Journal of Insect Physiology

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

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.

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.

Heat shock proteins (HSPs) are well known to prevent and repair protein damage caused by various abiotic stressors, but their role in low temperature and freezing stress is not well-characterized compared to other thermal challenges. Ice formation in and around cells is hypothesized to cause protein damage, yet many species of insects can survive freezing, suggesting HSPs may be an important mechanism in freeze tolerance. Here, we studied HSP70 in a freeze-tolerant cricket Gryllus veletis to better understand the role of HSPs in this phenomenon. We measured expression of one heat-inducible HSP70 isoform at the mRNA level (using RT-qPCR), as well as the relative abundance of total HSP70 protein (using semi-quantitative Western blotting), in five tissues from crickets exposed to a survivable heat treatment (2 h at 40{degrees}C), a 6-week fall-like acclimation that induces freeze tolerance, and a survivable freezing treatment (1.5 h at -8{degrees}C). While HSP70 expression was upregulated by heat at the mRNA or protein level in all tissues studied (fat body, Malphigian tubules, midgut, femur muscle, nervous system ganglia), no tissue exhibited HSP70 upregulation within 2 - 24 h following a survivable freezing stress. During fall-like acclimation to mild low temperatures, we only saw moderate upregulation of HSP70 at the protein level in muscle, and at the RNA level in fat body and nervous tissue. Although HSP70 is important for responding to a wide range of stressors, our work suggests that this chaperone may be less critical in the preparation for, and response to, moderate freezing stress. HighlightsO_LIHeat shock protein 70 (HSP70) may not contribute substantially to freeze tolerance C_LIO_LIHeat stress caused HSP70 mRNA and protein upregulation in the spring field cricket C_LIO_LIAcclimation prior to freezing was correlated with slight HSP70 upregulation C_LIO_LIHSP70 was not upregulated after freezing in this freeze-tolerant insect C_LIO_LIFurther work is needed to determine whether freezing causes protein damage C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/621172v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@170fda7org.highwire.dtl.DTLVardef@11cf432org.highwire.dtl.DTLVardef@1e41be9org.highwire.dtl.DTLVardef@e46c4d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Several studies have demonstrated that diapausing insects upregulate glycerol production in response to cold temperatures. However, relatively few studies have investigated whether diapause can independently trigger increased glycerol production. In other words, little research has been dedicated to analyzing whether diapause and glycerol upregulation are linked. If a linkage between these two elements in insects exists, then photoinduced diapausing insects should upregulate glycerol production, even without exposure to cold temperatures. The current study examines whether this linkage exists in the tobacco hornworm pupae (Manduca sexta). Tobacco hornworms will enter a pupal diapause if presented with consistently short photoperiods, most notably 12 hours of daylight followed 12 hours of darkness. Regarding temperature, photoinduction of diapause in the tobacco hornworm is optimal at 26 {degrees}C. Using a spectrophotometric assay, glycerol levels in hemolymph were assessed in non-diapausing pupae shortly after pupation and in diapausing pupae after 16 days of pupation. Interestingly, non-diapausing pupae contained significantly higher glycerol levels in hemolymph compared to diapausing pupae. Furthermore, non-diapausing pupae produced approximately .099 M glycerol and diapausing pupae produced .085 M glycerol. This study provides evidence that glycerol upregulation and diapause are not linked in Manduca sexta, but are rather two separate events with two distinct causes. Nevertheless, the results are valuable since non-diapause specimens produced more glycerol than diapausing specimens, and moderately high concentrations of glycerol were found in both diapausing pupae and non-diapausing pupae.

Insects living in temperate regions often accumulate a large amount of glycerol during winter to avoid freezing. This seasonal accrual of glycerol is generally produced from glycogen reserves through the pentose phosphate pathway. An alternative pathway to produce glycerol is through glycolysis, normally used for pyruvate production for eventual ATP synthesis. Aside from seasonal accumulation, some insects will also rapidly increase glycerol production as a short-term response to a sudden cold event, thereby increasing cold hardiness when necessary. In the eastern spruce budworm Choristoneura fumiferana, this plasticity in cold hardiness is locally adapted, where northern populations produce more glycerol upon cold shock. Here we investigate how glycerol is produced during the rapid plastic response to fluctuating cold conditions, and whether this pathway could be a target of local adaptation. After a period of repeated cold exposure, we found evidence of increased enzyme activity and increased mRNA abundance of several proteins associated with glycolysis, and a downregulation in expression of glucose-6-phosphate dehydrogenase, associated with pentose phosphate. Pyruvate production is prevented through downregulation of glyceraldehyde-3-phosphate dehydrogenase. We found higher overall enzyme activity and glycerol accumulation in a northern population from Alberta, although there was no evidence of an interaction effect between population and cold shock treatment. This is one the first studies to show a mechanistic basis of such plasticity in cold hardiness.

