Within-population plastic responses to combined thermal-nutritional stress differ from those in response to single stressors, and are genetically independent across traits in both males and females.

Phenotypic plasticity helps animals to buffer the effects of increasing thermal and nutritional stress created by climate change. Plastic responses to single and combined stressors can vary among genetically diverged populations. However, less is known about how plasticity in response to combined stress varies among individuals within a population or whether such variation changes across life-history traits. This is important because individual variation within populations shapes population-level responses to environmental change. Here, we used isogenic lines of Drosophila melanogaster to assess the plasticity of egg-to-adult viability and sex-specific body size for combinations of 2 temperatures (25 °C or 28 °C) and 3 diets (standard diet, low caloric diet, or low protein:carbohydrate ratio diet). Our results reveal substantial within-population genetic variation in plasticity for egg-to-adult viability and wing size in response to combined thermal-nutritional stress. This genetic variation in plasticity was a result of cross-environment genetic correlations that were often < 1 for both traits, as well as changes in the expression of genetic variation across environments for egg-to-adult viability. Cross-sex genetic correlations for body size were weaker when the sexes were reared in different conditions, suggesting that the genetic basis of traits may change with the environment. Furthermore, our results suggest that plasticity in egg-to-adult viability is genetically independent from plasticity in body size. Importantly, plasticity in response to diet and temperature individually differed from plastic shifts in response to diet and temperature in combination. By quantifying plasticity and the expression of genetic variance in response to combined stress across traits, our study reveals the complexity of animal responses to environmental change, and the need for a more nuanced understanding of the potential for populations to adapt to ongoing climate change.

Blind measurements did not confirm effects of forest fragmentation on fluctuating asymmetry of a tropical butterfly Morpho helenor.

Re-evaluation of photographs of the tropical butterfly Morpho helenor from a previous study (Pignataro et al. 2023) revealed that its conclusion regarding increased wing fluctuating asymmetry in forest edge habitats compared to forest interior habitats could not be replicated. This discrepancy likely arises from (i) original measurements not being conducted blindly, (ii) insufficient photograph quality hindering accurate landmark selection, and (iii) a lack of detailed description of the measurement protocol. The likelihood of false positive discoveries within the published data concerning the impacts of environmental stress on the fluctuating asymmetry of plants and animals is probably higher than previously assumed.

Size does matter: intraspecific geometric morphometric analysis of wings of the blowfly Chrysomya albiceps (Diptera: Calliphoridae).

Blowflies have forensic, sanitary and veterinary importance, as well as being pollinators, parasitoids and ecological bioindicators. There is still little work with real data and from experiments assessing the relationship between blowflies' morphologic features and environmental and demographic factors. The present work tests whether the variation, in the shape and size, of Chrysomya albiceps (Wiedemann, 1819) wings is influenced by the following factors: 1) time; 2) temperature; 3) sex and; 4) different types of carcasses (pig, dog/cat and whale). Male and female wings from four different sites collected in six different years were used to obtain wing size and shape of C. albiceps. Analyses between wing shape and the variables tested had low explanatory power, even though they had statistical support. However, it was possible to identify differences in wing shape between males and females, with good returns in sex identification. The comparison between wing size and the variables tested showed that wing size has a negative relationship with temperature, significant differences between sexes, slight variation over time and no influence by carcass types. Furthermore, wing size influenced wing shape. Understanding population-specific characteristics of C. albiceps provide important insights about how the species reacts under specific conditions.

Sublethal effects of a pyrethroid insecticide on cuticle thickness, wing size and shape in the main vector Triatoma infestans.

