Reversible mitophagy drives metabolic suppression in diapausing beetles.

Many insects enter a state of dormancy (diapause) during winter in which they lower their metabolism to save energy. Metabolic suppression is a hallmark of diapause, yet we know little about the mechanisms underpinning metabolic suppression in winter or how it is reversed in the spring. Here, we show that metabolic suppression in dormant Colorado potato beetles results from the breakdown of flight muscle mitochondria via mitophagy. Diapausing Colorado potato beetles suppress their metabolism by 90%, and this lowered metabolic rate coincides with a similar reduction in flight muscle mitochondrial function and density. During early diapause, beetles increase the expression of mitophagy-related transcripts (Parkin and ATG5) in their flight muscle coincident with an increase in mitophagy-related structures in the flight muscle. Knocking down Parkin expression with RNA interference in diapausing beetles prevented some mitochondrial breakdown and partially restored the whole animal metabolic rate, suggesting that metabolic suppression in diapausing beetles is driven by mitophagy. In other animals and in models of disease, such large-scale mitochondrial degradation is irreversible. However, we show that as diapause ends, beetles reverse mitophagy and increase the expression of PGC1α and NRF1 to replenish flight muscle mitochondrial pools. This mitochondrial biogenesis is activated in anticipation of diapause termination and in the absence of external stimuli. Our study provides a mechanistic link between mitochondrial degradation in insect tissues over the winter and whole-animal metabolic suppression.

Metabolic cost of freeze-thaw and source of CO2 production in the freeze-tolerant cricket Gryllus veletis.

Freeze-tolerant insects can survive the conversion of a substantial portion of their body water to ice. While the process of freezing induces active responses from some organisms, these responses appear absent from freeze-tolerant insects. Recovery from freezing likely requires energy expenditure to repair tissues and re-establish homeostasis, which should be evident as elevations in metabolic rate after thaw. We measured carbon dioxide (CO2) production in the spring field cricket (Gryllus veletis) as a proxy for metabolic rate during cooling, freezing and thawing and compared the metabolic costs associated with recovery from freezing and chilling. We hypothesized that freezing does not induce active responses, but that recovery from freeze-thaw is metabolically costly. We observed a burst of CO2 release at the onset of freezing in all crickets that froze, including those killed by either cyanide or an insecticide (thiacloprid), implying that the source of this CO2 was neither aerobic metabolism nor a coordinated nervous system response. These results suggest that freezing does not induce active responses from G. veletis, but may liberate buffered CO2 from hemolymph. There was a transient 'overshoot' in CO2 release during the first hour of recovery, and elevated metabolic rate at 24, 48 and 72 h, in crickets that had been frozen compared with crickets that had been chilled (but not frozen). Thus, recovery from freeze-thaw and the repair of freeze-induced damage appears metabolically costly in G. veletis, and this cost persists for several days after thawing.

Predator exposure-induced immunosuppression: trade-off, immune redistribution or immune reconfiguration?

Although predator exposure increases the risk of wound infections, it typically induces immunosuppression. A number of non-mutually exclusive hypotheses have been put forward to explain this immunosuppression, including: trade-offs between the immune system and other systems required for anti-predator behaviour, redistribution of immune resources towards mechanisms needed to defend against wound infections, and reconfiguration of the immune system to optimize defence under the physiological state of fight-or-flight readiness. We tested the ability of each hypothesis to explain the effects of chronic predator stress on the immune system of the caterpillar Manduca sexta Predator exposure induced defensive behaviours, reduced mass gain, increased development time and increased the concentration of the stress neurohormone octopamine. It had no significant effect on haemocyte number, melanization rate, phenoloxidase activity, lysozyme-like activity or nodule production. Predator stress reduced haemolymph glutathione concentrations. It also increased constitutive expression of the antimicrobial peptide attacin-1 but reduced attacin-1 expression in response to an immune challenge. These results best fit the immune reconfiguration hypothesis, although the other hypotheses are also consistent with some results. Interpreting stress-related changes in immune function may require an examination at the level of the whole organism.

Reconfiguration of the immune system network during food limitation in the caterpillar Manduca sexta.

Dwindling resources might be expected to induce a gradual decline in immune function. However, food limitation has complex and seemingly paradoxical effects on the immune system. Examining these changes from an immune system network perspective may help illuminate the purpose of these fluctuations. We found that food limitation lowered long-term (i.e. lipid) and short-term (i.e. sugars) energy stores in the caterpillar Manduca sexta. Food limitation also: altered immune gene expression, changed the activity of key immune enzymes, depressed the concentration of a major antioxidant (glutathione), reduced resistance to oxidative stress, reduced resistance to bacteria (Gram-positive and -negative bacteria) but appeared to have less effect on resistance to a fungus. These results provide evidence that food limitation led to a restructuring of the immune system network. In severely food-limited caterpillars, some immune functions were enhanced. As resources dwindled within the caterpillar, the immune response shifted its emphasis away from inducible immune defenses (i.e. those responses that are activated during an immune challenge) and increased emphasis on constitutive defenses (i.e. immune components that are produced consistently). We also found changes suggesting that the activation threshold for some immune responses (e.g. phenoloxidase) was lowered. Changes in the configuration of the immune system network will lead to different immunological strengths and vulnerabilities for the organism.

