Effects of nutrient and water restriction on thermal tolerance: A test of mechanisms and hypotheses.

Nutritional deprivation or desiccation can influence thermal tolerance by impacting the insects' ability to evaporatively cool, maintain cell membrane integrity and conduct protective or repair processes. Recovery from chilling is also linked to the re-establishment of iono- and osmo-regulatory homeostasis. Here, using Mediterranean fruit fly (Ceratitis capitata, Diptera: Tephritidae), we manipulated water and nutrient availability to test the mechanistic expectation that changes in whole organism lipid and water content can elicit variation in cold or heat tolerance (scored as chill coma recovery time and heat knockdown time). We measured body condition (body water and lipid content) as well as heat shock protein 70 gene (hsp70) and protein (HSP70) levels. A significant reduction in body water content with water restriction did not translate into differences in chill coma recovery. When nutrient restriction was coupled with water deprivation, this resulted in a significant reduction (-54%) of heat knockdown time in females but male flies were unaffected. There was no evidence for an hsp70 or HSP70 response under any of the stress treatments and therefore no correlation with heat or cold tolerance. Heat hardening decreased all hsp levels. Therefore, although body water and total body lipid content differed between the treatment groups, the contribution of these factors to thermal tolerance was inconsistent with mechanistic expectations in heat knockdown time and insignificant for chill coma recovery. These results therefore highlight that the effects of resource restriction on thermal limits in insects are mechanistically more complex than previous models of stress resistance have suggested.

Physiological mechanisms of dehydration tolerance contribute to the invasion potential of Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) relative to its less widely distributed congeners.

BACKGROUND: The Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a highly invasive species now with an almost cosmopolitan distribution. Two other damaging, polyphagous and closely-related species, the marula fruit fly, Ceratitis cosyra (Walker), and the Natal fly, Ceratitis rosa Karsch, are not established outside of sub-Saharan Africa. In this study, adult water balance traits and nutritional body composition were measured in all three species at different temperatures and levels of relative humidity to determine whether tolerance of water stress may partially explain their distribution. RESULTS: Adult C. capitata exhibited higher desiccation resistance than C. rosa but not C. cosyra. Desiccation resistance of C. capitata was associated with lower rates of water loss under hot and dry conditions, higher dehydration tolerance, and higher lipid reserves that were catabolised during water stress. In comparison with C. capitata, C. cosyra and C. rosa lost water at significantly higher rates under hot, dry conditions, and did not catabolise lipids or other sources of metabolic water during water stress. CONCLUSIONS: These results suggest that adult physiological traits permitting higher tolerance of water stress play a role in the success of C. capitata, particularly relative to C. rosa. The distribution of C. cosyra is likely determined by the interaction of temperature with water stress, as well as the availability of suitable hosts for larval development.

The closed spiracle phase of discontinuous gas exchange predicts diving duration in the grasshopper Paracinema tricolor.

The discontinuous gas exchange (DGE) pattern of respiration shown by many arthropods includes periods of spiracle closure (C-phase) and is largely thought to serve as a physiological adaptation to restrict water loss in terrestrial environments. One major challenge to this hypothesis is to explain the presence of DGE in insects in moist environments. Here, we show a novel ecological correlate of the C-phase, namely, diving behaviour in mesic Paracinema tricolor grasshoppers. Notably, maximal dive duration is positively correlated with C-phase length, even after accounting for mass scaling and absolute metabolic rate. Here, we propose that an additional advantage of DGE may be conferred by allowing the tracheal system to act as a sealed underwater oxygen reservoir. Spiracle closure may facilitate underwater submersion, which, in turn, may contribute to predator avoidance, the survival of accidental immersion or periodic flooding and the exploitation of underwater resources.

The speed and metabolic cost of digesting a blood meal depends on temperature in a major disease vector.

