genome complexity, polymorphic inversions, spruce bark beetle,
• MeSH
• Coleoptera genetics   MeSH
• Chromosome Inversion * MeSH
• Genome, Insect * MeSH
• Forests MeSH
• Polymorphism, Genetic * MeSH
• Receptors, Odorant genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Europe MeSH
• Names of Substances
• Receptors, Odorant MeSH

Beetles are ubiquitous cave invertebrates worldwide that adapted to scarce subterranean resources when they colonized caves. Here, we investigated the potential role of gut microbiota in the adaptation of beetles to caves from different climatic regions of the Carpathians. The beetles' microbiota was host-specific, reflecting phylogenetic and nutritional adaptation. The microbial community structure further resolved conspecific beetles by caves suggesting microbiota-host coevolution and influences by local environmental factors. The detritivore species hosted a variety of bacteria known to decompose and ferment organic matter, suggesting turnover and host cooperative digestion of the sedimentary microbiota and allochthonous-derived nutrients. The cave Carabidae, with strong mandibula, adapted to predation and scavenging of animal and plant remains, had distinct microbiota dominated by symbiotic lineages Spiroplasma or Wolbachia. All beetles had relatively high levels of fermentative Carnobacterium and Vagococcus involved in lipid accumulation and a reduction of metabolic activity, and both features characterize adaptation to caves.

• Keywords
• Adaptation, Carpathians, Cave beetles, Gut microbiome, Leptodirini, Sediments, Trechinae,
• Publication type
• Journal Article MeSH

The accumulation of trehalose has been suggested as a mechanism underlying insect cross-tolerance to cold/freezing and drought. Here we show that exposing diapausing larvae of the drosophilid fly, Chymomyza costata to dry conditions significantly stimulates their freeze tolerance. It does not, however, improve their tolerance to desiccation, nor does it significantly affect trehalose concentrations. Next, we use metabolomics to compare the complex alterations to intermediary metabolism pathways in response to three environmental factors with different ecological meanings: environmental drought (an environmental stressor causing mortality), decreasing ambient temperatures (an acclimation stimulus for improvement of cold hardiness), and short days (an environmental signal inducing diapause). We show that all three factors trigger qualitatively similar metabolic rearrangement and a similar phenotypic outcome-improved larval freeze tolerance. The similarities in metabolic response include (but are not restricted to) the accumulation of typical compatible solutes and the accumulation of energy-rich molecules (phosphagens). Based on these results, we suggest that transition to metabolic suppression (a state in which chemical energy demand is relatively low but need for stabilization of macromolecules is high) represents a common axis of metabolic pathway reorganization towards accumulation of non-toxic cytoprotective compounds, which in turn stimulates larval freeze tolerance.

• Keywords
• cold, cross-tolerance, cytoprotectants, drought, freezing, metabolism,
• MeSH
• Acclimatization physiology   MeSH
• Drosophilidae * MeSH
• Insecta MeSH
• Larva physiology   MeSH
• Cold Temperature MeSH
• Droughts * MeSH
• Trehalose MeSH
• Freezing MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Trehalose MeSH

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.

• Keywords
• Lepidoptera: Nymphalidae, butterfly physiology, carbohydrate, cold hardiness, cryoprotectants, elevation, mountains, temperate zone, winter survival,
• Publication type
• Journal Article MeSH

