Journal of Insect Physiology

Mechanisms aimed at recovering from heat-induced damage are closely associated with the ability of ectotherms to survive exposition to stressful temperatures. Among these mechanisms the respective contribution of autophagy, a ubiquitous stress-responsive catabolic process, has more recently come to light. By increasing the turnover of cellular structures as well as the clearance of long-lived protein and protein aggregates, the induction of autophagy has been linked to increased tolerance to range of abiotic stressors in diverse ectothermic organisms. Since our understanding of the relationship between autophagy and heat-tolerance currently remains limited in insect models, we hypothesized that (1) heat-stress would cause an increase of autophagy in Drosophila melanogaster tissues and (2) rapamycin exposure would trigger a detectable autophagic response in flies and increase their heat-tolerance. In line with our hypothesis, we report that flies exposed to heat-stress present signs of protein aggregation and appears to trigger an autophagy-related homoeostatic response as a result. We further show that rapamycin feeding causes the systemic effect associated with TOR inhibition, induces autophagy at least locally in the fly gut, and increase the heat-stress tolerance of individuals. This points toward a likely substantial contribution of this autophagy to cope with stressful temperatures in insects.

We examined water balance characteristics and influence of desiccating conditions on adult western flower thrips (Frankliniella occidentalis) physiology and behavior. Western flower thrips are globally invasive and likely to contend with shifts in water availability across their expansive geographic range. Basic water balance characteristics, including water mass and dry mass, were established for adult males and females, revealing a distinct sexual dimorphism wherein females are larger, but males retain a larger percentage of their mass as body water. Males lose relative water mass more quickly and their survival times are shorter when compared to females. RNA-seq analysis identified significant enrichment of factors associated with carbohydrate transport and metabolism in dehydrated males and females. A reduction of glycogen reserves was confirmed during dehydration. The probability of thrips feeding significantly increased when desiccation was a factor. Lastly, infection with Tomato spotted wilt orthotospovirus (TSWV), a principal plant-pathogenic virus transmitted by F. occidentalis, did not have a consistent and apparent influence on desiccation tolerance; however, a reduction in glycogen reserves, and an increase in feeding activity in infected thrips, very similar to that observed in dehydrated thrips, was observed. Our results establish the fundamental water balance characteristics of adult thrips, and indicate that dehydration significantly influences the survivorship and feeding behavior of thrips; crucial factors that contribute to their capacity to spread disease.

The insect gut, which plays a role in ion and water balance, has been shown to leak solutes in the cold. Cold stress can also activate insect immune systems, but it is unknown if the leak of the gut microbiome is a possible immune trigger in the cold. We developed a novel feeding protocol to load the gut of locusts (Locusta migratoria) with fluorescent bacteria before exposing them to -2{degrees}C for up to 48 h. No bacteria were recovered from the hemolymph of cold-exposed locusts, regardless of exposure duration. To examine this further, we used an ex vivo gut sac preparation to re-test cold-induced fluorescent FITC-dextran leak across the gut and found no increased rate of leak. These results question not only the validity of FITC-dextran as a marker of paracellular barrier permeability in the gut, but also to what extent the insect gut becomes leaky in the cold.

Kissing bugs are the primary vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Kissing bugs are exposed to thermal variability, including short periods of heat stress, which can induce mortality or exert sublethal effects. This study investigated Rhodnius prolixus following brief periods of high thermal stress with respect to survival, blood feeding, developmental processes, and T. cruzi infection, with a focus on sublethal effects. Our results demonstrated a significant decrease in survival for R. prolixus at 42 {degrees}C for 8 hours. When exposed to sub-lethal thermal stress (40{degrees}C for 8 hours), blood ingestion (amount and proportion) was reduced after 24 hours of recovery from thermal stress. Among the bugs that fed after 24 hours, molting was not impacted by temperature exposure. The infection rate decreased after heat exposure, likely due to reduced blood volume ingested when feeding 24 hours after heat stress. A week of recovery after exposure to higher temperatures improved feeding and increased infection rates to levels comparable to those of kissing bugs not exposed to thermal stress. Our findings offer insights into how extreme temperature events may influence Chagas disease. Specifically, these studies highlight the need to clarify how temperature, particularly at sublethal levels, interacts with vector biology to alter parasite transmission.