The energetics of processing a meal is crucial for understanding energy budgets of animals in the wild. Given that digestion and its associated costs may be dependent on environmental conditions, it is necessary to obtain a better understanding of these costs under diverse conditions and identify resulting behavioural or physiological trade-offs. This study examines the speed and metabolic costs - in cumulative, absolute and relative energetic terms - of processing a bloodmeal for a major zoonotic disease vector, the tsetse fly Glossina brevipalpis, across a range of ecologically relevant temperatures (25, 30 and 35°C). Respirometry showed that flies used less energy digesting meals faster at higher temperatures but that their starvation tolerance was reduced, supporting the prediction that warmer temperatures are optimal for bloodmeal digestion while cooler temperatures should be preferred for unfed or post-absorptive flies. (13)C-Breath testing revealed that the flies oxidized dietary glucose and amino acids within the first couple of hours of feeding and overall oxidized more dietary nutrients at the cooler temperatures, supporting the premise that warmer digestion temperatures are preferred because they maximize speed and minimize costs. An independent test of these predictions using a thermal gradient confirmed that recently fed flies selected warmer temperatures and then selected cooler temperatures as they became post-absorptive, presumably to maximize starvation resistance. Collectively these results suggest there are at least two thermal optima in a given population at any time and flies switch dynamically between optima throughout feeding cycles.

Does oxygen limit thermal tolerance in arthropods? A critical review of current evidence.

Over the last decade, numerous studies have investigated the role of oxygen in setting thermal tolerance in aquatic animals, and there has been particular focus on arthropods. Arthropods comprise one of the most species-rich taxonomic groups on Earth, and display great diversity in the modes of ventilation, circulation, blood oxygen transport, with representatives living both in water (mainly crustaceans) and on land (mainly insects). The oxygen and capacity limitation of thermal tolerance (OCLTT) hypothesis proposes that the temperature dependent performance curve of animals is shaped by the capacity for oxygen delivery in relation to oxygen demand. If correct, oxygen limitation could provide a mechanistic framework to understand and predict both current and future impacts of rapidly changing climate. In arthropods, most studies testing the OCLTT hypothesis have considered tolerance to thermal extremes. These studies likely operate from the philosophical viewpoint that if the model can predict these critical thermal limits, then it is more likely to also explain loss of performance at less extreme, non-lethal temperatures, for which much less data is available. Nevertheless, the extent to which lethal temperatures are influenced by limitations in oxygen supply remains unresolved. Here we critically evaluate the support and universal applicability for oxygen limitation being involved in lethal temperatures in crustaceans and insects. The relatively few studies investigating the OCLTT hypothesis at low temperature do not support a universal role for oxygen in setting the lower thermal limits in arthropods. With respect to upper thermal limits, the evidence supporting OCLTT is stronger for species relying on underwater gas exchange, while the support for OCLTT in air-breathers is weak. Overall, strongest support was found for increased anaerobic metabolism close to thermal maxima. In contrast, there was only mixed support for the prediction that aerobic scope decreases near critical temperatures, a key feature of the OCLTT hypothesis. In air-breathers, only severe hypoxia (<2 kPa) affected heat tolerance. The discrepancies for heat tolerance between aquatic and terrestrial organisms can to some extent be reconciled by differences in the capacity to increase oxygen transport. As air-breathing arthropods are unlikely to become oxygen limited under normoxia (especially at rest), the oxygen limitation component in OCLTT does not seem to provide sufficient information to explain lethal temperatures. Nevertheless, many animals may simultaneously face hypoxia and thermal extremes and the combination of these potential stressors is particularly relevant for aquatic organisms where hypoxia (and hyperoxia) is more prevalent. In conclusion, whether taxa show oxygen limitation at thermal extremes may be contingent on their capacity to regulate oxygen uptake, which in turn is linked to their respiratory medium (air vs. water). Fruitful directions for future research include testing multiple predictions of OCLTT in the same species. Additionally, we call for greater research efforts towards studying the role of oxygen in thermal limitation of animal performance at less extreme, sub-lethal temperatures, necessitating studies over longer timescales and evaluating whether oxygen becomes limiting for animals to meet energetic demands associated with feeding, digestion and locomotion.

Oxygen safety margins set thermal limits in an insect model system.

A mismatch between oxygen availability and metabolic demand may constrain thermal tolerance. While considerable support for this idea has been found in marine organisms, results from insects are equivocal and raise the possibility that mode of gas exchange, oxygen safety margins and the physico-chemical properties of the gas medium influence heat tolerance estimates. Here, we examined critical thermal maximum (CTmax) and aerobic scope under altered oxygen supply and in two life stages that varied in metabolic demand in Bombyx mori (Lepidoptera: Bombycidae). We also systematically examined the influence of changes in gas properties on CTmax. Larvae have a lower oxygen safety margin (higher critical oxygen partial pressure at which metabolism is suppressed relative to metabolic demand) and significantly higher CTmax under normoxia than pupae (53°C vs 50°C). Larvae, but not pupae, were oxygen limited with hypoxia (2.5 kPa) decreasing CTmax significantly from 53 to 51°C. Humidifying hypoxic air relieved the oxygen limitation effect on CTmax in larvae, whereas variation in other gas properties did not affect CTmax. Our data suggest that oxygen safety margins set thermal limits in air-breathing invertebrates and the magnitude of this effect potentially reconciles differences in oxygen limitation effects on thermal tolerance found among diverse taxa to date.