Many cold-acclimated insects accumulate high concentrations of low molecular weight cryoprotectants (CPs) in order to tolerate low subzero temperatures or internal freezing. The sources from which carbon skeletons for CP biosynthesis are driven, and the metabolic reprogramming linked to cold acclimation, are not sufficiently understood. Here we aim to resolve the metabolism of putative CPs by mapping relative changes in concentration of 56 metabolites and expression of 95 relevant genes as larvae of the drosophilid fly, Chymomyza costata transition from a freeze sensitive to a freeze tolerant phenotype during gradual cold acclimation. We found that C. costata larvae may directly assimilate amino acids proline and glutamate from diet to acquire at least half of their large proline stocks (up to 55 µg per average 2 mg larva). Metabolic conversion of internal glutamine reserves that build up in early diapause may explain the second half of proline accumulation, while the metabolic conversion of ornithine and the degradation of larval collagens and other proteins might be two additional minor sources. Next, we confirm that glycogen reserves represent the major source of glucose units for trehalose synthesis and accumulation (up to 27 µg per larva), while the diet may serve as an additional source. Finally, we suggest that interconversions of phospholipids may release accumulated glycero-phosphocholine (GPC) and -ethanolamine (GPE). Choline is a source of accumulated methylamines: glycine-betaine and sarcosine. The sum of methylamines together with GPE and GPC represents approximately 2 µg per larva. In conclusion, we found that food ingestion may be an important source of carbon skeletons for direct assimilation of, and/or metabolic conversions to, CPs in a diapausing and cold-acclimated insect. So far, the cold-acclimation- linked accumulation of CPs in insects was considered to be sourced mainly from internal macromolecular reserves.

• Keywords
• betaine, cryoprotectant metabolites, metabolic pathways, metabolomics, proline, transcriptomics, trehalose,
• Publication type
• Journal Article MeSH

The European corn borer Ostrinia nubilalis is a pest species, whose fifth instar larvae gradually develop cold hardiness during diapause. The physiological changes underlying diapause progression and cold hardiness development are still insufficiently understood in insects. Here, we follow a complex of changes related to energy metabolism during cold acclimation (5°C) of diapausing larvae and compare this to warm-acclimated (22°C) and non-diapause controls. Capillary electrophoresis of nucleotides and coenzymes has shown that in gradually cold-acclimated groups concentrations of ATP/ADP and, consequently, energy charge slowly decrease during diapause, while the concentration of AMP increases, especially in the first months of diapause. Also, the activity of cytochrome c oxidase (COX), as well as the concentrations of NAD+ and GMP, decline in cold-acclimated groups, until the latter part of diapause, when they recover. Relative expression of NADH dehydrogenase (nd1), coenzyme Q-cytochrome c reductase (uqcr), COX, ATP synthase (atp), ADP/ATP translocase (ant), and prohibitin 2 (phb2) is supressed in cold-acclimated larvae during the first months of diapause and gradually increases toward the termination of diapause. Contrary to this, NADP+ and UMP levels significantly increased in the first few months of diapause, after gradual cold acclimation, which is in connection with the biosynthesis of cryoprotective molecules, as well as regeneration of small antioxidants. Our findings evidence the existence of a cold-induced energy-saving program that facilitates long-term maintenance of larval diapause, as well as gradual development of cold hardiness. In contrast, warm acclimation induced faster depletion of ATP, ADP, UMP, NAD+, and NADP+, as well as higher activity of COX and generally higher expression of all energy-related genes in comparison to cold-acclimated larvae. Moreover, such unusually high metabolic activity, driven by high temperatures, lead to premature mortality in the warm-acclimated group after 2 months of diapause. Thus, our findings strongly support the importance of low temperature exposure in early diapause for gradual cold hardiness acquisition, successful maintenance of the resting state and return to active development. Moreover, they demonstrate potentially adverse effects of global climate changes and subsequent increase in winter temperatures on cold-adapted terrestrial organisms in temperate and subpolar regions.

• Keywords
• ATP, COX, NAD+, NADP+, diapause, energy metabolism, gene expression,
• Publication type
• Journal Article MeSH

