Estimation of genetic parameters for the implementation of selective breeding in commercial insect production.

BACKGROUND: There is a burgeoning interest in using insects as a sustainable source of food and feed, particularly by capitalising on various waste materials and by-products that are typically considered of low value. Enhancing the commercial production of insects can be achieved through two main approaches: optimising environmental conditions and implementing selective breeding strategies. In order to successfully target desirable traits through selective breeding, having a thorough understanding of the genetic parameters pertaining to those traits is essential. In this study, a full-sib half-sib mating design was used to estimate variance components and heritabilities for larval size and survival at day seven of development, development time and survival from egg to adult, and to estimate correlations between these traits, within an outbred population of house flies (Musca domestica), using high-throughput phenotyping for data collection. RESULTS: The results revealed low to intermediate heritabilities and positive genetic correlations between all traits except development time and survival to day seven of development and from egg to adulthood. Surprisingly, larval size at day seven exhibited a comparatively low heritability (0.10) in contrast to development time (0.25), a trait that is believed to have a stronger association with overall fitness. A decline in family numbers resulting from low mating success and high overall mortality reduced the amount of available data which resulted in large standard errors for the estimated parameters. Environmental factors made a substantial contribution to the phenotypic variation, which was overall high for all traits. CONCLUSIONS: There is potential for genetic improvement in all studied traits and estimates of genetic correlations indicate a partly shared genetic architecture among the traits. All estimates have large standard errors. Implementing high-throughput phenotyping is imperative for the estimation of genetic parameters in fast developing insects, and facilitates age synchronisation, which is vital in a breeding population. In spite of endeavours to minimise non-genetic sources of variation, all traits demonstrated substantial influences from environmental components. This emphasises the necessity of thorough attention to the experimental design before breeding is initiated in insect populations.

Manipulated ants: inducing loyalty to sugar feeders with an alkaloid.

BACKGROUND: Wood ants are promising biocontrol agents in fruit plantations because they prey on pest insects and inhibit plant diseases. However, these ants also attend plant-feeding homopterans to harvest their honeydew secretions, thereby increasing their numbers. This problem can be solved by offering ants alternative sugar sources that are more attractive than honeydew. From natural interactions, it is known that some species manipulate mutualistic partners toward loyalty by adding alkaloids to the food they offer their mutualists. Inspired by this, the addition of alkaloids might be used to make ants loyal to artificial sugar feeders and thus used to reduce populations of ant-farmed homopterans in ant-mediated biological control. We aimed to explore whether wood ants (Formica polyctena) would develop a taste preference for morphine-containing sugar solutions in two-choice laboratory tests. RESULTS: After having fed on a morphine/sugar solution for 1 week, ants showed a significant preference for morphine solutions compared with equal concentration sugar solutions without morphine. Furthermore, ants lost this preference after 6-9 days on a morphine-free diet. CONCLUSION: The results show that wood ants react to morphine in their food, enabling chemical manipulation of their behavior, most likely through a taste preference. Thus, ants are susceptible to manipulation by mutualistic partners in natural interactions and furthermore may be manipulated artificially in biocontrol programs to avoid ant-mediated build-up of homopteran populations. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Exploring cross-protective effects between cold and immune stress in Drosophila melanogaster.

It is well established that environmental and biotic stressors like temperature and pathogens/parasites are essential for the life of small ectotherms. There are complex interactions between cold stress and pathogen infection in insects. Possible cross-protective mechanisms occur between both stressors, suggesting broad connectivity in insect stress responses. In this study, the functional significance of these interactions was tested, as well as the potential role of newly uncovered candidate genes, turandot. This was done using an array of factorial experiments exposing Drosophila melanogaster flies to a combination of different cold stress regimes (acute or chronic) and infections with the parasitic fungus Beauveria bassiana. Following these crossed treatments, phenotypic and molecular responses were assessed by measuring 1) induced cold tolerance, 2) immune resistance to parasitic fungus, and 3) activation of turandot genes. We found various responses in the phenotypic outcomes according to the various treatment combinations with higher susceptibility to infection following cold stress, but also significantly higher acute cold survival in flies that were infected. Regarding molecular responses, we found overexpression of turandot genes in response to most treatments, suggesting reactivity to both cold and infection. Moreover, maximum peak expressions were distinctly observed in the combined treatments (infection plus cold), indicating a marked synergistic effect of the stressors on turandot gene expression patterns. These results reflect the great complexity of cross-tolerance reactions between infection and abiotic stress, but could also shed light on the mechanisms underlying the activation of these responses.

