Terrestrial Isopods Porcellio scaber and Oniscus asellus (Crustacea: Isopoda) Increase Bacterial Abundance and Modify Microbial Community Structure in Leaf Litter Microcosms: a Short-Term Decomposition Study.

Invasive terrestrial isopods are likely to have altered leaf litter decomposition processes in North American forests, but the mechanisms underlying these alterations and the degree to which they differ among isopod species are poorly characterized. Using mixed-deciduous leaf litter microcosms, we quantified the effects of two common, invasive isopods (Oniscus asellus and Porcellio scaber) on short-term leaf litter decomposition and microbial community structure and function. Microcosms containing ground litter and a microbial inoculant were exposed to one of the two isopod species or no isopods for 21 days. Mass loss was then quantified as the change in litter dry mass after leaching, and microbial respiration was quantified as the mass of CO2 absorbed by soda lime. Litter leachates were plated on agar to quantify culturable bacterial and fungal abundance, and denaturing gradient gel electrophoresis of amplified leachate microbial DNA was used to characterize shifts in microbial community structure. Isopod presence increased litter mass loss by a modest ~ 6%, but did not affect litter microbial respiration. Bacterial abundance increased significantly in the presence of isopods, while fungal abundance was either unchanged or reduced. Overall litter microbial species richness was reduced by isopods, with O. asellus specifically reducing fungal abundance and diversity. Isopods modified the microbial community structure by suppressing four bacterial and one fungal species, while promoting growth of four other bacterial species (two unique to each isopod species) and two fungal species (one which was unique to O. asellus).

Reversing sodium differentials between the hemolymph and hindgut speeds chill coma recovery but reduces survival in the fall field cricket, Gryllus pennsylvanicus.

Chill-susceptible insects enter the reversible state of chill coma at their critical thermal minimum (CTmin). During chill coma, movement of Na+ and water from the hemolymph to the gut lumen disrupt ion and water balance. Recovery from cold exposure requires re-establishment of this balance, and failure to do so results in chilling injury or death. We hypothesized that the passive leak of Na+ and consequently water during cold exposure is driven by the [Na+] differential between the gut and hemolymph. To determine the extent to which this [Na+] differential affects cold tolerance, we used artificial diets to load the guts of fall field crickets (Gryllus pennsylvanicus) with various concentrations of Na+. Manipulating [Na+] differentials had no effect on the CTmin, agreeing with recent studies demonstrating that chill coma onset precedes loss of ion balance in the cold. A high [Na+] diet reversed the direction of the [Na+] differential between the gut and hemolymph. Crickets fed a high [Na+] diet recovered from 12 h of chill coma nearly twice as fast as those fed low [Na+] diets. However, the high [Na+] diet was detrimental to survival after prolonged cold exposure (three days at 0 °C). Therefore, while a reduced [Na+] differential helps crickets recover from short-term cold exposure, an increased gut Na+ load itself appears to carry longer-term costs and promotes irreversible chilling injury.

Transcriptional analysis of insect extreme freeze tolerance.

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.

How crickets become freeze tolerant: The transcriptomic underpinnings of acclimation in Gryllus veletis.

Some ectotherms can survive internal ice formation. In temperate regions, freeze tolerance is often induced by decreasing temperature and/or photoperiod during autumn. However, we have limited understanding of how seasonal changes in physiology contribute to freeze tolerance, and how these changes are regulated. During a six week autumn-like acclimation, late-instar juveniles of the spring field cricket Gryllus veletis (Orthoptera: Gryllidae) become freeze tolerant, which is correlated with accumulation of low molecular weight cryoprotectants, elevation of the temperature at which freezing begins, and metabolic rate suppression. We used RNA-Seq to assemble a de novo transcriptome of this emerging laboratory model for freeze tolerance research. We then focused on gene expression during acclimation in fat body tissue due to its role in cryoprotectant production and regulation of energetics. Acclimated G. veletis differentially expressed >3000 transcripts in fat body. This differential expression may contribute to metabolic suppression in acclimated G. veletis, but we did not detect changes in expression that would support cryoprotectant accumulation or enhanced control of ice formation, suggesting that these latter processes are regulated post-transcriptionally. Acclimated G. veletis differentially regulated transcripts that likely coordinate additional freeze tolerance mechanisms, including upregulation of enzymes that may promote membrane and cytoskeletal remodelling, cryoprotectant transporters, cytoprotective proteins, and antioxidants. Thus, while accumulation of cryoprotectants and controlling ice formation are commonly associated with insect freeze tolerance, our results support the hypothesis that many other systems contribute to surviving internal ice formation. Together, this information suggests new avenues for understanding the mechanisms underlying insect freeze tolerance.