Body size is the major factor to the flight mechanics in animals. To fly at low Reynolds numbers, miniature insects have adaptations in kinematics and wing structure. Many microinsects have bristled wings, which reduce inertia and power requirements when providing good aerodynamic efficiency. But both bristled and membranous-winged microinsects fly at Reynolds numbers of about 10. Yet, the kinematics of the smallest known membranous-winged species have not been studied sufficiently. The available data are limited to the forewings of a relatively large parasitoid wasp Encarsia formosa. We studied kinematics of wings and body and flight performance in one of the smallest membranous-winged wasps, Trichogramma telengai (0.5 mm body length, Re = 12). T. telengai reaches 29 cm s-1 speed and 7 m s-2 acceleration in horizontal flight which are comparable with the flight performance of other microinsects. The wingbeat cycle is characterized by high frequency (283 Hz) and stroke amplitude (149{degrees}) and includes U-shaped strokes at high angles of attack and prolonged clap-and-fling. The hindwings move with a slight phase shift and smaller amplitude than the forewings. T. telengai differs from large membranous-winged insects and miniature featherwing beetles in kinematics, but it is fundamentally similar to E. formosa (Re = 18, membranous wings) and thrips Frankliniella occidentalis (Re = 15, bristled wings). We showed that, at Re {approx} 101, both membranous and bristled-winged insects have sufficient flight performance. Further study of the bristled-winged insects will make it possible to define the size limits of effectiveness of different wing structures.

Understanding and characterizing how insects tolerate low temperatures is important for predicting their overwintering survival and subsequent geographic spread. This study characterized the cold tolerance of two members of the Rhagoletis genus in Colorado, U.S.A. Pupae were collected from infested fruit in late summer and early fall. For the first time, we show that the rosehip fly Rhagoletis basiola is freeze-avoidant; overwintering pupae could supercool to temperatures as low as -26{degrees}C and survive. Interestingly, the temperature at which ice forms (supercooling point; SCP) did not vary between R. basiola at high (c. 2900 m above sea level) and lower (c. 1650 m a.s.l.) elevations. We also report the apple maggot Rhagoletis pomonella infesting an unusual host fruit, the Dolgo crabapple, in close proximity to infested hawthorn trees. R. pomonella infesting hawthorn fruits and crabapples had similar SCPs, and survived temperatures as low as -21{degrees}C. Pupae from both host fruits also survived prolonged exposure (2 weeks or more) to mild low temperatures (0 to -5{degrees}C). Further study into the mechanisms underlying the impressive and conserved cold tolerance of R. pomonella and R. basiola is an interesting avenue for future research.

In the cold, chill susceptible insects lose the ability to regulate ionic and osmotic gradients. This leads to hemolymph hyperkalemia that drives a debilitating loss of cell membrane polarization, triggering cell death pathways and causing organismal injury. Biotic and abiotic factors can modulate insect cold tolerance by impacting the ability to mitigate or prevent this cascade of events. In the present study, we test the combined and isolated effects of dietary manipulations and thermal acclimation on cold tolerance in fruit flies. Specifically, we acclimated adult Drosophila melanogaster to 15 or 25{degrees}C and fed them either a K+-loaded diet or a control diet. We then tested the ability of these flies to recover from and survive a cold exposure, as well as their capacity to protect transmembrane K+ gradients, and intracellular Na+ concentration. As predicted, cold-exposed flies experienced hemolymph hyperkalemia and cold-acclimated flies had improved cold tolerance due to an improved maintenance of the hemolymph K+ concentration at low temperature. Feeding on a high-K+ diet improved cold tolerance additively, but paradoxically reduced the ability to maintain extracellular K+ concentrations. Cold-acclimation and K+-feeding additively increased the intracellular K+ concentration, aiding in maintenance of the transmembrane K+ gradient during cold exposure despite cold-induced hemolymph hyperkalemia. There was no effect of acclimation of diet on intracellular Na+ concentration. These findings suggest intracellular K+ loading and reduced muscle membrane K+ sensitivity as mechanisms through which cold-acclimated and K+-fed flies are able to tolerate hemolymph hyperkalemia. Highlights- Insect cold tolerance varies in relation to ionoregulatory capacity - Cold acclimation improves cold tolerance and K+ handling during cold exposure - A high K+ diet also improves cold tolerance, but reduces the K+-handling capacity - We highlight a novel mechanism for preventing K+ gradient disruption