Title: Exploration des effets de protection croisée entre le froid et le stress immunitaire chez Drosophila melanogaster. Abstract: Il est établi que les stress environnementaux et biotiques, tels que la température et les agents pathogènes ou parasites, sont essentiels à la survie des petits ectothermes. Il existe des interactions complexes entre le stress froid et l'infection par des pathogènes chez les insectes. Des mécanismes de protection croisée sont possibles entre les deux facteurs de stress, suggérant une grande connectivité entre ces réponses. Dans cette étude, ces interactions ont été testées, ainsi que l'implication potentielle de nouveaux gènes candidats, les gènes turandot. Cela a été fait via un ensemble d'expériences factorielles exposant des mouches Drosophila melanogaster à différents types de stress froid (aigu ou chronique) couplés à des infections par le champignon entomoparasite Beauveria bassiana. Suite à ces traitements croisés, les réponses phénotypiques et moléculaires ont été analysées en mesurant 1) la tolérance induite au froid, 2) la résistance immunitaire à l'infection pathogène et 3) l'activation des gènes turandot. Les résultats phénotypiques ont montré des réponses variées selon les différentes combinaisons de traitements, avec une plus grande susceptibilité à l'infection suite à un stress froid, mais aussi une survie au stress froid aigu significativement plus élevée chez les mouches infectées. Pour les réponses moléculaires, nous avons trouvé une surexpression des gènes turandot en réponse à la plupart des traitements, suggérant une réactivité à la fois au froid et à l'infection. De plus, les expressions maximales ont été distinctivement observées dans les traitements combinés (infection plus froid), indiquant un effet synergique marqué des facteurs de stress sur l'expression des gènes turandot. Ces résultats reflètent la complexité des réactions de tolérance croisée entre infection et stress abiotique, et pourraient également éclairer les mécanismes sous-jacents de l'activation de ces réponses.

Rapid Evolutionary Adaptation to Diet Composition in the Black Soldier Fly (Hermetia illucens).

Genetic adaptation of Hermetia illucens (BSF) to suboptimal single sourced waste streams can open new perspectives for insect production. Here, four BSF lines were maintained on a single sourced, low-quality wheat bran diet (WB) or on a high-quality chicken feed diet (CF) for 13 generations. We continuously evaluated presumed evolutionary responses in several performance traits to rearing on the two diets. Subsequently, we tested responses to interchanged diets, i.e., of larvae that had been reared on low-quality feed and tested on high-quality feed and vice versa to evaluate costs associated with adaptation to different diets. BSF were found to experience rapid adaptation to the diet composition. While performances on the WB diet were always inferior to the CF diet, the adaptive responses were stronger to the former diet. This stronger response was likely due to stronger selection pressure experienced by BSF fed on the low-quality single sourced diet. The interchanged diet experiment found no costs associated with diet adaptation, but revealed cross generational gain associated with the parental CF diet treatment. Our results revealed that BSF can rapidly respond adaptively to diet, although the mechanisms are yet to be determined. This has potential to be utilized in commercial insect breeding to produce lines tailored to specific diets.

Variation among populations of Trichogramma euproctidis (Hymenoptera: Trichogrammatidae) revealed by life table parameters: perspectives for biological control.

The successful mass-rearing of potential biological control agents is a prerequisite for sustainable pest control. In this study, the performance of 3 Trichogramma euproctidis (Girault) (Hymenoptera: Trichogrammatidae) populations collected from different locations in Khuzestan (Southwest Iran) were evaluated to optimize the egg parasitoid mass-rearing for augmentative biological control of lepidopteran pests. We aimed to investigate the effects of both population origin and host quality on biological traits of ovipositing females (number of parasitized eggs) and of their progeny (development time, survival rate, sex ratio, longevity, and fecundity). The effect of host quality was assessed by allowing the parasitoid to oviposit into 1, 2, 3, or 4-day-old Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) eggs. The 3 T. euproctidis populations developed successfully regardless the age of the host eggs. However, we found significant variation among populations and a strong influence of host quality on the traits investigated. Progeny performance in all populations decreased with increasing host age. The best-performing population (collected in Mollasani) showed the highest parasitization rate, highest survival rate, and progeny sex ratio with the greatest percentage of females. A life table corroborated these findings with superior estimates of the net reproductive rate (R0), intrinsic rate of increase (r), and reduced generation time (T) for the Mollasani population on 1-day-old host eggs. We conclude that ample variation exists among T. euproctidis populations and that rearing the Mollasani population on young rather than old eggs of E. kuehniella would be recommended to implement the biological control programs to target lepidopteran pests in Southwestern Iran.