Insect fat body cell morphology and response to cold stress is modulated by acclimation.

Mechanistic understanding about the nature of cellular cryoinjury and mechanisms by which some animals survive freezing while others do not is currently lacking. Here, we exploited the broadly manipulable freeze tolerance of larval malt flies (Chymomyza costata) to uncover cell and tissue morphological changes associated with freeze mortality. Diapause induction, cold acclimation and dietary proline supplementation generate malt fly variants ranging from weakly to extremely freeze tolerant. Using confocal microscopy and immunostaining of the fat body, Malpighian tubules and anterior midgut, we described tissue and cytoskeletal (F-actin and α-tubulin) morphologies among these variants after exposure to various cold stresses (from chilling at -5°C to extreme freezing at -196°C), and upon recovery from cold exposure. Fat body tissue appeared to be the most susceptible to cryoinjury: freezing caused coalescence of lipid droplets, loss of α-tubulin structure and apparent aggregation of F-actin. A combination of diapause and cold acclimation substantially lowered the temperature at which these morphological disruptions occurred. Larvae that recovered from a freezing challenge repaired F-actin aggregation but not lipid droplet coalescence or α-tubulin structure. Our observations indicate that lipid coalescence and damage to α-tubulin are non-lethal forms of freeze injury, and suggest that repair or removal (rather than protection) of actin proteins is a potential mechanism of acquired freeze tolerance.

Effects of cold acclimation on rectal macromorphology, ultrastructure, and cytoskeletal stability in Gryllus pennsylvanicus crickets.

Cold-acclimated insects maintain ion and water balance in the cold, potentially by reducing permeability or increasing diffusion distance across ionoregulatory epithelia such as the rectum. We explored whether cold acclimation induces structural modifications that minimize water and ion diffusion across the rectum and maintain rectal cell integrity. We investigated rectal structure and cytoskeletal stability in chill-susceptible adult Gryllus pennsylvanicus crickets acclimated for one week to either warm (25 °C) or cold (12 °C) conditions. After acclimation, we used light and transmission electron microscopy to examine rectal macromorphology and rectal pad paracellular ultrastructure. We also used fluorescence microscopy and a filamentous-actin (F-actin) specific phalloidin stain to compare the polymerization state of the actin cytoskeleton for each of the acclimation groups before and after a cold shock (1 h at -4 °C). Cold acclimation did not alter rectal pad cell density, or the thickness of the rectal pads, muscle, or cuticle. The tortuosity and width of the rectal pad paracellular channels also did not differ between warm- and cold-acclimated crickets. Rectal pad cells had clear basal and apical regions with differing densities of F-actin. Cold shock reduced the density of F-actin in warm-acclimated crickets, whereas cold-acclimated crickets appeared to have unchanged (basal) or enhanced (apical) F-actin density after cold shock. This suggests that while cold acclimation does not modify rectal permeability through structural modifications to increase diffusion distance for water and ions, cold-acclimated crickets have a modified cytoskeleton that resists the depolymerising effects of cold shock.

The effect of cold acclimation on active ion transport in cricket ionoregulatory tissues.

Cold-acclimated insects defend ion and water transport function during cold exposure. We hypothesized that this is achieved via enhanced active transport. The Malpighian tubules and rectum are likely targets for such transport modifications, and recent transcriptomic studies indicate shifts in Na+-K+ ATPase (NKA) and V-ATPase expression in these tissues following cold acclimation. Here we quantify the effect of cold acclimation (one week at 12°C) on active transport in the ionoregulatory organs of adult Gryllus pennsylvanicus field crickets. We compared primary urine production of warm- and cold-acclimated crickets in excised Malpighian tubules via Ramsay assay at a range of temperatures between 4 and 25°C. We then compared NKA and V-ATPase activities in Malpighian tubule and rectal homogenates from warm- and cold-acclimated crickets via NADH-linked photometric assays. Malpighian tubules of cold-acclimated crickets excreted fluid at lower rates at all temperatures compared to warm-acclimated crickets. This reduction in Malpighian tubule excretion rates may be attributed to increased NKA activity that we observed for cold-acclimated crickets, but V-ATPase activity was unchanged. Cold acclimation had no effect on rectal NKA activity at either 21°C or 6°C, and did not modify rectal V-ATPase activity. Our results suggest that an overall reduction, rather than enhancement of active transport in the Malpighian tubules allows crickets to maintain hemolymph water balance during cold exposure, and increased Malpighian tubule NKA activity may help to defend and/or re-establish ion homeostasis.