Seasonal variation in day length provides a reliable cue that allows insects to anticipate upcoming environmental challenges. Here, we demonstrate that photoperiod induces pronounced, sex-specific immune priming in the linden bug Pyrrhocoris apterus. Females exposed to short-day, diapause-inducing conditions exhibited broadly enhanced immune activity compared with long-day females, whereas immune parameters in males were largely unaffected by photoperiod. Short-day females showed increased immune cell abundance, elevated expression of immune-related genes, enhanced humoral immune activity, and increased resistance to bacterial infection. Importantly, photoperiod-induced immune priming depended on a functional m-cryptochrome gene, linking seasonal immune regulation to the photoperiodic timer. Consistent with laboratory results, females collected under natural short-day conditions also displayed enhanced immune parameters despite increased environmental variability. Together, our findings identify photoperiod as a key regulator of immune preparedness in female insects and reveal a sex-specific anticipatory immune strategy associated with seasonal timing.

Insect populations are experiencing a global decline due to a variety of human-linked environmental changes. Among these changes, how insects physiology might be affected by predicted upslope migration due to climate change is unknown. Being ectotherms, insect physiology is impacted by abiotic factors like ambient temperature that change with elevation. Here, we performed in situ experiments to assess the sensory and cardiac physiology of an important generalist pollinating hoverfly Eristalis tenax (Diptera: Syrphidae), across different elevations in the eco-sensitive and biodiverse Himalayan mountains. We built a portable physiology setup and measured hoverfly antennal responses towards common floral volatiles at 3600 masl and 4200 masl. We also recorded their heart rate at 3000 masl, 3500 masl and 4000 masl. We report the first in situ physiology experiments performed in the high-altitude Himalayas. Our results show a contrasting impact of elevation and temperature on the sensory and cardiac physiology of hoverflies, with antennal sensitivity decreasing with increasing elevation, while average heart rate increased with temperature, independent of elevation. With upslope migration and climate warming, consequent sensory mismatches and cardiac stress could have deleterious effects on the health of both hoverflies and the vulnerable Himalayan ecosystem.

The convergent lady beetle Hippodamia convergens is widespread in the Americas and considered an important beneficial insect due to its use in pest control. Early work on this species characterized the beetles as freeze-avoidant (freeze-intolerant), suggesting they survive low winter temperatures by physiologically preventing ice formation to temperatures as low as -15{degrees}C. Here, we show that H. convergens can be freeze-tolerant if ice formation occurs at relatively high temperatures. There was 100% survival following inoculative freezing at -0.5{degrees}C and exposure to -3{degrees}C for 20 hours, as well as freezing that spontaneously occurred in fed beetles exposed to -4{degrees}C for 4 hours. Males exposed to 0{degrees}C or -4{degrees}C for 4 hours had similar mating behaviours (latency to first mating, copulation duration) as control beetles exposed to 4{degrees}C, although sample sizes were too small to determine whether freezing itself had an effect on these behaviours. Several putative cryoprotectants were detected in fat body tissue of H. convergens: glycerol, proline, trehalose, and myo-inositol - in order of abundance. Exposure to -3{degrees}C for 20 hours, whether frozen or unfrozen, did not statistically affect cryoprotectant accumulation, although there was a trend towards increased glycerol following freezing. This study is the first to describe inoculative freeze tolerance in a lady beetle and establishes a baseline for future studies that examine the mechanisms underlying this freeze tolerance and the effects of freezing on reproductive behaviour. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/744689v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1c0382org.highwire.dtl.DTLVardef@12a5932org.highwire.dtl.DTLVardef@145f4adorg.highwire.dtl.DTLVardef@1c2cb22_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIHippodamia convergens can tolerate freezing at high subzero temperatures C_LIO_LIMating behaviour of males is normal after chilling or freezing C_LIO_LICryoprotectant accumulation may support overwintering survival C_LI

Rhodnius prolixus is able to cool down the ingested blood during feeding on a warm-blooded host. This is possible because of a counter-current heat exchanger located in its head, which transfers heat from the warm blood to the insect haemolymph and can dissipate through the head cuticle. Given the key role haemolymph circulation in thermoregulation, we investigated the modulation of the activity of the heart during the warmed meal intake. We evaluated the impact of meal temperature on the heart rate and found that feeding led to an increase in the frequency of heart contractions, which increases with increasing food temperature. We also found that females have a higher heart rate during feeding compare to males.\n\nHIGHLIGHTSO_LIFeeding increases the heart rate of Rhodnius prolixus\nC_LIO_LIThe higher the meal temperature, the higher the heart rate becomes\nC_LIO_LIFemales have a higher heart rate than males\nC_LI

