12 research outputs found
Mechanisms Underlying Variation in Insect Chill Tolerance: The Role of Ion and Water Transport
Water and ion homeostasis has emerged as an important factor limiting chill-susceptible insects at low temperatures; loss of this homeostasis in the cold likely contributes to chronic chilling injury, and reestablishment of homeostasis is required for recovery from chilling. Both plastic and interspecific variation in cold tolerance correlates with enhanced defense of water and ion homeostasis during cold exposure, however the mechanisms are poorly understood. Using Gryllus crickets, I generated and tested hypotheses about the mechanisms underlying this variation in transport function. I first related interspecific variation in cold tolerance to water and ion balance in early chill coma. A rapid influx of Na+ to the hemolymph suggests that Na+ first leaks from the tissues, and could drive migration of Na+ and water to the gut. Gryllus veletis (a more cold-tolerant species) may avoid or slow this Na+ leak by maintaining lower hemolymph Na+ content and lower osmotic pressure between the gut and hemolymph, compared to G. pennsylvanicus. Plasticity in defense of water and ion homeostasis during cold exposure is thought to involve enhanced active transport function and/or decreased permeability of ionoregulatory tissues. Using G. pennsylvanicus I identified specific candidate mechanisms related to these transport function modifications by comparing the hindgut and Malpighian tubule transcriptomes of warm- and cold-acclimated individuals. Cold acclimation modified the expression of hindgut and Malpighian tubule ion transporters, and hindgut structural (cytoskeletal and cell junction) genes. Rectal macromorphology and rectal pad scalariform complex ultrastructure were unchanged (suggesting that modified permeability does not involve these structural elements), however cytoskeletal modifications do protect rectal pad actin stability during cold shock. Cold acclimation decreases excretion rate (i.e. active transport) across the Malpighian tubules, which may be driven by modified activity of Na+-K+ ATPase but not of V-ATPase. Increased expression of hindgut Na+-K+ ATPase did not alter the activity of this enzyme in the rectum. Overall I show that cold acclimation modifies active transport function in the Malpighian tubules and modifies rectal pad structure to enhance cytoskeletal stability during cold exposure
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
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
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 more than 3,000 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
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
Dissection of Anoplophora glabripennis (Coleoptera: Cerambycidae) larval tissues for physiological and molecular studies
Many biological processes are partitioned among organs and tissues, necessitating tissue-specific or organ-specific analysis (particularly for comparative -omics studies). Standardised techniques for tissue identification and dissection are therefore imperative for comparing among studies. Here we describe dissection protocols for isolating six key tissues/organs from larvae of the Asian longhorned beetle, Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae): the supraoesophageal ganglion, posterior midgut, hindgut, Malpighian tubules, fat body, and thoracic muscle. We also describe how to extract haemolymph and preserve whole larvae for measurements such as protein, lipid, and carbohydrate content. We include dissection protocols for both fresh-killed and previously frozen specimens. Although this protocol is developed for A. glabripennis, it should allow standardised tissue collection from larvae of other cerambycids and be readily transferrable to other beetle taxa with similar larval morphology