A trade-off between desiccation resistance and developmental humidity for pupation height in the North Indian seasonal population of Drosophilid-Zaprionus indianus.

Drosophila larvae and pupae are vulnerable to seasonal abiotic stressors such as humidity and temperature. In wild low-humidity habitats, desiccation stress can occur as Drosophila larvae forsake wet food in search of a drier pupation site. Henceforth, the hypothesis that developmental humidity impacts pupation height, affecting larval and pupae water balance and fitness-related traits, was examined. Accordingly, warm-adapted Drosophilid- Zaprionus indianus from two seasons were reared under season-specific simulated conditions, with significantly varying relative humidity (summer RH: 40%; rainy RH: 80%), but nearly identical temperatures. A trade-off between pupation height and developmental humidity was observed. Drier summer conditions lead to pupae wandering farther from drier glass surfaces, resulting in higher pupation height (17.3 cm) while rainy pupae prefer wet food, resulting in lower pupation height (7.12 cm). Additionally, density-dependent pupation height was developmental humidity-specific, with most rainy-season pupae pupated on wetter food, while dry summer pupae pupated on glass surfaces or cotton. Nevertheless, flies from far pupation exhibited greater desiccation resistance, fecundity, and copulation duration than those from near pupation. The cuticular lipid mass of larvae and pupae was higher during far-than-near pupation, indicating decreased water loss rates compared to near-pupation. Finally, pupae eclosion (%) was unaffected by greater humidity (85%) in either season. Still, it considerably decreased at lower humidity (RH: 0% and 38%) for rainy pupae, further supporting the selection of low-humidity desiccation resistance in pupae. In conclusion, low humidity is crucial for survival of pre-adult stages of Zaprionus indianus under desiccation stress and for preference of pupation site.

Cross-tolerance effects due to adult heat hardening, desiccation and starvation acclimation of tropical drosophilid-Zaprionus indianus.

Some insect taxa from polar or temperate habitats have shown cross-tolerance for multiple stressors but tropical insect taxa have received less attention. Accordingly, we considered adult flies of a tropical drosophilid-Zaprionus indianus for testing direct as well as cross-tolerance effects of rapid heat hardening (HH), desiccation acclimation (DA) and starvation acclimation (SA) after rearing under warmer and drier season specific simulated conditions. We observed significant direct acclimation effects of HH, DA and SA; and four cases of cross-tolerance effects but no cross-tolerance between desiccation and starvation. Cross-tolerance due to heat hardening on desiccation showed 20% higher effect than its reciprocal effect. There is greater reduction of water loss in heat hardened flies (due to increase in amount of cuticular lipids) as compared with desiccation acclimated flies. However, cross-tolerance effect of SA on heat knockdown was two times higher than its reciprocal. Heat hardened and desiccation acclimated adult flies showed substantial increase in the level of trehalose and proline while body lipids increased due to heat hardening or starvation acclimation. However, maximum increase in energy metabolites was stressor specific i.e. trehalose due to DA; proline due to HH and total body lipids due to SA. Rapid changes in energy metabolites due to heat hardening seem compensatory for possible depletion of trehalose and proline due to desiccation stress; and body lipids due to starvation stress. Thus, observed cross-tolerance effects in Z. indianus represent physiological changes to cope with multiple stressors related to warmer and drier subtropical habitats.

Cold and desiccation stress induced changes in the accumulation and utilization of proline and trehalose in seasonal populations of Drosophila immigrans.

Changes in the levels of energy metabolites can limit survival ability of Drosophila species under stressful conditions but this aspect has received less attention in wild populations collected in different seasons. We tested cold or desiccation triggered changes in the accumulation or utilization of two energy metabolites (trehalose and proline) in Drosophila immigrans flies reared under season specific environmental conditions. Such D.immigrans populations were subjected to different durations of cold (0°C) or desiccation stress (5% RH) or dual stress. We found stress induced effects of cold vs desiccation on the levels of trehalose as well as for proline. Different durations of cold stress led to accumulation of trehalose while desiccation stress durations revealed utilization of trehalose. In contrast, there was accumulation of proline under desiccation and utilization of proline with cold stress. Since accumulation levels were higher than utilization of each energy metabolite, the effects of dual stress showed additive effect. However, there was no utilization of total body lipids under cold or desiccation stress. We observed significant season specific differences in the amount of energy metabolites but the rate of metabolism did not vary across seasons. Stress triggered changes in trehalose and proline suggest possible link between desiccation and cold tolerance. Finally, stress specific (cold or desiccation) compensatory changes in the levels of trehalose and proline suggest possible energetic homeostasis in D.immigrans living under harsh climatic conditions of montane localities.