The Mediterranean fruit fly (medfly) (Ceratitis capitata (Wiedemann, 1824) is a major agricultural pest, and egg desiccation is a critical constraint during handling and mass-rearing, as even short periods without moisture may compromise developmental success and downstream adult performance. The Wolbachia-medfly symbiosis is a relatively recently established artificial association, generated less than three decades ago using Rhagoletis cerasi as the Wolbachia donor. In this study, we evaluated the effects of egg-stage desiccation on developmental success and subsequent adult performance in three medfly lines differing in Wolbachia status: the uninfected Benakeion line, the wCer2-infected 88.6 line, and the wCer4-infected S10.3 line. Eggs were exposed to desiccation for 0-24 h at 4-h intervals before transfer to larval diet, and hatching, pupation, and adult emergence were recorded. We additionally assessed adult survival under stress for flies emerging from the 0, 8, and 10 h egg-desiccation treatments. Under control conditions, Benakeion showed the highest hatching and developmental recovery, S10.3 the lowest, and 88.6 intermediate performance. Across all strains, short desiccation exposures were comparatively well tolerated, whereas prolonged exposure sharply reduced hatching, pupation, and adult emergence, with the clearest decline at 20-24 h. Strain-dependent differences were expressed mainly at the hatching stage, while later developmental transitions were more similar among strains once larvae had hatched. In the adult follow-up, strain, rather than moderate egg-stage desiccation, was the main determinant of short-term survival and survival under extreme stress, with S10.3 again showing the weakest performance. These results indicate that Wolbachia-associated fitness costs in medfly are strain dependent and that egg-stage desiccation primarily acts at the embryonic bottleneck. Beyond providing insight into the Wolbachia-medfly artificial symbiosis, our findings are directly relevant to egg-handling and strain-evaluation protocols in medfly mass-rearing systems.

Winter poses harsh physiological challenges for insects living in temperate ponds due to the combination of low temperatures and reduced oxygen levels (hypoxia). Despite these stressors, water boatmen (Hesperocorixa sp.), backswimmers (Notonecta sp.) and diving beetles (Laccophilus maculosus) remain active year-round in eastern Nova Scotia, including in ice- covered ponds. However, the underlying mechanisms that allow pond insects to survive overwintering have not been widely studied. We hypothesized that cold and hypoxia tolerance would improve from September to March in these insects, with changes in whole-animal and biochemical correlates of tolerance to both stressors. We collected insects from the field and stocked them in outdoor freshwater mesocosms. Every two months, between September 2023 and April 2024, we characterized whole animal responses and biochemical changes. Whole insect cold tolerance assays indicated a trend of improved ability to sustain voluntary muscle control (CTmin) at lower temperatures in winter-collected insects and higher internal fluid freezing temperatures (SCP). There were no changes in whole animal correlates of hypoxia tolerance over time (surface respiration frequency, submersion and surface time). Potential cryoprotectants like proline, myo-inositol and trehalose increased in concentration in multiple insect taxa during winter but were relatively low compared to terrestrial insect studies. As lactate dehydrogenase activity did not change, there is little evidence that the insects experienced functional hypoxia sufficient to induce anaerobic metabolism. Overall, this research has established a baseline cold and hypoxia tolerance dataset in understudied pond insects. Summary StatementPond insects remain active during low temperature and low oxygen conditions associated with ice cover in winter, supported by physiological and biochemical changes.

Temperature is a critical environmental variable that affects the distribution, survival, and reproduction of most animals. Although temperature receptors have been identified in different animals, how these receptors respond to temperatures is largely unknown. Here we use modified single-fly thermotactic assays to analyze movements and temperature preferences of nine Drosophila species. The ability/inclination to move varies among these species and at different temperatures. Importantly, different species prefer various ranges of temperatures. While wild-type D. melanogaster flies avoid the warm temperature in the warm avoidance assay and the cool temperature in the cool avoidance assay, D. bipectinata and D. yakuba avoid neither warm nor cool temperatures and D. biarmipes and D. mojavensis do not avoid the warm temperature in the warm avoidance assay. These results demonstrate that Drosophila species have different mobilities and temperature preferences, thereby benefiting the research on molecular mechanisms of temperature responsiveness. Summary statementThe ability to move and the preference for temperatures vary among fly species when flies are exposed to steep temperature gradients.