Effects of cold-acclimation on gene expression in Fall field cricket (Gryllus pennsylvanicus) ionoregulatory tissues.

BACKGROUND: Cold tolerance is a key determinant of temperate insect distribution and performance. Chill-susceptible insects lose ion and water homeostasis during cold exposure, but prior cold acclimation improves both cold tolerance and defense of homeostasis. The mechanisms underlying these processes are mostly unknown; cold acclimation is thought to enhance ion transport in the cold and/or prevent leak of water and ions. To identify candidate mechanisms of cold tolerance plasticity we generated transcriptomes of ionoregulatory tissues (hindgut and Malpighian tubules) from Gryllus pennsylvanicus crickets and compared gene expression in warm- and cold-acclimated individuals. RESULTS: We assembled a G. pennsylvanicus transcriptome de novo from 286 million 50-bp reads, yielding 70,037 contigs (~44% of which had putative BLAST identities). We compared the transcriptomes of warm- and cold-acclimated hindguts and Malpighian tubules. Cold acclimation led to a ≥ 2-fold change in the expression of 1493 hindgut genes (733 downregulated, 760 upregulated) and 2008 Malpighian tubule genes (1009 downregulated, 999 upregulated). Cold-acclimated crickets had altered expression of genes putatively associated with ion and water balance, including: a downregulation of V-ATPase and carbonic anhydrase in the Malpighian tubules and an upregulation of Na+-K+ ATPase in the hindgut. We also observed acclimation-related shifts in the expression of cytoskeletal genes in the hindgut, including actin and actin-anchoring/stabilizing proteins, tubulin, α-actinin, and genes involved in adherens junctions organization. In both tissues, cold acclimation led to differential expression of genes encoding cytochrome P450s, glutathione-S-transferases, apoptosis factors, DNA repair, and heat shock proteins. CONCLUSIONS: This is the first G. pennsylvanicus transcriptome, and our tissue-specific approach yielded new candidate mechanisms of cold tolerance plasticity. Cold acclimation may reduce loss of hemolymph volume in the cold by 1) decreasing primary urine production via reduced expression of carbonic anhydrase and V-ATPase in the Malpighian tubules and 2) by increasing Na+ (and therefore water) reabsorption across the hindgut via increase in Na+-K+ ATPase expression. Cold acclimation may reduce chilling injury by remodeling and stabilizing the hindgut epithelial cytoskeleton and cell-to-cell junctions, and by increasing the expression of genes involved in DNA repair, detoxification, and protein chaperones.

Ion and water balance in Gryllus crickets during the first twelve hours of cold exposure.

Insects lose ion and water balance during chilling, but the mechanisms underlying this phenomenon are based on patterns of ion and water balance observed in the later stages of cold exposure (12 or more hours). Here we quantified the distribution of ions and water in the hemolymph, muscle, and gut in adult Gryllus field crickets during the first 12h of cold exposure to test mechanistic hypotheses about why homeostasis is lost in the cold, and how chill-tolerant insects might maintain homeostasis to lower temperatures. Unlike in later chill coma, hemolymph [Na(+)] and Na(+) content in the first few hours of chilling actually increased. Patterns of Na(+) balance suggest that Na(+) migrates from the tissues to the gut lumen via the hemolymph. Imbalance of [K(+)] progressed gradually over 12h and could not explain chill coma onset (a finding consistent with recent studies), nor did it predict survival or injury following 48h of chilling. Gryllus veletis avoided shifts in muscle and hemolymph ion content better than Gryllus pennsylvanicus (which is less chill-tolerant), however neither species defended water, [Na(+)], or [K(+)] balance during the first 12h of chilling. Gryllus veletis better maintained balance of Na(+) content and may therefore have greater tissue resistance to ion leak during cold exposure, which could partially explain faster chill coma recovery for that species.