Effects of within-generation thermal history on the flight performance of Ceratitis capitata: colder is better.

The influence of thermal history on temperature-dependent flight performance was investigated in an invasive agricultural pest insect, Ceratitis capitata (Diptera: Tephritidae). Flies were exposed to one of four developmental acclimation temperatures (Tacc: 15, 20, 25, 30°C) during their pupal stage and tested at these temperatures (Ttest) as adults using a full-factorial study design. Major factors influencing flight performance included sex, body mass, Ttest and the interaction between Ttest and Tacc. Successful flight performance increased with increasing Ttest across all acclimation groups (from 10% at 15°C to 77% at 30°C). Although Tacc did not affect flight performance independently, it did have a significant interaction effect with Ttest. Multiple comparisons showed that flies which had been acclimated to 15°C and 20°C performed better than those acclimated to 25°C and 30°C when tested at cold temperatures, but warm-acclimated flies did not outperform cold-acclimated flies at warmer temperatures. This provides partial support for the 'colder is better' hypothesis. To explain these results, several flight-related traits were examined to determine whether Tacc influenced flight performance as a consequence of changes in body or wing morphology, whole-animal metabolic rate or cytochrome c oxidase enzyme activity. Although significant effects of Tacc could be detected in several of the traits examined, with an emphasis on sex-related differences, increased flight performance could not be explained solely on the basis of changes in any of these traits. Overall, these results are important for understanding dispersal physiology despite the fact that the mechanisms of acclimation-related changes in flight performance remain unresolved.

Cross-talk between low temperature and other environmental factors.

Low temperatures are rarely experienced in isolation. The impacts of low temperatures on insects can be exacerbated or alleviated by the addition of other environmental factors, including, for example, desiccation, hypoxia, or infection. One way in which environmental factors can interact is through cross-talk where different factors enact common signaling pathways. In this review, I highlight the breadth of abiotic and biotic factors that can interact with low-temperature tolerance in both natural and artificial environments; and discuss some of the candidate pathways that are possibly responsible for cross-talk between several factors. Specifically, I discuss three interesting candidates: the neurohormone octopamine, circadian clock gene vrille, and microbes. Finally, I discuss applications of cross-talk studies, and provide recommendations for researchers.

High metabolic and water-loss rates in caterpillar aggregations: evidence against the resource-conservation hypothesis.

Several hypotheses have been proposed for explaining animal aggregation, including energy or water conservation. However, these physiological hypotheses have not been well investigated. Here, we report the effects of aggregation on metabolic ( ) and evaporative water-loss rates ( ) of the gregarious caterpillar Eutricha capensis, by comparing individuals and groups of individuals (N=10-100). Contrary to findings from previous physiological studies, we did not find an advantage to aggregation: unexpectedly, and did not decrease with increasing group size. and generally remained constant or increased in larger groups relative to individuals. The amount of water lost per unit of CO2 exchanged ( : ratio) showed a marked increase in grouped caterpillars, particularly in larger groups. Other benefits of aggregation (e.g. reduced predation or increased growth rates) likely outweigh these potential costs, because individuals of E. capensis aggregate voluntarily despite no obvious energetic or hygric advantage, and other potentially confounding group effects (e.g. increased thermoregulatory advantage or whole-animal activity) are inconsequential. The results of this study provide an important exception to physiological studies reporting enhanced energy or water conservation in animal groups.

Developing the radiation-based sterile insect technique (SIT) for controlling Aedes aegypti: identification of a sterilizing dose.