Molecular mechanisms underlying plasticity in a thermally varying environment.

Adaptation to environmental variability is a prerequisite for species' persistence in their natural environments. With climate change predicted to increase the frequency and severity of temperature fluctuations, ectothermic organisms may increasingly depend on acclimation capacity to accommodate thermal variability. To elucidate the molecular basis of fluctuating temperature-induced phenotypic plasticity, we investigated heat tolerance and the mechanisms induced by acclimation to thermal variability as compared to those seen at constant temperature. We ran genome-wide transcriptomic analysis on Drosophila melanogaster subjected to acclimation at constant (19 ± 0°C) and fluctuating (19 ± 8°C) temperatures and contrasted the induction of molecular mechanisms in adult males, adult females and larvae. We found life stage- and sex-specific dynamics of the acclimation responses to fluctuating temperatures. Adult flies exposed to temperature fluctuations showed a constitutive improvement in heat tolerance while heat tolerance of larvae tracked thermal fluctuations. A constitutive down-regulation of gene expression was observed for several genes in the larvae exposed to fluctuations. Our results for adult females showed that, for several genes, fluctuating temperature acclimation resulted in canalization of gene expression. Both transcriptional and post-transcriptional machinery were greatly affected by fluctuations in adult males. Gene ontology analysis showed enrichment of the heat stress response involving several major heat shock proteins in both larvae and adults exposed to fluctuating temperatures, even though fluctuations were in a benign range of temperatures. Finally, molecular mechanisms related to environmental sensing seem to be an important component of insect responses to thermal variability.

Harnessing thermal plasticity to enhance the performance of mass-reared insects: opportunities and challenges.

Insects are mass-reared for release for biocontrol including the sterile insect technique. Insects are usually reared at temperatures that maximize the number of animals produced, are chilled for handling and transport, and released into the field, where temperatures may be considerably different to those experienced previously. Insect thermal biology is phenotypically plastic (i.e. flexible), which means that there may exist opportunities to increase the performance of these programmes by modifying the temperature regimes during rearing, handling, and release. Here we synthesize the literature on thermal plasticity in relation to the opportunities to reduce temperature-related damage and increase the performance of released insects. We summarize how and why temperature affects insect biology, and the types of plasticity shown by insects. We specifically identify aspects of the production chain that might lead to mismatches between the thermal acclimation of the insect and the temperatures it is exposed to, and identify ways to harness physiological plasticity to reduce that potential mismatch. We address some of the practical (especially engineering) challenges to implementing some of the best-supported thermal regimes to maximize performance (e.g. fluctuating thermal regimes), and acknowledge that a focus only on thermal performance may lead to unwanted trade-offs with other traits that contribute to the success of the programme. Together, it appears that thermal physiological plasticity is well-enough understood to allow its implementation in release programmes.

Fungal infections lead to shifts in thermal tolerance and voluntary exposure to extreme temperatures in both prey and predator insects.

Pathogens can modify many aspects of host behavior or physiology with cascading impacts across trophic levels in terrestrial food webs. These changes include thermal tolerance of hosts, however the effects of fungal infections on thermal tolerances and behavioral responses to extreme temperatures (ET) across trophic levels have rarely been studied. We examined how a fungal pathogen, Beauveria bassiana, affects upper and lower thermal tolerance, and behavior of an herbivorous insect, Acyrthosiphon pisum, and its predator beetle, Hippodamia convergens. We compared changes in thermal tolerance limits (CTMin and CTMax), thermal boldness (voluntary exposure to ET), energetic cost (ATP) posed by each response (thermal tolerance and boldness) between healthy insects and insects infected with two fungal loads. Fungal infection reduced CTMax of both aphids and beetles, as well as CTMin of beetles. Fungal infection modified the tendency, or boldness, of aphids and predator beetles to cross either warm or cold ET zones (ETZ). ATP levels increased with pathogen infection in both insect species, and the highest ATP levels were found in individuals that crossed cold ETZ. Fungal infection narrowed the thermal tolerance range and inhibited thermal boldness behaviors to cross ET. As environmental temperatures rise, response to thermal stress will be asymmetric among members of a food web at different trophic levels, which may have implications for predator-prey interactions, food web structures, and species distributions.