Exposure to sublethal doses of insecticide may affect biological traits in triatomines. We investigated the effects of toxicological phenotype (pyrethroid resistance status) and exposure to sublethal doses of deltamethrin on two traits of Triatoma infestans Klug (Heteroptera: Reduviidae) using a phenotypic plasticity experimental design. First-instar nymphs from 14 and 10 full-sib families from pyrethroid-susceptible and pyrethroid-resistant populations, respectively, were used. For the susceptible population, we treated first instars topically with acetone (control) or deltamethrin (treatment) once. For the resistant population, instars were treated once, twice and three times as first, third or fifth-instar nymphs, respectively. We measured cuticle thickness, wing size and wing shape of 484 emerging adults, and tested for treatment effects using mixed ANOVA and MANOVA models. Toxicological phenotype, exposure to deltamethrin and full-sib family exerted significant effects on cuticle thickness, wing size and wing shape. Adult triatomines previously treated with deltamethrin developed significantly thicker cuticles than control triatomines only in the resistant population and significantly bigger wings in both populations. Mean cuticle thickness and wing size increased with increasing exposures to deltamethrin. Exposure to sublethal doses of deltamethrin generated morphological modifications that may affect insect survival and flight dispersal, and hence may have evolutionary and epidemiological consequences.

Genomic Shifts, Phenotypic Clines, and Fitness Costs Associated With Cold Tolerance in the Asian Tiger Mosquito.

Climatic variation is a key driver of genetic differentiation and phenotypic traits evolution, and local adaptation to temperature is expected in widespread species. We investigated phenotypic and genomic changes in the native range of the Asian tiger mosquito, Aedes albopictus. We first refine the phylogeographic structure based on genome-wide regions (1,901 double-digest restriction-site associated DNA single nucleotide polymophisms [ddRAD SNPs]) from 41 populations. We then explore the patterns of cold adaptation using phenotypic traits measured in common garden (wing size and cold tolerance) and genotype-temperature associations at targeted candidate regions (51,706 exon-capture SNPs) from nine populations. We confirm the existence of three evolutionary lineages including clades A (Malaysia, Thailand, Cambodia, and Laos), B (China and Okinawa), and C (South Korea and Japan). We identified temperature-associated differentiation in 15 out of 221 candidate regions but none in ddRAD regions, supporting the role of directional selection in detected genes. These include genes involved in lipid metabolism and a circadian clock gene. Most outlier SNPs are differently fixed between clades A and C, whereas clade B has an intermediate pattern. Females are larger at higher latitude yet produce no more eggs, which might favor the storage of energetic reserves in colder climate. Nondiapausing eggs from temperate populations survive better to cold exposure than those from tropical populations, suggesting they are protected from freezing damages but this cold tolerance has a fitness cost in terms of egg viability. Altogether, our results provide strong evidence for the thermal adaptation of A. albopictus across its wide temperature range.

Responses to Developmental Temperature Fluctuation in Life History Traits of Five Drosophila Species (Diptera: Drosophilidae) from Different Thermal Niches.

Temperature has profound effects on biochemical processes as suggested by the extensive variation in performance of organisms across temperatures. Nonetheless, the use of fluctuating temperature (FT) regimes in laboratory experiments compared to constant temperature (CT) regimes is still mainly applied in studies of model organisms. We investigated how two amplitudes of developmental temperature fluctuation (22.5/27.5 °C and 20/30 °C, 12/12 h) affected several fitness-related traits in five Drosophila species with markedly different thermal resistance. Egg-to-adult viability did not change much with temperature except in the cold-adapted D. immigrans. Developmental time increased with FT among all species compared to the same mean CT. The impact of FT on wing size was quite diverse among species. Whereas wing size decreased quasi-linearly with CT in all species, there were large qualitative differences with FT. Changes in wing aspect ratio due to FT were large compared to the other traits and presumably a consequence of thermal stress. These results demonstrate that species of the same genus but with different thermal resistance can show substantial differences in responses to fluctuating developmental temperatures not predictable by constant developmental temperatures. Testing multiple traits facilitated the interpretation of responses to FT in a broader context.

Punctuational ecological changes rather than global factors drive species diversification and the evolution of wing phenotypes in Morpho butterflies.