BACKGROUND: The sterile insect technique (SIT) is emerging as a tool to supplement traditional pesticide-based control of Aedes aegypti, a prominent mosquito vector of microbes that has increased the global burden of human morbidity and mortality over the past 50 years. SIT relies on rearing, sterilizing and releasing large numbers of male mosquitoes that will mate with fertile wild females, thus reducing production of offspring from the target population. In this study, we investigated the effects of ionizing radiation (gamma) on male and female survival, longevity, mating behavior, and sterility of Ae. aegypti in a dose-response design. This work is a first step towards developing an operational SIT field suppression program against Ae. aegypti in St. Augustine, Florida, USA. RESULTS: Exposing late-stage pupae to 50 Gy of radiation yielded 99% male sterility while maintaining similar survival of pupae to adult emergence, adult longevity and male mating competitiveness compared to unirradiated males. Females were completely sterilized at 30 Gy, and when females were dosed with 50 Gy, they had a lower incidence of blood-feeding than unirradiated females. CONCLUSION: Our work suggests that an ionizing radiation dose of 50 Gy should be used for future development of operational SIT in our program area because at this dose males are 99% sterile while maintaining mating competitiveness against unirradiated males. Furthermore, females that might be accidentally released with sterile males as a result of errors in sex sorting also are sterile and less likely to blood-feed than unirradiated females at our 50 Gy dose. © 2022 Society of Chemical Industry.

Reactive oxygen species production and discontinuous gas exchange in insects.

While biochemical mechanisms are typically used by animals to reduce oxidative damage, insects are suspected to employ a higher organizational level, discontinuous gas exchange mechanism to do so. Using a combination of real-time, flow-through respirometry and live-cell fluorescence microscopy, we show that spiracular control associated with the discontinuous gas exchange cycle (DGC) in Samia cynthia pupae is related to reactive oxygen species (ROS). Hyperoxia fails to increase mean ROS production, although minima are elevated above normoxic levels. Furthermore, a negative relationship between mean and mean ROS production indicates that higher ROS production is generally associated with lower . Our results, therefore, suggest a possible signalling role for ROS in DGC, rather than supporting the idea that DGC acts to reduce oxidative damage by regulating ROS production.

Investigating SAPAP3 variants in the etiology of obsessive-compulsive disorder and trichotillomania in the South African white population.

BACKGROUND: Obsessive-compulsive disorder (OCD) is a debilitating psychiatric disorder characterized by repeated obsessions and compulsions. Trichotillomania (TTM), a psychiatric disorder characterized by repetitive hairpulling, is presently classified as an impulse control disorder, but has also been viewed as an obsessive-compulsive spectrum disorder. Both conditions are complex disorders, with evidence from family and twin studies indicating that their etiology includes a genetic component. Results from a recent knockout animal model suggest that SAP90/PSD95-associated protein 3 (SAPAP3) may be involved in the pathophysiology of both disorders. METHODS: Seven polymorphic variants distributed across the gene encoding SAPAP3 were genotyped in South African white OCD (n = 172), TTM (n = 45), and control (n = 153) subjects. Single-locus and haplotype analyses were conducted to determine association between genetic variants and subjects with OCD, TTM, and controls. RESULTS: Although single-locus analysis revealed a significant association between rs11583978 in SAPAP3 and TTM, this association was nonsignificant after correction for multiple testing. In the OCD group, a significant association was observed between earlier age at onset and the A-T-A-T (rs11583978-rs7541937-rs6662980-rs4652867) haplotype compared with the C-G-G-G haplotype. CONCLUSIONS: This study generated preliminary evidence to link SAPAP3 variants to the development of earlier onset OCD. Future studies should concentrate on locating the susceptibility variant(s) by focusing on functional polymorphisms within SAPAP3.

Critical PO2 as a diagnostic biomarker for the effects of low-oxygen modified and controlled atmospheres on phytosanitary irradiation treatments in the cabbage looper Trichoplusia ni (Hübner).

BACKGROUND: Phytosanitary irradiation is a sustainable alternative to chemical fumigants for disinfesting fresh commodities from insect pests. However, irradiating insects in modified atmospheres with very low oxygen (<1 kPa O2 ) has repeatedly been shown to increase radioprotective response. Thus, there is a concern that modified atmosphere packaging could reduce the efficacy of phytosanitary irradiation. One hurdle slowing the widespread application of phytosanitary irradiation is a lack of knowledge about how moderate levels of hypoxia relevant to the modified atmosphere packaging of most fresh commodities (3-10 kPa O2 ) may affect phytosanitary irradiation treatments. Therefore, we hypothesize that critical PO2 (Pcrit ), the level of oxygen at which an insect's metabolism becomes impaired, can be used as a diagnostic biomarker to predict the induction of a radioprotective response. RESULTS: Using the cabbage looper Trichoplusia ni (Hübner), we show that there is a substantial increase in radiation resistance when larvae are irradiated in atmospheres more hypoxic than their Pcrit (3.3 kPa O2 ). These data are consistent with our hypothesis that Pcrit could be used as a diagnostic biomarker for what levels of hypoxia may induce radioprotective effects that could impact phytosanitary irradiation treatments. CONCLUSION: We propose that the relationship between Pcrit and radioprotective effects could allow us to build a framework for predicting the effects of low-oxygen atmospheres on the efficacy of phytosanitary irradiation. However, more widespread studies across pest species are still needed to test the generality of this idea.

