Genetic variability and phenotypic plasticity of metric thoracic traits in an invasive drosophilid in America.

Thermal phenotypic plasticity of 5 metric thoracic traits (3 related to size and 2 to pigmentation) was investigated in Zaprionus indianus with an isofemale line design. Three of these traits are investigated for the first time in a drosophilid, i.e. thorax width and width of pigmented longitudinal white and black stripes. The reaction norms of white and black stripes were completely different: white stripes were insensitive to growth temperature while the black stripes exhibited a strong linear decrease with increasing temperatures. Thorax width exhibited a concave reaction norm, analogous but not identical to those of wing length and thorax length: the temperatures of maximum value were different, the highest being for thorax width. All traits exhibited a significant heritable variability and a low evolvability. Sexual dimorphism was very variable among traits, being nil for white stripes and thorax width, and around 1.13 for black stripes. The ratio thorax length to thorax width (an elongation index) was always >1, showing that males have a more rounded thorax at all temperatures. Black stripes revealed a significant increase of sexual dimorphism with increasing temperature. Shape indices, i.e. ratios between size traits all exhibited a linear decrease with temperature, the least sensitive being the elongation index. All these results illustrate the complexity of developmental processes but also the analytical strength of biometrical plasticity studies in an eco-devo perspective.

Cellular basis of morphological variation and temperature-related plasticity in Drosophila melanogaster strains with divergent wing shapes.

Organ shape evolves through cross-generational changes in developmental patterns at cellular and/or tissue levels that ultimately alter tissue dimensions and final adult proportions. Here, we investigated the cellular basis of an artificially selected divergence in the outline shape of Drosophila melanogaster wings, by comparing flies with elongated or rounded wing shapes but with remarkably similar wing sizes. We also tested whether cellular plasticity in response to developmental temperature was altered by such selection. Results show that variation in cellular traits is associated with wing shape differences, and that cell number may play an important role in wing shape response to selection. Regarding the effects of developmental temperature, a size-related plastic response was observed, in that flies reared at 16 °C developed larger wings with larger and more numerous cells across all intervein regions relative to flies reared at 25 °C. Nevertheless, no conclusive indication of altered phenotypic plasticity was found between selection strains for any wing or cellular trait. We also described how cell area is distributed across different intervein regions. It follows that cell area tends to decrease along the anterior wing compartment and increase along the posterior one. Remarkably, such pattern was observed not only in the selected strains but also in the natural baseline population, suggesting that it might be canalized during development and was not altered by the intense program of artificial selection for divergent wing shapes.

Thermal Stress and Adult Fitness in a Drosophila suzukii Neotropical Propagule.

Drosophila suzukii (Matsumura 1931) is a cosmopolitan horticultural pest originally from temperate East Asia; yet, its recent introduction in southeast and central Brazil raises the possibility it might expand into warmer climatic zones. In theoretical terms, the adaptive potential of invasive species can be impaired by the lack of genetic variation, but, on the other hand, phenotypic plasticity might play an important role in the adaptation to the new environment. In this context, we investigated the effects of temperature variation (18°C, 22°C, and 28°C) on fitness traits and size of male reproductive organs (accessory glands and testis) in a natural D. suzukii population recently introduced in the neotropical region. Development time decreased significantly with increasing temperature, but egg-to-adult survival was not affected, attaining rates around 50% for the three temperatures. Development at 28°C affected differentially adult male and female biological performance: males displayed higher mortality and severe and permanent reduction in offspring production, whereas females showed the same mortality as controls and a temporary decrease in offspring production, followed of a clear recovery. Finally, reproductive organs size in immature and mature males was affected by developmental temperature variation in the following ways. Testis length decreased with body size (i.e., at higher temperatures) and increased with maturation time after adult hatching, whereas for accessory glands there was no significant difference between different temperatures, resulting in proportionally larger glands for smaller body sizes. These results show differences in developmental dynamics of reproductive tract structures due to temperature variation.

Getting real with real-time qPCR: a case study of reference gene selection for morphological variation in Drosophila melanogaster wings.