Assessing the relative importance of geographical and ecological drivers of evolution is paramount to understand the diversification of species and traits at the macroevolutionary scale. Here, we use an integrative approach, combining phylogenetics, biogeography, ecology and quantified phenotypes to investigate the drivers of both species and phenotypic diversification of the iconic Neotropical butterfly genus Morpho. We generated a time-calibrated phylogeny for all known species and inferred historical biogeography. We fitted models of time-dependent (accounting for rate heterogeneity across the phylogeny) and paleoenvironment-dependent diversification (accounting for global effect on the phylogeny). We used geometric morphometrics to assess variation of wing size and shape across the tree and investigated their dynamics of evolution. We found that the diversification of Morpho is best explained when considering variable diversification rates across the tree, possibly associated with lineages occupying different microhabitat conditions. First, a shift from understory to canopy was characterized by an increased speciation rate partially coupled with an increasing rate of wing shape evolution. Second, the occupation of dense bamboo thickets accompanying a major host-plant shift from dicotyledons towards monocotyledons was associated with a simultaneous diversification rate shift and an evolutionary 'jump' of wing size. Our study points to a diversification pattern driven by punctuational ecological changes instead of a global driver or biogeographic history.

Phenotypic plasticity, canalisation and developmental stability of Triatoma infestans wings: effects of a sublethal application of a pyrethroid insecticide.

BACKGROUND: Triatomine control campaigns have traditionally consisted of spraying the inside of houses with pyrethroid insecticides. However, exposure to sublethal insecticide doses after the initial application is a common occurrence and may have phenotypic consequences for survivors. Here, using Triatoma infestans (the main vector of Chagas disease in the Southern Cone of South America) as a model species, we quantified the effects of exposure to a sublethal dose of pyrethroid insecticide on wing morphology. We tested if the treatment (i) induced a plastic effect (change in the character mean); (ii) altered environmental canalisation (higher individual variation within genotypes); (iii) altered genetic canalisation (higher variation among genotypes); and (iv) altered developmental stability (higher fluctuating asymmetry [FA]). METHODS: Each of 25 full-sib families known to be susceptible to pyrethroid insecticides were split in two groups: one to be treated with a sublethal dose of deltamethrin (insecticide-treated group) and the other to be treated with pure acetone (control group). Wings of the emerging adults were used in a landmark-based geometric morphometry analysis to extract size and shape measurements. Average differences among treatments were measured. Levels of variation among families, among individuals within families and among sides within individuals were computed and compared among treatments. RESULTS: Wing size and shape were affected by a sublethal dose of deltamethrin. The treated insects had larger wings and a more variable wing size and shape than control insects. For both wing size and shape, genetic variation was higher in treated individuals. Individual variations and variations in FA were also greater in deltamethrin-treated insects than in control ones for all full-sib families; however, the patterns of shape variation associated with genetic variation, individual variation and FA were different. CONCLUSIONS: Insects exposed to a sublethal dose of deltamethrin presented larger, less symmetrical and less canalised wings. The insecticide treatment jointly impaired developmental stability and genetic and environmental canalisation. The divergent patterns of shape variation suggest that the related developmental buffering processes differed at least partially. The morphological modifications induced by a single sublethal exposure to pyrethroids early in life may impinge on subsequent flight performance and consequently affect the dynamics of house invasion and reinfestation, and the effectiveness of triatomine control operations.

Orchid bees (Apidae, Euglossini) from Oil Palm Plantations in Eastern Amazon Have Larger but Not Asymmetrical Wings.

Phenotypic variation in both morphology and symmetry of individuals may appear due to environmental stress caused by land-use changes. Here, we evaluated fluctuating asymmetry (FA) and wing size variations of two orchid bee species, Euglossa ignita Smith, 1874 and Eulaema meriana (Olivier, 1789), comparing 11 wing traits. We sampled the individuals from legal reserves (LR), areas of permanent protection (APP), and oil palm plantations (PALM) in Eastern Amazonia. We calculated FA as the absolute difference between the wing measurements made in the right and left wings of specimens and both species' wing size. We corrected each FA measure for possible directional asymmetry bias by subtracting the mean value of the mean FA signed difference to each FA measure. We compared FA and the size of each wing trait of each species between land-use types using one-way ANOVAs. We found no effect of FA between land-use types, but we observed individuals of both species from PALM areas having larger wings than those from LR areas. Our results demonstrate that there seems to be a pressure exerted by land-use change associated with palm oil cultivation favoring individuals with larger wings, although both species had shown substantial permeability of oil palm.

