Dietary nutrient balance shapes phenotypic traits of Drosophila melanogaster in interaction with gut microbiota.

The dietary nutrient composition can affect insects' phenotypes by modulating their physiology. Furthermore, diet can affect gut microbiota composition and abundance, with indirect consequences for the host. In this study, we reared Drosophila melanogaster on five different diets; three with balanced sugar:yeast ratio, but with increasing caloric content (2:2, 8:8, 16:16, in weight %), and two with imbalanced sugar:yeast ratio, either with low sugar and high yeast content (2:16) or vice-versa (16:2). In each of these diets, we compared flies with conventional vs. artificially altered gut microbiota with antibiotics that reduced the bacterial load. The antibiotic treatment also had the surprising effect of increasing the amount of live yeast associated with the flies. We characterized flies from these ten treatments (5 diets × 2 microbiota) in terms of development, body mass, food preference, body reserves, metabolic rate and a range of stress tolerance traits (heat, cold, starvation and desiccation tolerance). Diets, and to a lesser extent antibiotic treatment, affected development rate, weight, and cold tolerance of adult flies. Other traits such as energy reserves, metabolic rate, food preference, or starvation tolerance were affected by diet alone. When detected, the effect of antibiotic treatment was stronger in yeast-poor diets, suggesting that gut bacterial community might help to counterbalance nutritional deficiencies. These results show that changes in dietary factors lead to a global re-organization of fly's physiology and development while the manipulation of gut microorganisms had minor effects that were mainly seen in case of protein restriction.

Combined effect of temperature and ammonia on molecular response and survival of the freshwater crustacean Gammarus pulex.

Freshwater ecosystems are experiencing mounting pressures from agriculture, urbanization, and climate change, which could drastically impair aquatic biodiversity. As nutrient inputs increase and temperatures rise, ammonia (NH3) concentration is likely to be associated with stressful temperatures. To investigate the interaction between NH3 and temperature on aquatic invertebrate survival, we performed a factorial experiment on the survival and molecular response of Gammarus pulex, with temperature (10, 15, 20, and 25°C) and NH3 (0, 0.5, 1, 2, 3, and 4mg NH3/L) treatments. We observed an unexpected antagonistic interaction between temperature and NH3 concentration, meaning survival in the 4mg NH3/L treatment was higher at 25°C than at the control temperature of 10°C. A toxicokinetic-toxicodynamic (TK-TD) model was built to describe this antagonistic interaction. While the No Effect Concentration showed no significant variation across temperatures, the 50% lethal concentration at the end of the experiment increased from 2.7 (2.1-3.6) at 10°C to 5.5 (3.5- 23.4) mg NH3/L at 25°C. Based on qPCR data, we associated these survival patterns to variations in the expression of the hsp70 gene, a generic biomarker of stress. However, though there was a 14-fold increase in hsp70 mRNA expression for gammarids exposed to 25°C compared to controls, NH3 concentration had no effect on hsp70 mRNA synthesis across temperatures. Our results demonstrate that the effects of combined environmental stressors, like temperature and NH3, may strongly differ from simple additive effects, and that stress response to temperature can actually increase resilience to nutrient pollution in some circumstances.

Metabolic effects of CO(2) anaesthesia in Drosophila melanogaster.

Immobilization of insects is necessary for various experimental purposes, and CO(2) exposure remains the most popular anaesthetic method in entomological research. A number of negative side effects of CO(2) anaesthesia have been reported, but CO(2) probably brings about metabolic modifications that are poorly known. In this work, we used GC/MS-based metabolic fingerprinting to assess the effect of CO(2) anaesthesia in Drosophila melanogaster adults. We analysed metabolic variation of flies submitted to acute CO(2) exposure and assessed the temporal metabolic changes during short- and long-term recovery. We found that D. melanogaster metabotypes were significantly affected by the anaesthetic treatment. Metabolic changes caused by acute CO(2) exposure were still manifested after 14 h of recovery. However, we found no evidence of metabolic alterations when a long recovery period was allowed (more than 24 h). This study points to some metabolic pathways altered during CO(2) anaesthesia (e.g. energetic metabolism). Evidence of short-term metabolic changes indicates that CO(2) anaesthesia should be used with utmost caution in physiological studies when a short recovery is allowed. In spite of this, CO(2) treatment seems to be an acceptable anaesthetic method provided that a long recovery period is allowed (more than 24 h).