Effects of saturation deficit on desiccation resistance and water balance in seasonal populations of the tropical drosophilid Zaprionus indianus.

Seasonally varying populations of ectothermic insect taxa from a given locality are expected to cope with simultaneous changes in temperature and humidity through phenotypic plasticity. Accordingly, we investigated the effect of saturation deficit on resistance to desiccation in wild-caught flies from four seasons (spring, summer, rainy and autumn) and corresponding flies reared in the laboratory under season-specific simulated temperature and humidity growth conditions. Flies raised under summer conditions showed approximately three times higher desiccation resistance and increased levels of cuticular lipids compared with flies raised in rainy season conditions. In contrast, intermediate trends were observed for water balance-related traits in flies reared under spring or autumn conditions but trait values overlapped across these two seasons. Furthermore, a threefold difference in saturation deficit (an index of evaporative water loss due to a combined thermal and humidity effect) between summer (27.5 mB) and rainy (8.5 mB) seasons was associated with twofold differences in the rate of water loss. Higher dehydration stress due to a high saturation deficit in summer is compensated by storage of higher levels of energy metabolite (trehalose) and cuticular lipids, and these traits correlated positively with desiccation resistance. In Z. indianus, the observed changes in desiccation-related traits due to plastic effects of simulated growth conditions correspond to similar changes exhibited by seasonal wild-caught flies. Our results show that developmental plastic effects under ecologically relevant thermal and humidity conditions can explain seasonal adaptations for water balance-related traits in Z. indianus and are likely to be associated with its invasive potential.

Sex-specific divergence for body size and desiccation-related traits in Drosophila hydei from the western Himalayas.

Sex-specific-differences are a widespread source of genetic variation in various Drosophila species. In the present study, we have examined desiccation survival in males and females of Drosophila hydei from colder and drier montane conditions of the western Himalayas (altitudinal populations; 600-2202 m). In contrast with most other studies in drosophilids, D. hydei males exhibited comparatively higher desiccation resistance despite smaller body size compared to females. Accordingly, we tested the physiological basis of such adaptations in both sexes of D. hydei. Body size traits (wing length, wet weight and dry weight) were ~1.2 fold higher in females than males. However, desiccation resistance was 10 to 13 h higher in males than females. These differences matched enhanced storage of trehalose content (~1.2 fold), higher hemolymph content (~1.2 fold) and enhanced cuticular lipid mass (~1.5 fold) in males than females. Water loss before succumbing to death (dehydration tolerance) was much higher in males (~81%) than females (~64%). A greater loss of hemolymph water until death under desiccation stress was associated with higher desiccation resistance in males. Further, there were lacks of differences in the rate of water loss, rate of trehalose utilization and rate of hemolymph depletion between the sexes in D. hydei. Therefore, sex-specific differences in desiccation resistance of D. hydei were independent of body size as well as the exhaustion of metabolite reserves and rather were caused by the higher dehydration tolerance as well as higher acquisition of hemolymph and trehalose contents.

Rapid effects of humidity acclimation on stress resistance in Drosophila melanogaster.

We tested the hypothesis whether developmental acclimation at ecologically relevant humidity regimes (40% and 75% RH) affects desiccation resistance of pre-adults (3rd instar larvae) and adults of Drosophila melanogaster Meigen (Diptera: Drosophilidae). Additionally, we untangled whether drought (40% RH) acclimation affects cold-tolerance in the adults of D. melanogaster. We observed that low humidity (40% RH) acclimated individuals survived significantly longer (1.6-fold) under lethal levels of desiccation stress (0-5% RH) than their counter-replicates acclimated at 75% RH. In contrast to a faster duration of development of 1st and 2nd instar larvae, 3rd instar larvae showed a delayed development at 40% RH as compared to their counterparts grown at 75% RH. Rearing to low humidity conferred an increase in bulk water, hemolymph content and dehydration tolerance, consistent with increase in desiccation resistance for replicates grown at 40% as compared to their counterparts at 75% RH. Further, we found a trade-off between the levels of carbohydrates and body lipid reserves at 40% and 75% RH. Higher levels of carbohydrates sustained longer survival under desiccation stress for individuals developed at 40% RH than their congeners at 75% RH. However, the rate of carbohydrate utilization did not differ between the individuals reared at these contrasting humidity regimes. Interestingly, our results of accelerated failure time (AFT) models showed substantial decreased death rates at a series of low temperatures (0, -2, or -4°C) for replicates acclimated at 40% RH as compared to their counter-parts at 75% RH. Therefore, our findings indicate that development to low humidity conditions constrained on multiple physiological mechanisms of water-balance, and conferred cross-tolerance towards desiccation and cold stress in D. melanogaster. Finally, we suggest that the ability of generalist Drosophila species to tolerate fluctuations in humidity might aid in their existence and abundance under expected changes in moisture level in course of global climate change.