How many scales on the wings? A case study based on Colias crocea (Geoffroy, 1785) (Hexapoda: Lepidoptera, Pieridae).

The covering by scales of the wings of Lepidoptera contributes to multiple functions that are critical for their survival and reproduction. In order to gain a better understanding about their distribution, we have exhaustively studied 4 specimens of Colias crocea (Geoffroy, 1785). We have quantified the sources of variability affecting scale density. The results indicate that the scale covering of butterfly wings may be remarkably heterogeneous, and that the importance of the sources of variability differs between forewings and hindwings. Thus, in forewing the greatest variability occurs between sectors, while in the hindwings it occurs between sides, with a higher density of scales on the underside, considerably higher (almost 19%) than on the upperside. It seems likely that this difference has an adaptive value, as the hindwing underside is more exposed (in resting position) to predators. These results are in contrast with the generally accepted notion that scale covering is uniform and homogeneous. Moreover, the cover scale density is independent of the size of the specimen and therefore an average density of scales can be attributed to this species. According to our measurements C. crocea has 312 scales/mm2 and the total number of scales per individual is about 520,000 on average.

Variation over time in wing size and shape of the coastal malaria vector Anopheles (Cellia) epiroticus Linton and Harbach (Diptera: Culicidae) in Samut Songkhram, Thailand.

OBJECTIVE: Anopheles (Cellia) epiroticus Linton & Harbach, a coastal mosquito (also called a brackish mosquito), is a secondary vector species of malaria distributed throughout eastern and southern regions of Thailand. This research aimed to investigate the differences of wing size and shape of this female Aonpheles species in Samut Songkhram Province, Thailand occurring over time between 2015 and 2017. MATERIALS AND METHODS: Coordinates of 13 landmarks were selected and digitized. Centroid size (CS) was used to estimate wing size. Shape variables were used to estimate wing shape and were calculated from the Generalized Procrustes Analysis following principal components of the partial warp. The statistically significant differences of the average wing size based on CS and wing shape based on Mahalanobis distances in each year were estimated using the non-parametric permutation testing with 1,000 cycles after Bonferroni correction with a significance level of 0.05 (p < 0.05). RESULTS: The A. epiroticus population in year 2016 had the highest average (3.61 mm), and the population in year 2017 had the lowest (3.47 mm). In this study, there was no difference in the size of wing between A. epiroticus population in the years 2015 and 2016 (p > 0.05). The A. epiroticus population in year 2017 was significantly smaller than the population in the years 2015 and 2016 (p < 0.05). All pairwise comparisons of wing shape Mahalanobis distances were significantly different in year 2017 compared with 2015 and 2016 (p < 0.01). CONCLUSION: These results indicate differences of wings occur over time that affect the morphological variability of A. epiroticus. The differences in weather conditions in each year affect the adaptive and morphological changes of mosquitoes in coastal areas.

Fluctuating asymmetry and exposure to pyrethroid insecticides in Triatoma infestans populations in northeastern Argentina.

Fluctuating asymmetry (FA), a phenotypic marker used as indicator of developmental stress or instability, is sometimes associated with insecticide application and resistance. Here we investigated the occurrence and amount of wing size and wing shape FA in Triatoma infestans females and males collected before and 4 months after a community-wide pyrethroid spraying campaign in a well-defined rural area of Pampa del Indio, Argentina. Moderate levels of pyrethroid resistance were previously confirmed for this area, and postspraying house infestation was mainly attributed to this condition. In the absence of insecticide-based selective pressures over the previous 12 years, we hypothesized that 1- if postspraying triatomines were mostly survivors to insecticide spraying (pyrethroid resistant), they would have higher levels of FA than prespraying triatomines. 2- if postspraying triatomines have a selective advantage, they would have lower FA levels than their prespraying counterparts, whereas if postspraying infestation was positively associated with immigrants not exposed to the insecticide, prespraying and postspraying triatomines would display similar FA levels. For 243 adult T. infestans collected at identified sites before insecticide spraying and 112 collected 4 months postspraying, wing size and wing shape asymmetry was estimated from landmark configurations of left and right sides of each individual. At population level, wing size and shape FA significantly decreased in both females and males after spraying. Males displayed greater wing size and shape FA than females. However, at a single peridomestic site that was persistently infested after spraying, FA declined similarly in females whereas the reverse pattern occurred in males. Our results suggest differential survival of adults with more symmetric wings. This pattern may be related to a selective advantage of survivors to insecticide spraying, which may be mediated or not by their pyrethroid-resistant status or to lower triatomine densities after insecticide spraying and the concomitant increase in feeding success.

