Insect Flight: State of the Field and Future Directions.

The evolution of flight in an early winged insect ancestral lineage is recognized as a key adaptation explaining the unparalleled success and diversification of insects. Subsequent transitions and modifications to flight machinery, including secondary reductions and losses, also play a central role in shaping the impacts of insects on broadscale geographic and ecological processes and patterns in the present and future. Given the importance of insect flight, there has been a centuries-long history of research and debate on the evolutionary origins and biological mechanisms of flight. Here, we revisit this history from an interdisciplinary perspective, discussing recent discoveries regarding the developmental origins, physiology, biomechanics, and neurobiology and sensory control of flight in a diverse set of insect models. We also identify major outstanding questions yet to be addressed and provide recommendations for overcoming current methodological challenges faced when studying insect flight, which will allow the field to continue to move forward in new and exciting directions. By integrating mechanistic work into ecological and evolutionary contexts, we hope that this synthesis promotes and stimulates new interdisciplinary research efforts necessary to close the many existing gaps about the causes and consequences of insect flight evolution.

Knock down to level up: Reframing RNAi for invertebrate ecophysiology.

Comparative ecophysiologists strive to understand physiological problems in non-model organisms, but molecular tools such as RNA interference (RNAi) are under-used in our field. Here, we provide a framework for invertebrate ecophysiologists to use RNAi to answer questions focused on physiological processes, rather than as a tool to investigate gene function. We specifically focus on non-model invertebrates, in which the use of other genetic tools (e.g., genetic knockout lines) is less likely. We argue that because RNAi elicits a temporary manipulation of gene expression, and resources to carry out RNAi are technically and financially accessible, it is an effective tool for invertebrate ecophysiologists. We cover the terminology and basic mechanisms of RNA interference as an accessible introduction for "non-molecular" physiologists, include a suggested workflow for identifying RNAi gene targets and validating biologically relevant gene knockdowns, and present a hypothesis-testing framework for using RNAi to answer common questions in the realm of invertebrate ecophysiology. This review encourages invertebrate ecophysiologists to use these tools and workflows to explore physiological processes and bridge genotypes to phenotypes in their animal(s) of interest.

Beclin-mediated Autophagy Drives Dorsal Longitudinal Flight Muscle Histolysis in the Variable Field Cricket, Gryllus lineaticeps.

Flight muscle histolysis is a widespread strategy used by insects to break down functional flight muscle and modulate the energetic costs associated with flight muscle use and maintenance. The variable field cricket, Gryllus lineaticeps, undergoes histolysis during their transition between dispersal flight and reproduction. Despite the importance of histolysis on insect reproduction and fitness, the molecular mechanisms driving this flight muscle breakdown are not well understood. Here, we show that beclin-mediated autophagy, a conserved lysosomal-dependent degradation process, drives breakdown of dorsal longitudinal flight muscle in female flight capable G. lineaticeps. We found that female G. lineaticeps activate autophagy in their dorsal longitudinal flight muscle (DLM), but to a greater extent than the neighboring dorsoventral flight muscle (DVM) during histolysis. RNA interference knockdown of beclin, a gene which encodes a critical autophagy initiation protein, delayed DLM histolysis, but did not affect DVM histolysis. This suggests that crickets selectively activate autophagy to break down the DLMs, while maintaining DVM function for other fitness-relevant activities such as walking. Overall, we confirmed that autophagy is a critical pathway used to remodel flight muscle cells during flight muscle histolysis, providing novel insights into the mechanisms underlying a major life history transition between dispersal and reproduction.

Chilled, starved or frozen: insect mitochondrial adaptations to overcome the cold.

Physiological adaptations to tackle cold exposure are crucial for insects living in temperate and arctic environments and here we review how cold adaptation is manifested in terms of mitochondrial function. Cold challenges are diverse, and different insect species have evolved metabolic and mitochondrial adaptations to i) energize homeostatic regulation at low temperatures ii) stretch energy reserves during prolonged cold exposure, and iii) preserve the structural organization of organelles following extracellular freezing. While the literature is still sparse, our review suggests that cold-adapted insects preserve ATP production at low temperatures by maintaining preferred mitochondrial substrate oxidation, which is otherwise challenged in cold-sensitive species. Chronic cold exposure and metabolic depression during dormancy are linked to reduced mitochondrial metabolism and may involve mitochondrial degradation. Finally, adaptation to extracellular freezing could be associated with the superior structural integrity of the mitochondrial inner membrane following freezing which is linked to cellular and organismal survival.

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.

Diapause differentially modulates the transcriptomes of fat body and flight muscle in the Colorado potato beetle.

Many temperate insects, such as the Colorado potato beetle, enter diapause in winter, during which they arrest their development, suppress their metabolic rate and have high stress tolerance. Diapause phenotypes can be transcriptionally regulated, however many studies to date report only whole animal gene expression rather than tissue-specific processes during diapause. We used RNA-seq to measure gene expression in fat body and flight muscle of diapausing and non-diapausing beetles. We used differential expression and GO enrichment analyses to evaluate longstanding hypotheses about the mechanisms that drive arrested development, changes in energy metabolism, and increased stress tolerance during diapause. We found evidence of G2/M cell cycle arrest, juvenile hormone catabolism, increased antioxidant metabolism, epigenetic modification, transposable element regulation, and cytoskeletal remodeling in both the fat body and flight muscle of diapausing beetles. Beetles differentially modulated the fat body and flight muscle transcriptomes during diapause with fat body playing a larger role in the hypoxia response and immunity, whereas flight muscle had higher abundance of transcripts related to the chaperone response and proteostasis. Our transcriptome provides evidence for distinct roles and responses of fat body and flight muscle during diapause in the Colorado potato beetle, and we provide testable hypotheses for biological processes that appear to drive diapause phenotypes in insects.

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.

Loss of ion homeostasis is not the cause of chill coma or impaired dispersal in false codling moth Thaumatotibia leucotreta (Lepidoptera: Tortricidae).

Dispersal is a central requirement of a successful sterile insect release programme, but field-released false codling moth (FCM) typically suffer from poor dispersal ability, especially at low ambient temperatures. Here we test the hypothesis that poor activity and dispersal in FCM is caused by delayed or perturbed recovery of ion and/or water homeostasis after chilling for handling and transport prior to field release. Hemolymph and flight muscle were collected from two treatment groups at three time points that targeted thermal conditions above and below the chill coma induction threshold of ~ 6 °C: 1) control moths kept at 25 °C, 2) moths exposed to 3 °C or 9 °C for 4 h, and 3) moths allowed to recover at 25 °C for 24 h after exposure to either 3 °C or 9 °C. We measured concentrations of Na+, K+ and Mg2+ in the hemolymph and muscle collected at each time point. Exposure to a chill-coma inducing temperature had little effect overall on ion balance in the hemolymph and flight muscle of false codling moth, but hemolymph [Na+] decreased from 10.4 ±â€¯0.4 mM to 6.9 ±â€¯0.7 mM as moths were chilled to 3 °C and then increased to 10.4 ±â€¯0.9 mM after the 24 h recovery period. In the 9 °C cooling treatment, [K+] increased from 8.2 ±â€¯0.5 mM during chilling to 14.1 ±â€¯1.9 mM after the 24 h recovery period. No changes were seen in equilibrium potentials in either of the ions measured. Thus, we did not find evidence that water and ion homeostasis are lost by the moths in chill coma and conclude that reduced dispersal in field-released moths is not direct a consequence of the costs of re-establishment of homeostasis.