Disruption of ATP homeostasis during chronic cold stress and recovery in the chill susceptible beetle (Alphitobius diaperinus).

This study examined the impact of fluctuating thermal regimes (FTRs) on cold tolerance of the polyphagous beetle Alphitobius diaperinus. Daily pulses of elevated temperatures can provide breaks in chronic cold stress, potentially allowing for physiological recovery and improving survival. Perturbations in central metabolism appear to be a common physiological response in insects exposed to low temperatures. It has been suggested that energy supplies, which may be depleted during cold exposure, can be regenerated during the warming pulses of FTRs. This study tested the assumption that chronic cold stress may induce ATP depletion and that recovery during FTR warming pulses may allow re-establishment of ATP supplies. In this study, A. diaperinus were exposed to cold stress under different thermal regimes (constant or fluctuating). The results did not confirm the aforementioned assumption. No cold-induced ATP depletion was observed. The lowest ATP levels were repeatedly detected in the untreated controls. The data show that homoeostasis of ATP is lost when adults A. diaperinus are exposed to cold stress, whatever thermal regime (constant or fluctuating). ATP accumulation may be viewed as a symptom of a production/consumption imbalance under cold stress conditions. Periodic short (2-h) warming pulses clearly improved cold survival. Cellular homeostasis, however, probably requires a longer recovery period to be fully restored.

Larval density affects phenotype and surrounding bacterial community without altering gut microbiota in Drosophila melanogaster.

Larval crowding represents a complex stressful situation arising from inter-individual competition for time- and space-limited resources. The foraging of a large number of individuals may alter the chemical and bacterial composition of food and in turn affect individual's traits. Here we used Drosophila melanogaster to explore these assumptions. First, we used a wide larval density gradient to investigate the impact of crowding on phenotypical traits. We confirmed that high densities increased development time and pupation height, and decreased viability and body mass. Next, we measured concentrations of common metabolic wastes (ammonia, uric acid) and characterized bacterial communities, both in food and in larvae, for three contrasting larval densities (low, medium and high). Ammonia concentration increased in food from medium and high larval densities, but remained low in larvae regardless of the larval density. Uric acid did not accumulate in food but was detected in larvae. Surprisingly, bacterial composition remained stable in guts of larvae whatever their rearing density, although it drastically changed in the food. Overall, these results indicate that crowding deeply affects individuals, and also their abiotic and biotic surroundings. Environmental bacterial communities likely adapt to altered nutritional situations resulting from crowding, putatively acting as scavengers of larval metabolic wastes.

Male reproductive potential of Aphidius colemani (Hymenoptera: Aphidiinae) exposed to constant or fluctuating thermal regimens.

Prolonged exposure to low temperature generally induces deleterious effects on survival and reproduction of insects. Reproduction costs are well documented in cold-exposed female parasitoids, but there is little information concerning males. In some species, low temperature is suspected to cause male sterility. Mummies of the aphid parasitoid Aphidius colemani Viereck (Hymenoptera: Aphidiinae) were exposed to either fluctuating thermal regimens (FTR: 4 degrees C, 22 h; 20 degrees C, 2 h) or constant low temperature (CLT: 4 degrees C) for 15 d. We verified whether cold exposure can sterilize males and evaluated treatment-related survival, reproductive potential, and mobility parameters. Sterility trials showed that cold-exposed males were all fertile. Survival and reproductive potential of males (e.g., mating success, premating period, and competition for mating) were negatively affected when individuals were exposed to CLT. These alterations were associated with a reduction in locomotion performances during premating period. When parasitoids were exposed to FTR, survival, reproductive potential, and mobility parameters were unaffected. The reduced survival and mobility under CLT, probably results physiological perturbations: processes that may have a limited impact on individuals exposed to FTR. The consequence of mobility reduction on partner acceptance and competitive mating ability is discussed.

