Tommaso Manenti

How to assess Drosophila cold tolerance: chill coma temperature and lower lethal temperature are the best predictors of cold distribution limits

Summary 1. Thermal tolerance may limit and therefore predict ectotherm geographic distributions. However, which of the many metrics of thermal tolerance best predict distribution is often unclear, even for drosophilids, which constitute a popular and well-described animal model. 2. Five metrics of cold tolerance were measured for 14 Drosophila species to determine which metrics most strongly correlate with geographic distribution. The species represent tropical to temperate regions but all were reared under similar (common garden) conditions (20 °C). The traits measured were: chill coma temperature (CTmin), lethal temperature (LTe50), lethal time at low temperature (LTi50), chill coma recovery time (CCRT) and supercooling point (SCP). 3. Measures of CTmin, LTe50 and LTi50 proved to be the best predictors to describe the variation in realized latitudinal distributions (R 2 =0 699, R 2 =0 741 and 0550, respectively) and estimated environmental cold exposure (R 2 =0 633, R 2 =0 641 and 0511, respectively). Measures of CCRT also correlated significantly with estimated minimum temperature (R 2 =0 373), while the SCP did not. These results remained consistent after phylogenetically independent analysis or when applying nonlinear regression. Moreover, our findings were supported by a similar analysis based on existing data compiled from the Drosophila cold tolerance literature. 4. Trait correlations were strong between LTe50, LTi50 and CTmin, respectively (083 > R 2 >0 55). However, surprisingly, there was only a weak correlation between the entrance into coma (CTmin) and the recovery from chill coma (CCRT) (R 2 =0 256). 5. Considering the findings of the present study, data from previous studies and the logistical constraints of each measure of cold tolerance, we conclude that CTmin and LTe50 are superior measures when estimating the ecologically relevant cold tolerance of drosophilids. Of these two traits, CTmin requires less equipment, time and animals and thereby presents a relatively fast, simple and dynamic measure of cold tolerance.

Predictability rather than amplitude of temperature fluctuations determines stress resistance in a natural population of Drosophila simulans

The adaptability of organisms to novel environmental conditions depends on the amount of genetic variance present in the population as well as on the ability of individuals to adjust their phenotype through phenotypic plasticity. Here, we investigated the phenotypic plasticity induced by a single generations exposure to three different temperature regimes with respect to several life‐history and stress‐resistance traits in a natural population of Drosophila simulans. We studied a constant as well as a predictably and an unpredictably fluctuating temperature regime. We found high levels of phenotypic plasticity among all temperature regimes, suggesting a strong influence of both temperature fluctuations and their predictability. Increased heat tolerance was observed for flies developed in both types of fluctuating thermal environments compared with flies developed in a constant environment. We suggest that this was due to beneficial hardening when developing in either fluctuating temperature environment. To our surprise, flies that developed in constant and predictably changing environments were similar to each other in most traits when compared to flies from the unpredictably fluctuating environment. The unpredictably changing thermal environment imposed the most stressful condition, resulting in the lowest performance for stress‐related traits, even though the absolute temperature changes never exceeded that of the predictably fluctuating environment. The overall decreased stress resistance of flies in the unpredictably fluctuating environment may be the consequence of maladaptive phenotypic plasticity in this setting, indicating that the adaptive value of plasticity depends on the predictability of the environment.

Phenotypic plasticity is not affected by experimental evolution in constant, predictable or unpredictable fluctuating thermal environments

The selective past of populations is presumed to affect the levels of phenotypic plasticity. Experimental evolution at constant temperatures is generally expected to lead to a decreased level of plasticity due to presumed costs associated with phenotypic plasticity when not needed. In this study, we investigated the effect of experimental evolution in constant, predictable and unpredictable daily fluctuating temperature regimes on the levels of phenotype plasticity in several life history and stress resistance traits in Drosophila simulans. Contrary to the expectation, evolution in the different regimes did not affect the levels of plasticity in any of the traits investigated even though the populations from the different thermal regimes had evolved different stress resistance and fitness trait means. Although costs associated with phenotypic plasticity are known, our results suggest that the maintenance of phenotypic plasticity might come at low and negligible costs, and thus, the potential of phenotypic plasticity to evolve in populations exposed to different environmental conditions might be limited.

The Role of Storage Lipids in the Relation between Fecundity, Locomotor Activity, and Lifespan of Drosophila melanogaster Longevity-Selected and Control Lines

The contribution of insect fat body to multiple processes, such as development, metamorphosis, activity, and reproduction results in trade-offs between life history traits. In the present study, age-induced modulation of storage lipid composition in Drosophila melanogaster longevity-selected (L) and non-selected control (C) lines was studied and the correlation between total body fat mass and lifespan assessed. The trade-offs between fecundity, locomotor activity, and lifespan were re-evaluated from a lipid-related metabolic perspective. Fewer storage lipids in the L lines compared to the C lines supports the impact of body fat mass on extended lifespan. The higher rate of fecundity and locomotor activity in the L lines may increase the lipid metabolism and enhance the lipolysis of storage lipids, reducing fat reserves. The correlation between neutral lipid fatty acids and fecundity, as well as locomotor activity, varied across age groups and between the L and C lines. The fatty acids that correlated with egg production were different from the fatty acids that correlated with locomotor activity. The present study suggests that fecundity and locomotor activity may positively affect the lifespan of D. melanogaster through the inhibition of fat accumulation.