Chill susceptible insects are thought to be injured through different mechanisms depending on the duration and severity of chilling. While chronic chilling causes "indirect" injury through disruption of metabolic and ion homeostasis, acute chilling is suspected to cause "direct" injury, in part through phase transitions of cell membrane lipids. Dietary supplementation of cholesterol can reduce acute chilling injury in Drosophila melanogaster, but the generality of this effect and the mechanisms underlying it remain unclear. To better understand how and why cholesterol has this effect, we assessed how a high cholesterol diet and thermal acclimation independently and interactively impact several measures of chill tolerance in both male and female flies. Cholesterol supplementation positively affected tolerance to acute chilling in warm-acclimated flies (as reported previously). Conversely, feeding on the high-cholesterol diet negatively affected tolerance to chronic chilling in both cold and warm acclimated flies, as well as tolerance to acute chilling in cold acclimated flies. Cholesterol had no effect on the ability of flies to remain active in the cold or recover movement after a cold stress. Our findings support the idea that dietary cholesterol reduces mechanical injury to membranes caused by direct chilling injury, and that acute and chronic chilling are associated with distinct mechanisms of injury. Feeding on a high-cholesterol diet may interfere with mechanisms involved in cold acclimation, leaving cholesterol augmented flies more susceptible to chilling injury under some conditions. HighlightsO_LICholesterol improves cold shock tolerance of warm-acclimated flies C_LIO_LICold acclimation and chronic cold instead lead to negative effects of cholesterol on chill tolerance C_LIO_LICholesterol did not affect the ability of flies to remain active in the cold C_LIO_LIBoth sexes were similarly affected by a high cholesterol diet C_LI

The majority of insects can acclimate to changes in their thermal environment and counteract temperature effects on neuromuscular function. At the critical thermal minimum a spreading depolarization (SD) event silences central neurons, but the temperature at which this event occurs can be altered through acclimation. SD is triggered by an inability to maintain ion homeostasis in the extracellular space in the brain and is characterized by a rapid surge in extracellular K+ concentration, implicating ion pump and channel function. Here, we focused on the role of the Na+/K+-ATPase specifically in lowering the SD temperature in cold-acclimated D. melanogaster. After first confirming cold acclimation altered SD onset, we investigated the dependency of the SD event on Na+/K+-ATPase activity by injecting an inhibitor, ouabain, into the head of the flies to induce SD over a range of temperatures. Latency to SD followed the pattern of a thermal performance curve, but cold acclimation resulted in a left-shift of the curve to an extent similar to its effect on the SD temperature. With Na+/K+-ATPase activity assays and immunoblots, we found that cold-acclimated flies have ion pumps that are less sensitive to temperature, but do not differ in their overall abundance in the brain. Combined, these findings suggest a key role for plasticity in Na+/K+-ATPase thermal sensitivity in maintaining central nervous system function in the cold, and more broadly highlight that a single ion pump can be an important determinant of whether insects can respond to their environment to remain active at low temperatures.

Salts are essential nutrients required for many physiological processes, and deficient or excessive salt results in adverse health problems. Taste is the ultimate sensory modality involved in resource quality assessment, resulting in acceptance or rejection. Here, we show that detection of high-salt substrates by a salt-sensitive antennal gustatory receptor neuron, S1-GRN, results in feeding avoidance in the hematophagous bug Rhodnius prolixus. Knock-down of two antennal-expressed amiloride-sensitive pickpocket channel receptors (PPKs; RproPPK014276 and RproPPK28) using RNA interference, prevents avoidance of bugs to high-salt substrates. Tracing antennal GRNs to the central nervous system reveals the antennal lobes as a gustatory processing center. The identification of the gustatory basis of high-salt detection in a blood feeder provides novel targets to prevent biting and feeding, as well as to promote substrate avoidance in a relevant disease vector. Significance StatementDetection of aversive gustatory stimuli induces avoidance responses in animals. Avoidance acquires particular interest if it reduces the biting rates of blood-feeding insects of medical relevance. Here we describe the molecular and physiological basis of high-salt detection in the blood-sucking disease vector Rhodnius prolixus. We show that detection of high-salt substrates through two PPK receptors expressed in an antennal gustatory receptor neuron produces feeding avoidance. Understanding these gustatory-driven aversive responses allows the hitherto overlooked use of gustatory molecules as a complement to known olfactory repellents.