Insects live in varied habitats and experience different kinds of environmental stresses. These stresses can impair neural performance, leading to spreading depolarization (SD) of nerve cells and neural shutdown underlying coma. The sensitivity of an insects nervous system to stress (e.g., anoxia) can be modulated by acute pre-treatment. Rapid cold hardening (RCH) is a form of preconditioning, in which a brief exposure to low temperature can enhance the stress tolerance of insects. SD is associated with a sudden loss of ion, notably K+, homeostasis. We used a pharmacological approach to investigate whether RCH affects anoxia-induced SD in the locust, L. migratoria, via one or more of the following homeostatic mechanisms: (1) Na+/K+-ATPase (NKA), (2) Na+/K+/2Cl- co-transporter (NKCC), and (3) voltage-gated K+ (Kv) channels. We also assessed abundance and phosphorylation of NKCC using immunoblotting. We found that inhibition of NKA or Kv channels delayed the onset of anoxia-induced SD in both control and RCH preparations. However, NKCC inhibition preferentially abrogated the effect of RCH. Additionally, we observed a higher abundance of NKCC in RCH preps but no statistical difference in its phosphorylation level, indicating the involvement of NKCC expression or degradation as part of the RCH mechanism.

Extreme temperatures associated with climate change are expected to impact the physiology and fertility of a variety of insects, including honey bees. Most previous work has focused on female honey bees, and comparatively little research has investigated how heat exposure affects males (drones). To address this gap, we tested how body mass, viral infections, Africanization, and geographic origin (including stocks from Australia, California, and Ukraine as well as diverse locations within British Columbia, Canada) influenced drone and sperm heat tolerance. We found that individual body size was highly influential, with heavier drones being more likely to survive a heat challenge than smaller drones. Drones originating from feral colonies in Southern California (which are enriched for African genetics) were also more likely to survive a heat challenge than drones originating from commercially-supplied Californian stock. We found no association between drone mass and thermal tolerance of sperm over time in an in vitro challenge assay, but experimental viral infection decreased the heat tolerance of sperm. Overall, there is ample variation in sperm heat tolerance, with sperm from some groups displaying remarkable heat resilience and sperm from others being highly sensitive, with additional factors influencing heat tolerance of the drones themselves.

Dietary quantity and quality are key determinants for insect development from egg to adult. When nutritional deficiency is sub-optimal, development is completed, albeit resulting in an adult insect that is smaller than normal in size. If now fed a normal diet, would the smaller adults be similar to normally developed flies? To begin to answer this question, we characterised a few physiological and musculoskeletal readouts. Larvae were subject to acute starvation in late stages of development, and the resulting adults (Early Life Starved; ELS) maintained on a normal diet, were tested for biochemistry, gut physiology and locomotor activity. In females, no significant difference was observed in biochemical readouts for the whole-body or hemolymph, between control and ELS flies. In males, whole-body glucose and hemolymph trehalose were significantly reduced in ELS flies. Interestingly, ELS flies of both sexes respond with a disproportionally higher accumulation of triacylglycerides (TAGs) when on a high-fat diet. Age-related changes in the adult gut were compared between control and ELS flies: these revealed an increased proportion of ELS flies with loss of gut barrier integrity, deviant number of intestinal stem cells and no difference in enteroendocrine cells. The rate of antimicrobial peptide gene expression to an enteric infection challenge was also slower in ELS flies. For musculoskeletal readouts, climbing and flight behaviour were measured. In population assays, both male and female ELS flies showed climbing deficits. In a fine-scale climbing assay on individual flies, female but not male ELS flies showed higher climbing speed, while males but not females, showed lower geotactic index. This collection of phenotypic assessments show that firstly, larval undernutrition, even when not lethal, continues to impact adult functioning. Secondly, for some phenotypes, a normal diet in adults exposed to early life malnutrition is insufficient to restore optimal functioning. Thirdly, larval dietary loss affects adult insects in a sex-dependent manner. This study lays the framework to uncover the molecular, cellular and hormonal mechanisms that are altered by early life malnutrition. Furthermore, these readouts may be used to develop Drosophila as a high-throughput model for interrogating the efficacy of diet therapies to address malnutrition.

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.