Accurate estimation of gene expression differences during development requires sensitive techniques combined with gold-standard normalization procedures. This is particularly true in the case of quantitative traits, where expression changes might be small. Nevertheless, systematic selection and validation of reference genes has been overlooked, even in Drosophila studies. Here, we tested the stability of six traditional reference genes across samples of imaginal wing disks from morphologically divergent strains of Drosophila melanogaster, in a two-class comparison: quantitative or qualitative variation in wing morphology. Overall, we identified and validated a pair of genes (RpL32 and Tbp) as being stably expressed in both experimental comparisons. These genes might be considered as a bona fide pair of reference genes for gene expression analyses of morphological divergence in D. melanogaster wings. They might also be taken as good candidates for experimental identification of stable reference genes in other morphological comparisons using Drosophila or other insect species. Besides, we found that some genes traditionally used as reference in qPCR experiments were not stably expressed in wing disks from the different fly strains. In fact, a significant bias was observed when the expression of three genes of interest, which are involved in the regulation of growth and patterning during imaginal wing development, was normalized with such putative reference genes. Our results demonstrate how inaccurate findings and opposite conclusions might be drawn if traditional reference genes are arbitrarily used for internal normalization without proper validation in the given experimental condition, a practice still common in qPCR experiments.

Mesosternal bristle number in a cosmopolitan drosophilid: an X-linked variable trait independent of sternopleural bristles.

Mesosternal (MS) bristles in Drosophila are a pair of machrochaetae found at the sternal end of the sternopleural (STP) microchaetae, and are thought to be invariable. In a closely related drosophilid genus, Zaprionus, their number is four and, in contrast to Drosophila, they show interspecific and intraspecific variability. The genetic basis of MS bristle number variability was studied in Z. indianus, the only cosmopolitan species of the genus. The trait responded rapidly to selection and two lines were obtained, one lacking any bristles (0-0) and the other bearing the normal phenotype (2-2). Other symmetrical phenotypes, (1-1) and (3-3), could also be selected for, but with lesser success. By contrast, STP bristle number did not vary significantly between the two lines (0-0) and (2-2), revealing its genetic independence from MS bristle number. Reciprocal crosses between these two lines showed that MS bristle number is mainly influenced by a major gene on the X chromosome (i.e. F(1) males always resembled their mothers) with codominant expression (i.e. heterozygous F(1) females harboured an average phenotype of 2 bristles). However, trait penetrance was incomplete and backcrosses revealed that this variability was partly due to genetic modifiers, most likely autosomal. The canalization of MS bristle number was investigated under different temperatures, and the increased appearance of abnormal phenotypes mainly occurred at extreme temperatures. There was a bias, however, towards bristle loss, as shown by a liability (developmental map) analysis. Finally, when ancestral and introduced populations were compared, the latter were far less stable, suggesting that genetic bottlenecks may perturb the MS bristle number canalization system. MS bristle number, thus, appears to be an excellent model for investigating developmental canalization at both the quantitative and the molecular level.

Diversity and pathogenic potential of vibrios isolated from Abrolhos Bank corals.

We performed the first taxonomic characterization of vibrios and other culturable microbiota from apparently healthy and diseased Brazilian-endemic corals at the Abrolhos reef bank. The diseases affecting corals were tissue necrosis in Phyllogorgia dillatata, white plague and bleaching in Mussismilia braziliensis and bleaching in Mussismilia hispida. Bacterial isolates were obtained from mucus of 22 coral specimens originated from the Abrolhos Bank (i.e. Itacolomis reef, Recife de Fora reef and Santa Barbara Island) in 2007. Vibrios counts in the water and coral mucus were approximately 104 cfu ml(-1) and 106 cfu ml(-1) respectively. One hundred and thirty-one representative vibrio isolates were identified. Most vibrio isolates (n = 79) fell into the core group using the pyrH identification marker. According to our analysis, diseased corals did not possess a unique vibrio microbiota. Vibrio species encompassed strains originated from both apparently healthy and diseased corals. The pathogenic potential of representative vibrio isolates (V. alginolyticus 40B, V. harveyi-like 1DA3 and V. coralliilyticus 2DA3) were evaluated in a standardized bioassay using the animal model Drosophila melanogaster and caused 25-88% mortality. This is the first taxonomic characterization of the culturable microbiota from the Brazilian-endemic corals. Endemic Brazilian corals are a reservoir of the vibrio core group. Vibrio alginolyticus, V. harveyi and V. coralliilyticus are dominant in the mucus of these corals and may be a normal component of the holobiont.