Acclimation, duration and intensity of cold exposure determine the rate of cold stress accumulation and mortality in Drosophila suzukii.

The spotted wing drosophila (SWD), Drosophila suzukii, is a major invasive fruit pest. There is strong consensus that low temperature is among the main drivers of SWD population distribution, and the invasion success of SWD is also linked to its thermal plasticity. Most studies on ectotherm cold tolerance focus on exposure to a single stressful temperature but here we investigated how cold stress intensity affected survival duration across a broad range of low temperatures (-7 to +3 °C). The analysis of Lt50 at different stressful temperatures (Thermal Death Time curve - TDT) is based on the suggestion that cold injury accumulation rate increases exponentially with the intensity of thermal stress. In accordance with the hypothesis, Lt50 of SWD decreased exponentially with temperature. Further, comparison of TDT curves from flies acclimated to 15, 19 and 23 °C, respectively, showed an almost full compensation with acclimation such that the temperature required to induce mortality over a fixed time decreased almost 1 °C per °C lowering of acclimation temperature. Importantly, this change in cold tolerance with acclimation was uniform across the range of moderate to intense cold stress exposures examined. To understand if cold stress at moderate and intense exposures affects the same physiological systems we examined how physiological markers/symptoms of chill injury developed at different intensities of the cold stress. Specifically, hsp23 expression and extracellular [K+] were measured in flies exposed to different intensities of cold stress (-6, -2 and +2 °C) and at various time points corresponding to the same progression of injury (equivalent to 1/3, 2/3 or 3/3 of Lt50). The different cold stress intensities all triggered hsp23 expression following 2 h of recovery, but patterns of expression differed. At the most intense cold stress (-6 and -2 °C) a gradual increase with time was found. In contrast, at +2 °C an initial increase was followed by a dissipating expression. A gradual perturbation of ion balance (hyperkalemia) was also found at all three cold stress intensities examined, with only slight dissimilarities between treatment temperatures. Despite some differences between the three cold intensities examined, the results generally support the hypothesis that intense and moderate cold stress induces the same physiological perturbation. This suggests that cold stress experienced during natural fluctuating conditions is additive and the results also illustrate that the rate of injury accumulation increases dramatically (exponentially) with decreasing temperature (increasing stress).

Evidence for genetic isolation and local adaptation in the field cricket Gryllus campestris.

Understanding how species can thrive in a range of environments is a central challenge for evolutionary ecology. There is strong evidence for local adaptation along large-scale ecological clines in insects. However, potential adaptation among neighbouring populations differing in their environment has been studied much less. We used RAD sequencing to quantify genetic divergence and clustering of ten populations of the field cricket Gryllus campestris in the Cantabrian Mountains of northern Spain, and an outgroup on the inland plain. Our populations were chosen to represent replicate high and low altitude habitats. We identified genetic clusters that include both high and low altitude populations indicating that the two habitat types do not hold ancestrally distinct lineages. Using common-garden rearing experiments to remove environmental effects, we found evidence for differences between high and low altitude populations in physiological and life-history traits. As predicted by the local adaptation hypothesis, crickets with parents from cooler (high altitude) populations recovered from periods of extreme cooling more rapidly than those with parents from warmer (low altitude) populations. Growth rates also differed between offspring from high and low altitude populations. However, contrary to our prediction that crickets from high altitudes would grow faster, the most striking difference was that at high temperatures, growth was fastest in individuals from low altitudes. Our findings reveal that populations a few tens of kilometres apart have independently evolved adaptations to their environment. This suggests that local adaptation in a range of traits may be commonplace even in mobile invertebrates at scales of a small fraction of species' distributions.

Contrasting Manual and Automated Assessment of Thermal Stress Responses and Larval Body Size in Black Soldier Flies and Houseflies.