Females of the Northern house mosquito, Culex pipiens, enter an overwintering dormancy, or diapause, in response to short day lengths and low environmental temperatures. Diapausing female mosquitoes feed exclusively on sugar-rich products rather than human or animal blood, thereby reducing disease transmission. During diapause, Major Royal Jelly Protein 1 (MRJP1) is upregulated in females of Cx. pipiens. This protein is highly abundant in royal jelly, a substance produced by honey bees (Apis mellifera), that is fed to future queens throughout larval development and stimulates longevity and fecundity. However, the role of MRJP1 in Cx. pipiens is unknown. We investigated how supplementing the diets of both diapausing and nondiapausing females of Cx. pipiens with royal jelly affects gene expression, egg follicle length, fat content, protein content, longevity, and metabolic profile. We found that feeding royal jelly to long day-reared females significantly reduced the egg follicle lengths of females and switched their metabolic profiles to be similar to diapausing females. In contrast, feeding royal jelly to short day-reared females significantly reduced lifespan and switched their metabolic profile to be similar nondiapausing mosquitoes. Moreover, RNAi directed against MRJPI significantly increased egg follicle length of short day-reared females, suggesting that these females averted diapause, although RNAi against MRJP1 also extended the lifespan of short day-reared females. Taken together, our data show that consuming royal jelly reverses the seasonal responses of Cx. pipiens and that these responses are likely mediated in part by MRJP1. Summary StatementConsuming royal jelly reversed seasonal differences in physiological states, lifespan and metabolic profiles in females of the Northern house mosquito, a major vector of West Nile virus.

Gypsy moth Lymantria dispar (GM) is a polyphagous insect and one of the most significant pests in the forests of Eurasia and North America. Accurate information on GM cold hardiness is needed to improve methods for the prediction of population outbreaks, as well as for forecasting possible GM range displacements due to climate change. As a result of laboratory and field studies, we found that the lower lethal temperature (at which all L. dispar asiatica eggs die) range from -29.0 {degrees}C to -29.9 {degrees}C for three studied populations, and no egg survived cooling to -29.9 {degrees}C. These limits agree to within one degree with the previously established cold hardiness limits of the European subspecies L. dispar dispar, which is also found in North America. This coincidence indicates that the lower lethal temperature of L. dispar is conservative. Thus, we found that the Siberian populations of GM inhabit an area where winter temperatures go beyond the limits of egg physiological tolerance, because temperature often fall below -30 {degrees}C. Apparently, it is due to the flexibility of ovipositional behavior that L. dispar asiatica survives in Siberia: the lack of physiological tolerance of eggs is compensated by choosing warm biotopes for oviposition. One of the most important factors contributing to the survival of GM eggs in Siberia is the stability of snow cover. SummaryWithin the geographical range of Siberian gypsy moth populations, extreme temperatures go beyond the limits of the physiological tolerance of wintering eggs (-29.9 {degrees}C), and their survival depends on the choice of warm biotopes for oviposition.

One of the most stressful factors for insects is increasing temperature because of the risk of potentially fatal dehydration linked to their small size. We used respirometry to study the effect of both temperature and body mass on water loss and metabolic rate in individual workers of the polymorphic ant species Messor barbarus. As expected, we found that large ants exposed to increasing temperatures have a lower rate of water loss than small ants and that their mass-specific metabolic rate increases more slowly. However, counterintuitively, the measure of worker sensitivity to changes in temperature, as assessed by the instantaneous Q10 value (i.e., the rate of change across 10{degrees}C temperature intervals), shows that large ants are more sensitive than small ants to changes in temperature in terms of both water loss and metabolic rate. Such differential thermal sensitivity allows to make testable predictions on the temporal distribution of foraging activity among workers of different sizes in polymorphic ant species, as well as how these species may alter their colony demographics in response to rising temperatures.

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

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.

Understanding the role of nutrients in microbial population dynamics relies on a sound appreciation of their nutritional environment and how this may vary in different habitats. For microbial pathogens and commensals, this can be especially challenging because the microbe may share nutritional resources with its host. Here we design a series of 20 synthetic haemolymphs (nutribloods) that mimic haemolymph nutrient profiles of caterpillars fed on one of 20 chemically-defined diets, that vary in their protein:carbohydrate (P:C) ratio and caloric density. Using these, we are able to simulate the range of nutritional conditions that insect blood pathogens might face, providing a model system for understanding the role of nutrition in microbial growth. We tested this using the entomopathogen, Xenorhabdus nematophila, a gram-positive extracellular bacterium of insect hosts. This revealed that whilst bacterial fitness peaked in nutriblood nutrient space that was high in carbohydrates and low in proteins, levels of amino acids in the nutribloods also appear to be an important driving force for bacterial growth. Using synthetic haemolymphs that had average levels of all nutrients other than carbohydrate, protein or amino acids, we also established that bacterial growth is generally enhanced by carbohydrate and amino acids but reduced by proteins. Here, we have established a tractable model system for examining the role that nutrition plays in the growth of an entomopathogenic bacterium. In future work, this model host-pathogen system can be used to test a range of nutritionally-driven processes, including competition during co-infection and interactions with the host microbiome, as well as comparative studies of other entomopathogens.