Gamma-Irradiation Reduces Survivorship, Feeding Behavior, and Oviposition of Female Aedes aegypti.

Aedes aegypti is a prominent disease vector that is difficult to control through traditional integrated vector management due to its cryptic peridomestic immature-stage habitat and adult resting behavior, increasing resistance to pesticide formulations approved by the US Environmental Protection Agency, escalating deregistration of approved pesticides, and slow development of new effective chemical control measures. One novel method to control Ae. aegypti is the sterile insect technique (SIT) that leverages the mass release of irradiated (sterilized) males to overwhelm mate choice of natural populations of females. However, one potential liability of SIT is sex sorting errors prior to irradiation, resulting in accidental release of females. Our goal in this study was to test the extent to which irradiation affects female life-history parameters to assess the potential impacts of releasing irradiated females accidentally sorted with males. In this study, we determined that a radiation dose ≥30 Gy-a dose sufficient to sterilize males while preserving their mating competitiveness-may substantially impact longevity, bloodfeeding, oviposition, and egg hatch rate of female Ae. aegypti after being irradiated as pupae. These findings could reduce public concern for accidental release of females alongside irradiated males in an operational Ae. aegypti SIT control program.

The complete mitochondrial genome of Apis mellifera jemenitica (Insecta: Hymenoptera: Apidae), the Arabian honey bee.

The mitochondrial genome of a worker Apis mellifera jemenitica was 16,623 bp. It consisted of 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs and a control region. Phylogenetic analyses suggest a close relationship between A. m. jemenitica, A. m. lamarckii and A. m. syriaca.

The mitochondrial genome of the Maltese honey bee, Apis mellifera ruttneri (Insecta: Hymenoptera: Apidae).

The mitochondrial genome of Apis mellifera ruttneri consisted of 13 protein-coding genes, two rRNAs, 22 tRNAs, an AT-rich control region, and was 16,577 bp long. The phylogenetic analyses suggested that A. m. ruttneri was closely related to two North African subspecies: A. m. sahariensis and A. m. intermissa.

The complete mitochondrial genome of Apis mellifera unicolor (Insecta: Hymenoptera: Apidae), the Malagasy honey bee.

The complete mitochondrial genome of the endemic Malagasy honey bee Apis mellifera unicolor is 16,373 bp and comprises 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a control region. The mitochondrial genome closely resembles mitogenomes of other published Apis mellifera subspecies, and the phylogenetic analysis suggests that A. m. unicolor is distinct from other African (A) lineage honey bees but is most closely related to the honey bees from southern African: A. m. scutellata and A. m. capensis.

The mitochondrial genome of the Carniolan honey bee, Apis mellifera carnica (Insecta: Hymenoptera: Apidae).

Sequencing the mitochondrial genome of the Carniolan honey bee, Apis mellifera carnica, revealed 16,358 bp, consisting of 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and a control region. Phylogenetic analysis supported a close relationship to another south-eastern European (C-lineage) honey bee, A. m. ligustica.

The mitochondrial genome of Apis mellifera simensis (Hymenoptera: Apidae), an Ethiopian honey bee.

The complete mitochondrial genome of Apis mellifera simensis was 16,523 bp long. The 13 protein-coding genes, two rRNAs, and 22 tRNAs resembled other Apis mitogenomes. The location of this Apis subspecies in our phylogenetic tree supported the hypothesis that this subspecies is distinct, and is most closely related to A. m. scutellata and A. m. monticola.

The complete mitochondrial genome of the West African honey bee Apis mellifera adansonii (Insecta: Hymenoptera: Apidae).

The complete mitochondrial genome of the West African honey bee Apis mellifera adansonii consisted of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a control region. It was 16,466 bp and consisted of 84.7% AT nucleotides. This subspecies had a similar mitogenome to those of other southern African honey bees, namely A. m. scutellata, A. m. capensis, and A. m. monticola.