Morphological variation of Aphidius ervi Haliday (Hymenoptera: Braconidae) associated with different aphid hosts.

BACKGROUND: Parasitoids are frequently used in biological control due to the fact that they are considered host specific and highly efficient at attacking their hosts. As they spend a significant part of their life cycle within their hosts, feeding habits and life history of their host can promote specialization via host-race formation (sequential radiation). The specialized host races from different hosts can vary morphologically, behaviorally and genetically. However, these variations are sometimes inconspicuous and require more powerful tools in order to detect variation such as geometric morphometrics analysis. METHODS: We examined Aphidius ervi, an important introduced biological control agent in Chile associated with a great number of aphid species, which are exploiting different plant hosts and habitats. Several combinations (biotypes) of parasitoids with various aphid/host plant combinations were analyzed in order to obtain measures of forewing shape and size. To show the differences among defined biotypes, we chose 13 specific landmarks on each individual parasitoid wing. The analysis of allometric variation calculated in wing shape and size over centroid size (CS), revealed the allometric changes among biotypes collected from different hosts. To show all differences in shape of forewings, we made seven biotype pairs using an outline-based geometric morphometrics comparison. RESULTS: The biotype A. pis_pea (Acyrthosiphon pisum on pea) was the extreme wing size in this study compared to the other analyzed biotypes. Aphid hosts have a significant influence in the morphological differentiation of the parasitoid forewing, splitting biotypes in two groups. The first group consisted of biotypes connected with Acyrthosiphon pisum on legumes, while the second group is composed of biotypes connected with aphids attacking cereals, with the exception of the R. pad_wheat (Rhopalosiphum padi on wheat) biotype. There was no significant effect of plant species on parasitoid wing size and shape. DISCUSSION: Although previous studies have suggested that the genotype of parasitoids is of greater significance for the morphological variations of size and shape of wings, this study indicates that the aphid host on which A. ervi develops is the main factor to alter the structure of parasitoid forewings. Bigger aphid hosts implied longer and broader forewings of A. ervi.

Protocols to Study Growth and Metabolism in Drosophila.

Signaling pathways such as the insulin/insulin-like growth factor pathway concurrently regulate organismal growth and metabolism. Drosophila has become a popular model system for studying both organismal growth and metabolic regulation. Care must be taken, however, when assessing such phenotypes because they are quantitative in nature, and influenced by environment. This chapter first describes how to control animal age and nutrient availability, since growth and metabolism are sensitive to these parameters. It then provides protocols for measuring tissue growth, cell size, and metabolic parameters such as stored lipids and glycogen, and circulating sugars.

Wing trait-inversion associations in Drosophila subobscura can be generalized within continents, but may change through time.

Clinal variation is one of the most emblematic examples of the action of natural selection at a wide geographical range. In Drosophila subobscura, parallel clines in body size and inversions, but not in wing shape, were found in Europe and South and North America. Previous work has shown that a bottleneck effect might be largely responsible for differences in wing trait-inversion association between one European and one South American population. One question still unaddressed is whether the associations found before are present across other populations of the European and South American clines. Another open question is whether evolutionary dynamics in a new environment can lead to relevant changes in wing traits-inversion association. To analyse geographical variation in these associations, we characterized three recently laboratory founded D. subobscura populations from both the European and South American latitudinal clines. To address temporal variation, we also characterized the association at a later generation in the European populations. We found that wing size and shape associations can be generalized across populations of the same continent, but may change through time for wing size. The observed temporal changes are probably due to changes in the genetic content of inversions, derived from adaptation to the new, laboratory environment. Finally, we show that it is not possible to predict clinal variation from intrapopulation associations. All in all this suggests that, at least in the present, wing traits-inversion associations are not responsible for the maintenance of the latitudinal clines in wing shape and size.