Phenotypic variability of natural populations of an invasive drosophilid, Zaprionus indianus, on different continents: comparison of wild-living and laboratory-grown flies.

Phenotypic variability in nature is the most important feature for Darwinian adaptation, yet it has been rarely investigated in invasive species. Zaprionus indianus is an Afrotropical drosophilid species that have recently invaded the Palearctic and the Neotropical regions. Here, we compared the variability of three size-related traits and one meristic trait the sternopleural (STP) bristle number, between wild-collected flies living under different conditions: a stressful Mediterranean environment in Egypt, and a benign tropical environment in Brazil. From each population, a F(1) generation was also grown under the stable conditions of the laboratory. Variability of size in nature had a variance 13 times greater than in the laboratory, but not affected by different climates. By contrast, STP variability was identical in nature and in the laboratory. Sexual dimorphism was also investigated with contrasting results between traits. It is suggested that the very high invasiveness of Z. indianus might be related to a better capacity to survive adverse conditions.

Sexual dimorphism of body size and sternopleural bristle number: a comparison of geographic populations of an invasive cosmopolitan drosophilid.

Zaprionus indianus is a cosmopolitan drosophilid, of Afrotropical origin, which has recently colonized South America. The sexual dimorphism (SD) of body size is low, males being almost as big as females. We investigated 10 natural populations, 5 from America and 5 from Africa, using the isofemale line technique. Three traits were measured on each fly: wing and thorax length and sternopleural bristle number. Two indices of SD were compared, and found to be highly correlated (r > 0.99). For the sake of simplicity, only the female/male (F/M) ratio was further considered. A significant genetic variability of SD was found in all cases, although with a low heritability (intra-class correlation of 0.13), about half the value found for the traits themselves. For size SD, we did not find any variation among continents or any latitudinal trend, and average values were 1.02 for wing length and 1.01 for thorax length. Bristle number SD was much greater (1.07). Among mass laboratory strains, SD was genetically much more variable than in recently collected populations, a likely consequence of laboratory drift. Altogether, SD, although genetically variable and prone to laboratory drift, is independent of size variations and presumably submitted to a stabilizing selection in nature.

Variability of wing size and shape in three populations of a recent Brazilian invader, Zaprionus indianus (Diptera: Drosophilidae), from different habitats.

Zaprionus indianus (Diptera: Drosophilidae) is an African species that was introduced in Brazil near the end of the 1990's decade. To evaluate the adaptive potential of morphological traits in natural populations of this recently introduced species, we have investigated wing size and shape variation at Rio de Janeiro populations only two years after the first record of Z. indianus in Brazil. Significant genetic differences among populations from three distinct ecological habitats were detected. The heritability and evolvability estimates show that, even with the population bottleneck that should have occurred during the invasion event, an appreciable amount of additive genetic variation for wing size and shape was retained. Our results also indicated a greater influence of environmental variation on wing size than on wing shape. The importance of quantitative genetic variability and plasticity in the successful establishment and dispersal of Z. indianus in the Brazilian territory is then discussed.

Chromosomal inversion polymorphism in Drosophila mediopunctata: seasonal, altitudinal, and latitudinal variation

The most polymorphic chromosome for inversions in Drosophila mediopunctata is the chromosome II, where 17 inversions have been found, eight of which occurring in the distal region and nine in the proximal region. We present an analysis of the chromosome II inversion polymorphism with respect to seasonal, altitudinal and latitudinal variation. In D. mediopunctata from the Parque Nacional do Itatiaia (southeastern Brazil), the frequencies of three of the distal inversions (namely DA, DS, and DP) vary seasonally. These inversions also show altitudinal clines in their frequencies. This microgeographic pattern was not observed on a macrogeographic scale. D. mediopunctata from Porto Alegre are less polymorphic for inversions than other populations, the most remarkable reduction occurring in the proximal region of chromosome II. There is a considerable difference between D. mediopunctata from Campinas and specimens from Serra do Japi, which are separated by only 50 km. In contrast, D. mediopunctata from Serra do Japi are much more similar to specimens from the Parque Nacional do Itatiaia, which is 200 km far.