Climatic changes, such as heatwaves, pose unprecedented challenges for insects, as escalated temperatures above the thermal optimum alter insect reproductive strategies and energy metabolism. While thermal stress responses have been reported in different insect species, thermo-induced developmental abnormalities in phloem-feeding pests are largely unknown. In this laboratory study, we raised two groups of first instar nymphs belonging to two clones of the pea aphid Acyrthosiphon pisum, on fava beans Vicia faba. The instars developed and then asexually reproduced under constant exposure to a sub-lethal heatwave (27{degrees}C) for 14 days. Most mothers survived but their progenies showed abnormalities, as stillbirths and appendageless or weak nymphs with folded appendages were delivered. Clone N116 produced more deceased and appendageless embryos, contrary to N127, which produced fewer dead and more malformed premature embryos. Interestingly, the expression of the HSP70 and HSP83 genes differed in mothers between the clones. Moreover, noticeable changes in metabolism, e.g., lipids, were also detected and that differed in response to stress. Deformed offspring production after heat exposure may be due to heat injury and differential HSP gene expression, but may also be indicative of a conflict between maternal and offspring fitness. Reproductive altruism might have occurred to ensure some of the genetically identical daughters survive. This is because maintaining homeostasis and complete embryogenesis could not be simultaneously fulfilled due to the high costs of stress. Our findings shine new light on pea aphid responses to heatwaves and merit further examination across different lineages and species. HighlightsO_LIParthenogenetic aphids were resilient under heatwave yet produced deformed progeny C_LIO_LIDevelopmental deformities by clones imply conflicting maternal/offspring fitness C_LIO_LIClone N127 produced fewer dead and more malformed premature embryos than clone N116 C_LIO_LIExpression of two heat shock protein genes differed across clones under heat stress C_LIO_LIExposure to heat stress led to metabolic differences in the exposed aphids C_LI

In the era of global warming, much attention has been paid to the possibility that high temperature may influence diverse insects not only directly but also indirectly via effects on their symbiotic microorganisms. The stinkbug Plautia stali develops a midgut symbiotic organ that harbors a specific bacterial symbiont indispensable for its growth and survival. Being maintainable in laboratory and tractable experimentally, P. stali is recently highlighted as a model system to investigate the mechanisms underpinning insect-microbe symbiotic interactions. In this study, we reared newly-emerged adult insects of P. stali under different temperature conditions for 8 days and monitored how their symbiotic organs and symbiotic bacteria are affected. While all insects survived at temperatures from 25{degrees}C to 37{degrees}C, some insects died at 38{degrees}C, 39{degrees}C and 40{degrees}C, wherein mortality rates increased as temperature elevated. While the symbiotic organs of the normal insects exhibited vivid yellow color, the symbiotic organs of the insects reared at 35{degrees}C or higher frequently exhibited abnormal colors such as pale yellow, yellowish white, or white, the extent of which became more severe as temperature elevated. Symbiont quantification revealed that, while the symbiont titers were almost constant for 8 days at 25{degrees}C and 30{degrees}C, the symbiont titers on the 8th day drastically declined to 1/100 at 35 and 1/10000 at 37{degrees}C and 39{degrees}C. Based on these results, we propose that rearing at 37{degrees}C for a week is a recommended treatment regime by which the symbiont is effectively suppressed with minimal damage to the host insect.

Iron is an essential nutrient for all types of organisms, including insects and the microbes that infect them. We predicted that insects fed an iron-supplemented diet would accumulate more iron in their hemolymph, and, because infectious microbes acquire iron from their hosts, that this extra iron would increase the severity of bacterial infections. To test this hypothesis, we studied the effects of dietary iron supplementation on infection outcomes in Manduca sexta (tobacco hornworm). Larvae were fed an artificial diet, with or without antibiotics, or the same diets supplemented with 10 mM iron. Control and iron-treated larvae were inoculated with non-pathogenic Escherichia coli or the entomopathogenic Enterococcus faecalis, and bacterial load and larval survival were measured. We found that dietary iron supplementation increased the iron content of hemolymph by approximately 20 fold; however, contrary to our prediction, this increase in iron did not result in an increase in the bacterial load of either E. coli or E. faecalis. The effect of iron supplementation on survival was more complicated. As expected, for larvae inoculated with nonpathogenic E. coli, iron supplementation had no effect. For larvae inoculated with E. faecalis, the effect of iron supplementation depended on whether antibiotics were present in the diet. Without antibiotics, iron supplementation prolonged larval survival; with antibiotics, iron supplementation decreased larval survival. The results of this study do not support the hypothesis that dietary iron supplementation increases infection severity in M. sexta. Instead, the results support the viewpoint that the relationship between dietary iron and infection outcome is complex.