Within ecophysiological and genetic studies on insects, morphological and physiological traits are commonly assessed and phenotypes are typically obtained from manual measurements on numerous individuals. Manual observations are, however, time consuming, can introduce observer bias and are prone to human error. Here, we contrast results obtained from manual assessment of larval size and thermal tolerance traits in black soldier flies (Hermetia illucens) and houseflies (Musca domestica) that have been acclimated under three different temperature regimes with those obtained automatically using an image analysis software (Noldus EthoVision XT). We found that (i) larval size estimates of both species, obtained by manual weighing or by using the software, were highly correlated, (ii) measures of heat and cold tolerance using manual and automated approaches provided qualitatively similar results, and (iii) by using the software we obtained quantifiable information on stress responses and acclimation effects of potentially higher ecological relevance than the endpoint traits that are typically assessed when manual assessments are used. Based on these findings, we argue that automated assessment of insect stress responses and largescale phenotyping of morphological traits such as size will provide new opportunities within many disciplines where accurate and largescale phenotyping of insects is required.

Temperature Affects Biological Control Efficacy: A Microcosm Study of Trichogramma achaeae.

Current quality control of mass-reared biological control agents (BCAs) is usually performed in the laboratory and often fails to include behavioural aspects of the BCAs. As a result, the use of efficacy measurements determined solely under laboratory conditions to predict field efficacy can be questioned. In this study, microcosms were designed to estimate biological control efficacy (realised parasitisation efficiency) of Trichogramma achaeae Nagaraja and Nagarkatti (Hymenoptera: Trichogrammatidae) parasitising Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) eggs across the operational temperature range (15-30 °C). Temperature greatly affected the success of females in finding and parasitising E. kuehniella eggs, with parasitisation being reduced at 15 and 20 °C, as both the percentage of parasitised host eggs and the percentage of leaves per plant with parasitised host eggs decreased sharply compared with higher temperatures. Graphing previous data on laboratory fecundity against parasitisation efficiency shows that the laboratory-measured fecundity of T. achaeae was unlikely to predict field efficacy across temperatures. Results also showed that leaf side had no effect on the preference of T. achaeae in parasitising E. kuehniella eggs; however, T. achaeae preferred to lay their eggs on the top tier of plants. These findings suggest that more complex assays, which include behavioural responses, might be developed for optimised quality control of BCAs intended for field application.

Cold acclimation increases depolarization resistance and tolerance in muscle fibers from a chill-susceptible insect, Locusta migratoria.

Cold exposure depolarizes cells in insects due to a reduced electrogenic ion transport and a gradual increase in extracellular K+ concentration ([K+]). Cold-induced depolarization is linked to cold injury in chill-susceptible insects, and the locust, Locusta migratoria, has been shown to improve cold tolerance following cold acclimation through depolarization resistance. Here we investigate how cold acclimation influences depolarization resistance and how this resistance relates to improved cold tolerance. To address this question, we investigated if cold acclimation affects the electrogenic transport capacity and/or the relative K+ permeability during cold exposure by measuring membrane potentials of warm- and cold-acclimated locusts in the presence and absence of ouabain (Na+-K+ pump blocker) or 4-aminopyridine (4-AP; voltage-gated K+ channel blocker). In addition, we compared the membrane lipid composition of muscle tissue from warm- and cold-acclimated locust and the abundance of a range transcripts related to ion transport and cell injury accumulation. We found that cold-acclimated locusts are depolarization resistant due to an elevated K+ permeability, facilitated by opening of 4-AP-sensitive K+ channels. In accordance, cold acclimation was associated with an increased abundance of Shaker transcripts (gene encoding 4-AP-sensitive voltage-gated K+ channels). Furthermore, we found that cold acclimation improved muscle cell viability following exposure to cold and hyperkalemia even when muscles were depolarized substantially. Thus cold acclimation confers resistance to depolarization by altering the relative ion permeability, but cold-acclimated locusts are also more tolerant to depolarization.

Drawing the line: Linear or non-linear reaction norms in response to adult acclimation on lower thermal limits.

Estimates of lower thermal limits are widely used to infer sensitivity to climate variability, local adaptation and adaptive acclimation responses in ectotherms. These inferences build on the ecological relevance of the tolerance estimates and assume that estimates can be extrapolated to relevant conditions. Methodological effects for upper thermal limits have been extensively investigated, with different ramping rates and acclimation regimes giving rise to varying, and even disparate, conclusions. However, methodological effects have received much less attention for lower thermal limits. In this study, we explicitly test whether methodology could affect estimates of lower thermal limits in interaction with acclimation temperature and thermal variability, by acclimating adult Drosophila melanogaster to different constant and fluctuating temperature regimes and generating reaction norms at different ramping rates. We find that ramping rates have no significant effect on the lower thermal limits. Constant temperature acclimation resulted in non-linear reaction norms, while the introduction of thermal variability during adult life result in linear reaction norms. Thus, applying ecologically relevant conditions (here thermal variability) potentially impacts the results and conclusions of insect low temperature tolerance and acclimation capacity.