Scorpions regulate their energy metabolism towards increased carbohydrate oxidation in response to dehydration.

Scorpions successfully inhabit some of the most arid habitats on earth. During exposure to desiccating stress water is mobilized from the scorpion hepatopancreas to replenish the hemolymph and retain hydration and osmotic stability. Carbohydrate catabolism is advantageous under these conditions as it results in high metabolic water production rate, as well as the release of glycogen-bound water. Hypothesizing that metabolic fuel utilization in scorpions is regulated in order to boost body water management under stressful conditions we used a comparative approach, studying energy metabolism during prolonged desiccation in four species varying in resistance performance. We used respirometry for calculating respiratory gas exchange ratios, indicative of metabolic fuel utilization, and measured metabolic fuel contents in the scorpion hepatopancreas. We found that hydrated scorpions used a mixture of metabolic fuels (respiratory exchange rates, RER~0.9), but a shift towards carbohydrate catabolism was common during prolonged desiccation stress. Furthermore, the timing of metabolic shift to exclusive carbohydrate oxidation (RER not different from 1.0) was correlated with desiccation resistance of the respective studied species, suggesting triggering by alterations to hemolymph homeostasis.

Divergence of water balance mechanisms in two melanic Drosophila species from the western Himalayas.

Drosophila busckii is more abundant under colder and drier montane habitats in the western Himalayas as compared to Drosophila melanogaster but the mechanistic basis of such climatic adaptations is largely unknown. We tested the hypothesis whether genetic variation or phenotypic plasticity of cuticular traits confer adaptive protection against desiccation stress in two melanic Drosophila species living under drier montane localities. For D. melanogaster, changes in melanisation are known to be associated with reduced water loss but there are no data on D. busckii. We investigated changes in body melanisation, cuticular lipids, desiccation resistance, water loss, extractable hemolymph volume (%), and dehydration tolerance in six sympatric populations of D. busckii and D. melanogaster over an altitudinal range of 640-2236 m. D. busckii is a melanic species but changes in cuticular water loss are negatively correlated with cuticular lipid mass and not with body melanisation. In D. melanogaster, there are no plastic effects (14-28 °C) for cuticular lipid mass but variation in body melanisation is associated with desiccation-related traits. Effects of organic solvents (hexane or chloroform: methanol), developmental plasticity and seasonal variation in cuticular lipids affect body water loss in D. busckii but no such changes occur in D. melanogaster. Thus, sympatric populations of D. busckii and D. melanogaster have evolved different water balance mechanisms under shared environmental conditions in the western Himalayas. Multiple measures of desiccation resistance in these species show clinal variation with altitude, consistent with adaptation to increased desiccation stress.

Sexual dimorphism for water balance mechanisms in montane populations of Drosophila kikkawai.

Conservation of water is critical to the ecological success of Drosophila species living in the drier montane localities of the Western Himalayas. We observed clinal variation in desiccation resistance for both sexes of Drosophila kikkawai from an altitudinal transect (512-2226 m above sea level). Since more than 90 per cent of body water is lost through cuticular transpiration, the target of selection may be cuticular lipids or cuticular melanization. We tested whether melanic females and non-melanic males of D. kikkawai have similar mechanisms of desiccation resistance. There is clinal variation in the amount of cuticular lipids per fly in males, but not in females. By contrast, for females, elevational increase in melanization is positively correlated with desiccation resistance and negatively with cuticular water loss, but there is no variation in the amount of cuticular lipids. Thus, sexual dimorphism for the mechanism of desiccation resistance in D. kikkawai matches the water proofing role of body melanization as well as cuticular lipids.