Cold exposure and associated metabolic changes in adult tropical beetles exposed to fluctuating thermal regimes.

Environmental stress deleteriously affects every aspect of an ectotherm's biological function. Frequent exposure of terrestrial insects to temperature variation has thus led to the evolution of protective biochemical and physiological mechanisms. However, the physiological mechanisms underlying the positive impact of fluctuating thermal regimes (FTRs) on the fitness and survival of cold-exposed insects have not been studied. We have thus investigated the metabolic changes in adults of the beetle Alphitobius diaperinus in order to determine whether FTRs trigger the initiation of a metabolic response involving synthesis of protective compounds, such as free amino acids (FAAs) and polyols. The metabolic profile was analyzed during constant fluctuating thermal regimes (the beetles had daily pulses at higher temperatures that enabled them to recover) and compared with constant cold exposure and untreated controls. The increase of several essential amino acids (Lys, Iso, Leu, Phe and Trp) in cold-exposed beetles supports the conclusion that it results from the breakdown of proteins. Some FAAs have been shown to have cryoprotective properties in insects, but the relationship between FAAs, cold tolerance and survival has not yet been well defined. Instead of considering FAAs only as a part of the osmo- and cryoprotective arsenal, they should also be regarded as main factors involved in the multiple regulatory pathways activated during cold acclimation. Under FTRs, polyol accumulation probably contributes to the increased duration of survival in A. diaperinus.

Seasonal changes in the cold hardiness of the two-spotted spider mite females (Acari: Tetranychidae).

The twospotted spider mite Tetranychus urticae Koch (Acari: Tetranychidae) is an important agricultural pest. Population dynamics and pest outbreaks highly depend on the overwintering success of the mite specimens; therefore, it is necessary to assess winter survival dynamics of this pest. Seasonal changes in supercooling point (SCP) and acute cold tolerance (2-h exposure at -5, -10, -15, -20, -23, or -25°C) were assessed in field-collected females during the winter in 2010-2011 in Iran. The SCP values varied from a minimum of -30.5°C (January 2011) to a maximum of -12.6°C (April 2011). Significant differences were recorded in the SCP distribution patterns between autumn- and winter-sampled females, depicting the acquisition of cold hardiness over the winter. The mean ambient air temperature was the lowest in January (4°C), when the females showed the highest supercooling ability. Correlated patterns between monthly temperatures and acute cold tolerance also were found. At -20°C, the survival of the mites was very low (10%) when they were sampled in October 2010; whereas it was high (97.5%) in January 2011, before decreasing to 5% in April 2011. The present data show that T. urticae females are chill tolerant and capable of adjusting their cold tolerance over the winter season. Acute cold tolerance (-15 and -20°C) and SCP represent valuable metrics that can be used for predicting the seasonal changes of the cold hardiness of T. urticae females.

Manipulation of parasitoid size using the temperature-size rule: fitness consequences.

The phenotypic effects of rearing temperature on several fitness components of the koinobiont parasitoid, Aphidius colemani, were examined. Temperatures experienced during development induced a plastic linear response in the dry and fat masses of the immature stage and a non-linear response in the growth rate as well as in the size of adults. We investigated if the phenotypic morphometrical plasticity exhibited by parasitoids reared at different temperatures can induce variations in fitness-related traits in females. We did not find any difference in immature (pupal) mortality in accordance to rearing temperature. However, when examining adult longevity, we found an inverse linear relation with developmental temperature, confirming the usual rule that larger and fatter wasps live longer than smaller ones. The pattern of female fecundity was non-linear; wasps that developed at high and low temperatures were less productive. We suggest that when development is short, the accumulated reserves are not adequate to support both fecundity and survival. By manipulating adult size through changes in the rearing temperature, we showed that the usual shape of the size/fitness function is not always linear as expected. Developmental temperature induced a plasticity in energy reserves which affected the functional constraints between survival and reproduction.