Testing the thermal limits: Non-linear reaction norms drive disparate thermal acclimation responses in Drosophila melanogaster.

Critical thermal limits are important ecological parameters for studying thermal biology and for modelling species' distributions under current and changing climatic conditions (including predicting the risk of extinction for species from future warming). However, estimates of the critical thermal limits are biased by the choice of assay and assay conditions, which differ among studies. Furthermore, estimates of the potential for phenotypic plasticity (thermal acclimation) to buffer against thermal variability are usually based on single assay conditions and (usually linear) extrapolation from a few acclimation temperatures. We produced high resolution estimates of adult acclimation capacity for upper tolerance limits at different assay conditions (ramping rates and knock-down temperatures) using CTmax (dynamic) and knock-down (static) thermal assays in the model species Drosophila melanogaster. We found the reaction norms to be highly dependent on assay conditions. We confirmed that progressively lower ramping rates or higher knock-down temperatures led to overall lower tolerance estimates. More surprisingly, extended assays (lower ramping rates or lower knock-down temperatures) also led to increasingly non-linear reaction norms for upper thermal tolerance across adult acclimation temperatures. Our results suggest that the magnitude (capacity) and direction (beneficial or detrimental) of acclimation responses are highly sensitive to assay conditions. The results offer a framework for comparison of acclimation responses between different assay conditions and a potential for explaining disparate acclimation capacity theories. We advocate cautious interpretation of acclimation capacities and careful consideration of assay conditions, which should represent realistic environmental conditions based on species' ecological niches.

Expression of thermal tolerance genes in two Drosophila species with different acclimation capacities.

Heat tolerance increases at higher acclimation temperatures in D. melanogaster, but not in D. subobscura. The two species represent separate lineages of the subgenus Sophophora of Drosophila with contrasting tropical African and temperate Palearctic evolutionary histories. D. melanogaster has five copies of the inducible hsp70 gene distributed in two clusters, named A (with two copies) and B (three copies), while D. subobscura has only two copies arranged similarly to cluster A of D. melanogaster. The hsp70s of the two species also differ in their cis-regulatory regions, with D. melanogaster exhibiting features of a faster and more productive promoter. We predicted that the interspecific variation in acclimation capacity of heat tolerance is explained by evolved variation in expression of the major group of heat shock proteins. To test this prediction, we compared basal levels of gene expression at different developmental temperatures within each of the two species. Furthermore, we explored the heat hardening dynamics by measuring the induction of gene expression during a ramping assay. The prediction of a stronger heat shock protein response in D. melanogaster as compared to D. subobscura was confirmed for both long-term acclimation and short-term hardening. For D. melanogaster the upregulation with temperature ramping ranged from less than two fold (hsp26) to 2500 fold (hsp70A) increase. In all cases induction in D. melanogaster exceeded that of D. subobscura homologs. These differences correlate with structural differences in the regulatory regions of hsp70, and might explain differences in acclimation capacity among species. Finally, in D. melanogaster we found an indication of an inverse relationship between basal and induced levels of hsp70A and hsp83 expression, suggesting a divergent role for thermal adaptation of these genes at benign and stressful temperatures, respectively.

How much starvation, desiccation and oxygen depletion can Drosophila melanogaster tolerate before its upper thermal limits are affected?

Heat tolerance is commonly assessed as the critical thermal maximum (CTmax) using the dynamic method exposing organisms to a gradually increasing (ramping) temperature until organisms fall into a coma. The CTmax estimate is dependent on the ramping rate, with decreased rates leading to longer treatments and ultimately lower CTmax estimates. There is a current discussion surrounding the physiological dynamics of the effect of the time of exposure by temperature interaction on these estimates. Besides temperature the time of exposure to limited food (starvation), desiccation, and reduced levels of oxygen or increased levels of CO2 may, in interaction with ramping rate, act as confounding factors when assessing upper thermal limits using the dynamic method. Here we test the role of the different potentially confounding factors for assaying thermal tolerance using a ramping assay under four different ramping rates, varying from 0.01 °C/min to 0.2 °C/min. We find that CTmax values are higher at faster ramping rates and that oxygen or CO2 concentration does not show any negative effect on the CTmax values obtained within the experimental pre-treatment period (32 h). Both water (up to 6 h) and food (up to 42 h) deprivation prior to assay showed a negative correlation with thermal tolerance of the flies. For both traits, we found a significant interaction with ramping rate, most likely due to prolonged assays at lower rates. However, as little water was lost during the ramping assay and as food deprivation only modestly affected CTmax values, results were very robust to the conditions experienced during the assay (even at slow rates) and mainly affected by the conditions experienced prior to performing the assay. Thus, for the most commonly applied experimental conditions CTmax estimates are unlikely to be biased or confounded by ramping rate, starvation, desiccation or deteriorating atmospheric composition.