On farms, the conditions experienced by black soldier fly (BSF) prepupae may impact their development and survival, altering production goals by changing the fitness of breeding adults. Few studies have addressed the impact of environmental variables (such as substrate type or moisture content), or stressors like handling, on the development and survival of BSF as they transition from prepupae through adulthood. This study examined the effects of pupation substrate (corn cob grits, potting soil, vermiculite, wood chips, and frass), moisture content (20%, 60%, and 100%), and handling during the prepupal period (daily handling, no handling) on eclosion rate and timing, morphology (mass, head width, thorax length), and abdominal window fullness of adult BSF. Handling delayed adult emergence by over three days, reduced eclosion rates by 30.6%, reduced head width and wet mass in adult males, and reduced window fullness and head width in adult females. Frass resulted in the lowest eclosion rate (72.1 {+/-} 2.4%) while corn cob grits (82.2 {+/-} 2.3%) and wood chips (81.5 {+/-} 2.2%) had the highest. Wood chips also resulted in the highest wet and dry mass, head width, and thorax length for adult males and females. Wood chips may be the best pupation substrate for BSF as it enhances body size and has a good eclosion rate. Significant prepupal handling delays adult emergence, reduces eclosion/survival rates, and reduces adult body size; however, more research is necessary to determine if the less chronic handling regimes that are likely present on farms produce similar effects.

Nociception, the capacity to detect tissue-damaging stimuli such as noxious chemicals or high heat, is increasingly studied across insect orders and life stages, informing our understanding of its adaptive value and molecular mechanisms. The black soldier fly (Hermetia illucens; Diptera: Stratiomyidae) is widely recognized as the star of the growing insects as food and feed industry. Black soldier fly larvae (BSFL) are reared at high densities that, combined with their extraordinary metabolism, can generate lethal overheating on farms. Data on the thermal nocifensive capabilities of BSFL could inform our understanding of larval behaviors during overheating events (and potential welfare impacts of thermal slaughter), and provide a comparative datapoint to the well-studied vinegar fly (Drosophila melanogaster; Diptera: Drosophilidae). Accordingly, we adapted global and local thermal nociception methods from larval vinegar flies for use with BSFL. We find that global assays (akin to boiling) adapt easily to both first and sixth instar BSFL, that BSFL exhibit slightly different nocifensive behaviors than last instar vinegar fly larvae, and that BSFL have higher thresholds (thrashing begins at 39.70 {degrees}C in last instar BSFL versus 26.6 {degrees}C in D. melanogaster). In contrast, the classical local nociception assay did not adapt easily to BSFL; a modified version generated a unique, gradual pattern of increasing responsiveness to the probe in sixth instar BSFL (>95% responsiveness above 66 {degrees}C) rather than the sharp cutoff in responsiveness at 52 {degrees}C demonstrated in D. melanogaster. Altogether, these protocols open the door for standardized research on BSFL thermal nociception for fundamental and applied purposes.

Many insects enter diapause, a programmed state of developmental arrest that enables survival under adverse environmental conditions. In Drosophila melanogaster Meigen, 1830, diapause is characterized by reproductive arrest and reduced intestinal growth, accompanied by suppressed intestinal stem cell (ISC) activity. Juvenile Hormone (JH) promotes ISC proliferation under favorable conditions, but its capacity to modulate stem cell dynamics during cold-induced diapause remains unclear. Here, we investigated whether JH signaling can reactivate midgut remodeling in adult females maintained at 11. At this temperature, flies exhibited pronounced gut atrophy and elevated Phospho-histone H3 (PH3+) cell abundance, consistent with temperature-dependent G2/M phase arrest JH treatment significantly increased the proportion of Delta-positive progenitor cells in the anterior (R2) and posterior (R5) midgut regions at both 11 and 25, demonstrating that JH acts as a conserved mitogen for the ISC pool irrespective of thermal environment. A trend toward reduced PH3+ accumulation in the posterior midgut following JH treatment (p = 0.061) suggests possible facilitation of mitotic exit, though this effect did not reach statistical significance. Despite cellular-level changes, JH treatment did not restore overall gut size, indicating that the 72-84 hour exposure window was insufficient for subsequent tissue hypertrophy. Additionally, we identified a recurrent cold-induced pathology of gut distension, provisionally termed Lumen Obstruction Syndrome (LOS), which was independent of JH signaling. These findings reveal an uncoupling of JH-driven stem cell expansion from gross organ growth under diapause conditions, highlighting the selective sensitivity of the ISC compartment to endocrine signaling during environmental stress.