Trait-specific consequences of inbreeding on adaptive phenotypic plasticity.

Environmental changes may stress organisms and stimulate an adaptive phenotypic response. Effects of inbreeding often interact with the environment and can decrease fitness of inbred individuals exposed to stress more so than that of outbred individuals. Such an interaction may stem from a reduced ability of inbred individuals to respond plastically to environmental stress; however, this hypothesis has rarely been tested. In this study, we mimicked the genetic constitution of natural inbred populations by rearing replicate Drosophila melanogaster populations for 25 generations at a reduced population size (10 individuals). The replicate inbred populations, as well as control populations reared at a population size of 500, were exposed to a benign developmental temperature and two developmental temperatures at the lower and upper margins of their viable range. Flies developed at the three temperatures were assessed for traits known to vary across temperatures, namely abdominal pigmentation, wing size, and wing shape. We found no significant difference in phenotypic plasticity in pigmentation or in wing size between inbred and control populations, but a significantly higher plasticity in wing shape across temperatures in inbred compared to control populations. Given that the norms of reaction for the noninbred control populations are adaptive, we conclude that a reduced ability to induce an adaptive phenotypic response to temperature changes is not a general consequence of inbreeding and thus not a general explanation of inbreeding-environment interaction effects on fitness components.

Role of Drosophila retinoblastoma protein instability element in cell growth and proliferation.

The RB tumor suppressor, a regulator of the cell cycle, apoptosis, senescence, and differentiation, is frequently mutated in human cancers. We recently described an evolutionarily conserved C-terminal "instability element" (IE) of the Drosophila Rbf1 retinoblastoma protein that regulates its turnover. Misexpression of wild-type or non-phosphorylatable forms of the Rbf1 protein leads to repression of cell cycle genes. In contrast, overexpression of a defective form of Rbf1 lacking the IE (ΔIE), a stabilized but transcriptionally less active form of the protein, induced ectopic S phase in cell culture. To determine how mutations in the Rbf1 IE may induce dominant effects in a developmental context, we assessed the impact of in vivo expression of mutant Rbf1 proteins on wing development. ΔIE expression resulted in overgrowth of larval wing imaginal discs and larger adult wings containing larger cells. In contrast, a point mutation in a conserved lysine of the IE (K774A) generated severely disrupted, reduced wings. These contrasting effects appear to correlate with control of apoptosis; expression of the pro-apoptotic reaper gene and DNA fragmentation measured by acridine orange stain increased in flies expressing the K774A isoform and was suppressed by expression of Rbf1ΔIE. Intriguingly, cancer associated mutations affecting RB homologs p130 and p107 may similarly induce dominant phenotypes.

EXPERIMENTAL ANALYSES OF WING SIZE, FLIGHT, AND SURVIVAL IN THE WESTERN WHITE BUTTERFLY.

Butterflies have distinctively large wings relative to body size, but the functional and fitness consequences of wing size for butterflies are largely unknown. I use natural and experimentally generated variation in wing surface area to examine how decreased wing size affects flight and survival in a population of the western white butterfly, Pontia occidentalis. In the laboratory, experimental reductions in wing area (reduced-wings manipulation) significantly increased wingbeat frequencies of hovering butterflies, whereas a control manipulation had no detectable effects. In contrast, behavioral observations and mark-release-recapture (MRR) studies in the field detected no significant differences in flight activity, initial dispersal rates, or recapture probabilities among treatment groups. Estimated selection coefficients indicated that natural variation in wing size, body mass, and wing loading in the population were not significantly correlated with survival in the two MRR studies. In two mark-recapture studies with manipulated butterflies, survival probabilities were not significantly different for reduced-wings individuals compared with control or unmanipulated individuals. In summary, experimental reductions in wing area significantly altered aspects of flight in the laboratory, but did not detectably alter flight or survival in the field for this population. The large wing size typical of butterflies may reduce the functional and survival consequences of wing size variation within populations.