Constitutive up-regulation of Turandot genes rather than changes in acclimation ability is associated with the evolutionary adaptation to temperature fluctuations in Drosophila simulans.

Most research on thermal adaptation of ectotherms is based on experiments performed at constant temperatures. However, for short-lived insects daily fluctuations of temperature could be an important environmental parameter involved in evolutionary adaptation to thermal heterogeneity. In this study we investigated the mechanisms underlying evolutionary adaptation to daily fluctuating temperatures. We studied replicated selection lines of Drosophila simulans evolved in a constant or a daily fluctuating thermal regime. Previous studies of these lines have shown clear acclimation benefits to heat tolerance induced by the fluctuating regime. First, we tested the existence of an evolved circadian controlled adjustment of heat resistance in selected flies. This was done by investigating the daily variation in time to heat knockdown in flies from both selection regimes when exposed to either a constant or a daily fluctuating thermal regime for a single generation. While daily variation in heat resistance was found, the results suggest that there was neither an evolved adaptive circadian controlled adjustment of heat resistance nor a continuous acclimation response induced by fluctuating temperatures in these lines. Second, in order to reveal functional candidates for adaptation to the fluctuating thermal regime, we investigated the global transcriptomic response to a high temperature exposure in flies from both regimes. We found that flies selected both in constant and fluctuating thermal regimes responded similarly to increasing temperature. However, we found that evolutionary adaptation to the fluctuating thermal regime led to transcriptional enrichment of the GO terms eggshell chorion assembly and cellular response to heat. The latter category was constituted by a constitutive up-regulation of four Turandot genes and not heat shock protein genes, suggesting that Turandot genes could play a prominent role for adaptation to daily fluctuating thermal conditions.

Critical thermal limits affected differently by developmental and adult thermal fluctuations.

Means and variances of the environmental thermal regime play an important role in determining the fitness of terrestrial ectotherms. Adaptive phenotypic responses induced by heterogeneous temperatures have been shown to be mediated by molecular pathways independent of the classic heat shock responses; however, an in-depth understanding of plasticity induced by fluctuating temperatures is still lacking. We investigated high and low temperature acclimation induced by fluctuating thermal regimes at two different mean temperatures, at two different amplitudes of fluctuation and across the developmental and adult life stages of Drosophila melanogaster For developmental acclimation, we found mildly detrimental effects of high-amplitude fluctuations for critical thermal minima, while the critical thermal maxima showed a beneficial response to higher amplitude fluctuations. For adult acclimation involving shifts between fluctuating and constant regimes, cold tolerance was shown to be dictated by developmental temperature conditions irrespective of the adult treatments, while the acquired heat tolerance was readily lost when flies developed at fluctuating temperature were shifted to a constant regime as adults. Interestingly, we also found that the effect of fluctuations at any life stage was gradually lost with prolonged adult maintenance, suggesting a more prominent effect of fluctuations during developmental compared with adult acclimation in D.melanogaster.

Evolutionary and ecological patterns of thermal acclimation capacity in Drosophila: is it important for keeping up with climate change?

Phenotypic plasticity of temperature tolerance (thermal acclimation) is often highlighted as an important component of the acute and evolutionary adaptation to temperatures in insects. For this reason, it is often suggested that thermal acclimation ability could be important for buffering the consequences of climate change. Based on data from Drosophila we discuss if and how phenotypic plasticity is likely to mitigate the effects of climate change. We conclude that plasticity of upper thermal limits is small in magnitude, evolves slowly and that acclimation ability is weakly correlated with latitude and environmental heterogeneity. Accordingly plasticity in upper thermal limits is unlikely to effectively buffer effects of global warming for species already close to their upper thermal boundaries.