The parasitic wasp Cotesia congregata uses multiple mechanisms to control host (Manduca sexta) behaviour.

Some parasites alter the behaviour of their hosts. The larvae of the parasitic wasp Cotesia congregata develop within the body of the caterpillar Manduca sexta During the initial phase of wasp development, the host's behaviour remains unchanged. However, once the wasps begin to scrape their way out of the caterpillar, the caterpillar host stops feeding and moving spontaneously. We found that the caterpillar also temporarily lost sensation around the exit hole created by each emerging wasp. However, the caterpillars regained responsiveness to nociception in those areas within 1 day. The temporary reduction in skin sensitivity is probably important for wasp survival because it prevents the caterpillar from attacking the emerging wasp larvae with a defensive strike. We also found that expression of plasmatocyte spreading peptide (PSP) and spätzle genes increased in the fat body of the host during wasp emergence. This result supports the hypothesis that the exiting wasps induce a cytokine storm in their host. Injections of PSP suppressed feeding, suggesting that an augmented immune response may play a role in the suppression of host feeding. Injection of wasp larvae culture media into non-parasitized caterpillars reduced feeding, suggesting that substances secreted by the wasp larvae may help alter host behaviour.

Cold tolerance, water balance, energetics, gas exchange, and diapause in overwintering brown marmorated stink bugs.

Halyomorpha halys (Hemiptera: Pentatomidae) is an emerging pest which established in Ontario, Canada, in 2012. Halyomporpha halys overwinters in anthropogenic structures as an adult. We investigated seasonal variation in the cold tolerance, water balance, and energetics of H. halys in southwestern Ontario. We also induced diapause in laboratory-reared animals with short daylength at permissive temperatures and compared cold tolerance, water balance, energetics, and metabolism and gas exchange between diapausing and non-diapausing individuals. Halyomorpha halys that overwintered outside in Ontario all died, but most of those that overwintered in sheltered habitats survived. We confirm that overwintering H. halys are chill-susceptible. Over winter, Ontario H. halys depressed their supercooling point to c. -15.4 °C, and 50% survived a 1 h exposure to -17.5 °C. They reduce water loss rates over winter, and do not appear to significantly consume lipid or carbohydrate reserves to a level that might cause starvation. Overall, it appears that H. halys is dependent on built structures and other buffered microhabitats to successfully overwinter in Ontario. Laboratory-reared diapausing H. halys have lower supercooling points than their non-diapausing counterparts, but LT50 is not enhanced by diapause induction. Diapausing H. halys survive desiccating conditions for 3-4 times longer than those not in diapause, through decreases in both respiratory and cuticular water loss. Diapausing H. halys do not appear to accumulate any more lipid or carbohydrate than those not in diapause, but do have lower metabolic rates, and are slightly more likely to exhibit discontinuous gas exchange.

Laboratory acclimation to autumn-like conditions induces freeze tolerance in the spring field cricket Gryllus veletis (Orthoptera: Gryllidae).

Many temperate insects encounter temperatures low enough to freeze their body fluids. Remarkably, some insects are freeze-tolerant, surviving this internal ice formation. However, the mechanisms underlying freeze tolerance are not well-understood, in part due to a lack of tractable model organisms. We describe a novel laboratory model to study insect freeze tolerance, the spring field cricket Gryllus veletis (Orthopera: Gryllidae). Following acclimation to six weeks of decreasing temperature and photoperiod, G. veletis become freeze-tolerant, similar to those exposed to natural autumn conditions in London, Ontario, Canada. Acclimated crickets suppress their metabolic rate by c. 33%, and survive freezing for up to one week at -8 °C, and to temperatures as low as -12 °C. Freeze-tolerant G. veletis protect fat body cells from freeze injury in vivo, and fat body tissue from freeze-tolerant cricket survives brief freeze treatments when frozen ex vivo. Freeze-tolerant crickets freeze at c. -6 °C, which may be initiated by accumulation of ice-nucleating agents in hemolymph or gut tissue. Although we hypothesize that control of ice formation facilitates freeze tolerance, initiating ice formation at high subzero temperatures does not confer freeze tolerance on freeze-intolerant nymphs. Acclimation increases hemolymph osmolality from c. 400 to c. 650 mOsm, which may facilitate freeze tolerance by reducing ice content. Hemolymph ion concentrations do not change with acclimation, and we therefore predict that freeze-tolerant G. veletis elevate hemolymph osmolality by accumulating other molecules. Gryllus veletis is easily reared and manipulated in a controlled laboratory environment, and is therefore a suitable candidate for further investigating the mechanisms underlying freeze tolerance.