Impact of body melanisation on desiccation resistance in montane populations of D. melanogaster: Analysis of seasonal variation.

In the montane localities of subtropical regions, winter is the dry season and ectothermic drosophilids are expected to evolve desiccation resistance to cope with drier climatic conditions. An analysis of six montane populations (600-2226m) of D. melanogaster showed variations for body melanisation (i.e. pigmentation) and desiccation resistance across seasons as well as along altitude. During winter season, plastic changes for melanisation of three posterior abdominal segments (5th+6th+7th) correspond with higher desiccation resistance. Thus, we analyzed genetic and plastic effects for these ecophysiological traits by comparing wild-caught and laboratory reared individuals of D. melanogaster for autumn as well as winter season. A ratio of slope values in wild vs. laboratory populations has shown a 1.64-fold plastic effect during autumn; and a two-fold effect during winter. For body melanisation and desiccation resistance, evolutionary response to altitudinal environmental gradient is similar to the phenotypic response across seasons. Thus, our observations are in agreement with the co-gradient hypothesis. Further, we tested the hypothesis whether a thicker cuticle (either due to melanisation or cuticular lipids) leads to lesser cuticular water loss and higher desiccation resistance across seasons as well as according to altitude. Based on within and between population analyses, body melanisation was found to be positively correlated with desiccation resistance but negatively with cuticular water loss. Interestingly, there were no changes in the amount of cuticular lipids per fly across seasons as well as along altitude; and therefore cuticular lipids did not account for desiccation resistance. Cuticular water loss exhibited negative correlation with body melanisation but not with cuticular lipids as well as with changes in body size across seasons. Thus, our data suggest that seasonal changes in body melanisation confer desiccation resistance in montane populations of D. melanogaster.

Changes in cuticular lipids, water loss and desiccation resistance in a tropical drosophilid: analysis of variation between and within populations.

We investigated within as well as between population variability in desiccation resistance, cuticular lipid mass per fly and cuticular water loss in nine geographical populations of a tropical drosophilid, Zaprionus indianus. Interestingly, the amount of cuticular lipids and desiccation resistance in this non-melanic species are significantly higher as compared with melanic Drosophila melanogaster. On the basis of isofemale line analysis, within population trait variability in cuticular lipid mass per fly is positively correlated with desiccation resistance and negatively correlated with cuticular water loss but show lack of correlation with body size. We observed geographical variation in the amount of cuticular lipid mass per fly in Zaprionus indianus but no such divergence was found in D.melanogaster. In both the species, geographical variations in desiccation resistance are negatively correlated with cuticular water loss but the underlying mechanisms for changes in cuticular permeability are quite different. Thus, we may suggest that body melanisation and cuticular lipids may represent alternative strategies for coping with dehydration stress in melanic versus non-melanic drosophilids. For both the species, desiccation resistance and cuticular water loss are correlated with regular increase in aridity in the northern subtropical localities as compared with southern peninsular humid tropical localities. The role of climatic selection is evident from multiple regression analysis with seasonal changes in temperature and humidity (Tcv and RHcv) of the sites of origin of populations of Zaprionus indianus along latitude.

Climatic adaptations of body melanisation in Drosophila melanogaster from Western Himalayas.

We investigated population divergence in body melanisation in wild samples of Drosophila melanogaster across an elevational gradient (512-2202 m) in the Western Himalayas. Wild populations are characterized by higher phenotypic variability as compared with laboratory populations. Significant differences in elevational slope values for three posterior abdominal segments (fifth, sixth and seventh) in wild versus laboratory populations suggest plastic effects. However, elevational slope values do not differ for the three anterior abdominal segments (second, third and fourth). Thus, elevational changes in melanisation include genetic as well as plastic effects. Fitness consequences of within population variability were analyzed on the basis of assorted darker and lighter flies from two highlands as well as from two lowland localities. There is lack of correlation of melanisation with body size as well as ovariole number in assorted darker and lighter flies. For each population, darker flies showed higher desiccation resistance, lower rate of water loss, longer copulation duration and greater fecundity as compared with lighter flies. Phenotypic variations in body melanisation can be interpreted in relation with seasonal changes in temperature as well as humidity (Tcv and RHcv) of the sites of origin of populations. Thus, elevational changes in body melanisation may represent genetic response to selection pressures imposed by colder and drier climatic conditions in the Western Himalayas.