Genetic rescue of an endangered domestic animal through outcrossing with closely related breeds: A case study of the Norwegian Lundehund

Genetic rescue, outcrossing with individuals from a related population, is used to augment genetic diversity in populations threatened by severe inbreeding and extinction. The endangered Norwegian Lundehund dog underwent at least two severe bottlenecks in the 1940s and 1960s that each left only five inbred dogs, and the approximately 1500 dogs remaining world-wide today appear to descend from only two individuals. The Lundehund has a high prevalence of a gastrointestinal disease, to which all remaining dogs may be predisposed. Outcrossing is currently performed with three Nordic Spitz breeds: Norwegian Buhund, Icelandic Sheepdog, and Norrbottenspets. Examination of single nucleotide polymorphism (SNP) genotypes based on 165K loci in 48 dogs from the four breeds revealed substantially lower genetic diversity for the Lundehund (HE 0.035) than for other breeds (HE 0.209–0.284). Analyses of genetic structure with > 15K linkage disequilibrium-pruned SNPs showed four distinct genetic clusters. Pairwise FST values between Lundehund and the candidate breeds were highest for Icelandic Sheepdog, followed by Buhund and Norrbottenspets. We assessed the presence of outlier loci among candidate breeds and examined flanking genome regions (1 megabase) for genes under possible selection to identify potential adaptive differences among breeds; outliers were observed in flanking regions of genes associated with key functions including the immune system, metabolism, cognition and physical development. We suggest crossbreeding with multiple breeds as the best strategy to increase genetic diversity for the Lundehund and to reduce the incidence of health problems. For this project, the three candidate breeds were first selected based on phenotypes and then subjected to genetic investigation. Because phenotypes are often paramount for domestic breed owners, such a strategy could provide a helpful approach for genetic rescue and restoration of other domestic populations at risk, by ensuring the involvement of owners, breeders and managers at the start of the project.

Environmental heterogeneity does not affect levels of phenotypic plasticity in natural populations of three Drosophila species

Abstract Adaptation of natural populations to variable environmental conditions may occur by changes in trait means and/or in the levels of plasticity. Theory predicts that environmental heterogeneity favors plasticity of adaptive traits. Here we investigated the performance in several traits of three sympatric Drosophila species freshly collected in two environments that differ in the heterogeneity of environmental conditions. Differences in trait means within species were found in several traits, indicating that populations differed in their evolutionary response to the environmental conditions of their origin. Different species showed distinct adaptation with a very different role of plasticity across species for coping with environmental changes. However, geographically distinct populations of the same species generally displayed the same levels of plasticity as induced by fluctuating thermal regimes. This indicates a weak and trait‐specific effect of environmental heterogeneity on plasticity. Furthermore, similar levels of plasticity were found in a laboratory‐adapted population of Drosophila melanogaster with a common geographic origin but adapted to the laboratory conditions for more than 100 generations. Thus, this study does not confirm theoretical predictions on the degree of adaptive plasticity among populations in relation to environmental heterogeneity but shows a very distinct role of species‐specific plasticity.

Inbreeding Affects Locomotor Activity in Drosophila melanogaster at Different Ages

The ability to move is essential for many behavioural traits closely related to fitness. Here we studied the effect of inbreeding on locomotor activity (LA) of Drosophila melanogaster at different ages under both dark and light regimes. We expected to find a decreased LA in inbred lines compared to control lines. We also predicted an increased differentiation between lines due to inbreeding. LA was higher in the dark compared to the light regime for both inbred and outbred control lines. As expected, inbreeding increased phenotypic variance in LA, with some inbred lines showing higher and some lower LA than control lines. Moreover, age per se did not affect LA neither in control nor in inbred lines, while we found a strong line by age interaction between inbred lines. Interestingly, inbreeding changed the daily activity pattern of the flies: these patterns were consistent across all control lines but were lost in some inbred lines. The departure in the daily pattern of LA in inbred lines may contribute to the inbreeding depression observed in inbred natural populations.

Phospholipid fatty acid composition linking larval-density to lifespan of adult Drosophila melanogaster.

Pre-adult density-associated alterations in the composition of storage lipids may affect the cell membrane fatty acid profile (mainly phospholipids), membrane integrity, and cell function. The present study evaluated the impact of pre-adult density conditions, sex, and the selection regime on the composition of phospholipid fatty acids and lifespan of Drosophila melanogaster. The phospholipid profile of adult flies developed under larval crowding contained a higher proportion of polyunsaturated fatty acids, lower proportion of monounsaturated fatty acids, and greater risk of peroxidation. There was also a negative correlation between the peroxidation index (PI) and longevity. The longevity-selected females showed a lower PI compared with control lines under both densities. The present results indicate that pre-adult density may play a significant role in the lifespan of adult flies by altering the composition of phospholipids and shaping cell membrane bilayers with different susceptibilities to peroxidation.

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.

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.