Many insects survive internal freezing, but the great complexity of freezing stress hinders progress in understanding the ultimate nature of freezing-induced injury. Here, we use larvae of the drosophilid fly, Chymomyza costata to assess the role of mitochondrial responses to freezing stress. Respiration analysis revealed that fat body mitochondria of the freeze-sensitive (non-diapause) phenotype significantly decrease oxygen consumption upon lethal freezing stress, while mitochondria of the freeze-tolerant (diapausing, cold-acclimated) phenotype do not lose respiratory capacity upon the same stress. Using transmission electron microscopy, we show that fat body and hindgut mitochondria swell, and occasionally burst, upon exposure of the freeze-sensitive phenotype to lethal freezing stress. By contrast, mitochondrial swelling is not observed in the freeze-tolerant phenotype exposed to the same stress. We hypothesize that mitochondrial swelling results from permeability transition of the inner mitochondrial membrane and loss of its barrier function, which causes osmotic influx of cytosolic water into the matrix. We therefore suggest that the phenotypic transition to diapause and cold acclimation could be associated with adaptive changes that include the protection of the inner mitochondrial membrane against permeability transition and subsequent mitochondrial swelling. Accumulation of high concentrations of proline and other cryoprotective substances might be a part of such adaptive changes as we have shown that freezing-induced mitochondrial swelling was abolished by feeding the freeze-sensitive phenotype larvae on a proline-augmented diet.

• Keywords
• freeze tolerance, insects, mitochondrial morphology,
• MeSH
• Acclimatization MeSH
• Drosophilidae MeSH
• Insecta physiology   MeSH
• Larva physiology   MeSH
• Mitochondria * MeSH
• Freezing * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Few invertebrates can survive cryopreservation in liquid nitrogen, and the mechanisms by which some species do survive are underexplored, despite high application potential. Here, we turn to the drosophilid Chymomyza costata to strengthen our fundamental understanding of extreme freeze tolerance and gain insights about potential avenues for cryopreservation of biological materials. We first use RNAseq to generate transcriptomes of three C. costata larval phenotypic variants: those warm-acclimated in early or late diapause (weak capacity to survive cryopreservation), and those undergoing cold acclimation after diapause entry (extremely freeze tolerant, surviving cryopreservation). We identify mRNA transcripts representing genes and processes that accompany the physiological transition to extreme freeze tolerance and relate cryopreservation survival to the transcriptional profiles of select candidate genes using extended sampling of phenotypic variants. Enhanced capacity for protein folding, refolding and processing appears to be a central theme of extreme freeze tolerance and may allow cold-acclimated larvae to repair or eliminate proteins damaged by freezing (thus mitigating the toxicity of denatured proteins, endoplasmic reticulum stress and subsequent apoptosis). We also find a number of candidate genes (including both known and potentially novel, unannotated sequences) whose expression profiles tightly mirror the change in extreme freeze tolerance status among phenotypic variants.

• Keywords
• cold acclimation, cryopreservation, cryoprotectant, insect, transcriptome,
• MeSH
• Acclimatization genetics   MeSH
• Drosophilidae genetics   MeSH
• Insecta genetics   MeSH
• Transcriptome MeSH
• Freezing * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Physiological adjustments accompanying insect cold acclimation prior to cold stress have been relatively well explored. In contrast, recovery from cold stress received much less attention. Here we report on recovery of drosophilid fly larvae (Chymomyza costata) from three different levels of cold stress: supercooling to -10 °C, freezing at -30 °C, and cryopreservation at -196 °C. Analysis of larval CO2 production suggested that recovery from all three cold stresses requires access to additional energy reserves to support cold-injury repair processes. Metabolomic profiling (targeting 41 metabolites using mass spectrometry) and custom microarray analysis (targeting 1,124 candidate mRNA sequences) indicated that additional energy was needed to: clear by-products of anaerobic metabolism, deal with oxidative stress, re-fold partially denatured proteins, and remove damaged proteins, complexes and/or organelles. Metabolomic and transcriptomic recovery profiles were closely similar in supercooled and frozen larvae, most of which successfully repaired the cold injury and metamorphosed into adults. In contrast, the majority of cryopreseved larvae failed to proceed in ontogenesis, showed specific metabolic perturbations suggesting impaired mitochondrial function, and failed to up-regulate a set of 116 specific genes potentially linked to repair of cold injury.

• MeSH
• Drosophilidae * genetics   metabolism   MeSH
• Stress, Physiological * MeSH
• Cryopreservation * methods   MeSH
• Larva MeSH
• Metabolomics methods   MeSH
• Preservation, Biological MeSH
• Cold-Shock Response MeSH
• Gene Expression Profiling MeSH
• Freezing * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH