Altitudinal distribution of two sibling species of the Drosophila tripunctata group in a preserved tropical forest and their male sterility thermal thresholds.

Variation of ecophysiological traits may help to explain geographic distribution patterns of Drosophila sibling species. Many traits in ectotherms have optimal performance within specific temperature ranges. Altitudinal gradients are potentially informative for characterizing differences of sibling species distributions. We collected two sibling species of the tripunctata group - Drosophila mediopunctata (MPT) and D. unipunctata (UNI) - at eight altitudes (ranging from 593 to 1185m above sea level) located at a continuous Atlantic Rainforest reserve in consecutive years (2009-2011), with two collections at the hot-rainy season and two at the cold-dry season. Mean altitude was significantly different between species and seasons. Their distributions showed a consistent pattern with MPT always occurring at higher altitudes than UNI. A significant correlation was found between altitude and species relative abundance. We characterized the thermal range of fertility, an important fitness component, for each species and found evidence for differential thermal adaptation. Our results suggest that the two species altitudinal distributions and seasonal relative abundances are consistent with their differential thermal adaptations: MPT seems to be adapted to lower temperatures, occupies higher altitudes and occurs at higher relative abundances in the cold-dry season; while UNI tolerates higher temperatures and occurs at lower altitudes and higher relative abundances in the hot-rainy season. However, their thermal ranges overlap at most temperatures, suggesting that additional variables (e.g. habitat choice, competition, differential survival etc.) may also play a role to determine their distribution in the field.

Chromosomal localization of microsatellite loci in Drosophila mediopunctata.

Drosophila mediopunctata has been used as a model organism for genetics and evolutionary studies in the last three decades. A linkage map with 48 microsatellite loci recently published for this species showed five syntenic groups, which had their homology determined to Drosophila melanogaster chromosomes. Then, by inference, each of the groups was associated with one of the five major chromosomes of D. mediopunctata. Our objective was to carry out a genetic (chromosomal) analysis to increase the number of available loci with known chromosomal location. We made a simultaneous analysis of visible mutant phenotypes and microsatellite genotypes in a backcross of a standard strain and a mutant strain, which had each major autosome marked. Hence, we could establish the chromosomal location of seventeen loci; including one from each of the five major linkage groups previously published, and twelve new loci. Our results were congruent with the previous location and they open new possibilities to future work integrating microsatellites, chromosomal inversions, and genetic determinants of physiological and morphological variation.

Low temperature reveals genetic variability against male-killing Spiroplasma in Drosophila melanogaster natural populations.

Spiroplasma endosymbionts are maternally inherited microorganisms which infect many arthropod species. In some Drosophila species, it acts as a reproductive manipulator, spreading in populations by killing the sons of infected mothers. Distinct Drosophila melanogaster populations from Brazil exhibit variable male-killing Spiroplasma prevalences. In this study, we investigated the presence of variability for the male-killing phenotype among Drosophila and/or Spiroplasma strains and verified if it correlates with the endosymbiont prevalence in natural populations. For that, we analyzed the male-killing expression when Spiroplasma strains from different populations were transferred to a standard D. melanogaster line (Canton-S) and when a common Spiroplasma strain was transferred to different wild-caught D. melanogaster lines, both at optimal and challenging temperatures for the bacteria. No variation was observed in the male-killing phenotype induced by different Spiroplasma strains. No phenotypic variability among fly lines was detected at optimal temperature (23 °C), as well. Conversely, significant variation in the male-killing expression was revealed among D. melanogaster lines at 18.5 °C, probably caused by imperfect transmission of the endosymbiont. Distinct lines differed in their average sex ratios as well as in the pattern of male-killing expression as the infected females aged. Greater variation occurred among lines from one locality, although there was no clear correlation between the male-killing intensity and the endosymbiont prevalence in each population. Imperfect transmission or male killing may also occur in the field, thus helping to explain the low or intermediate prevalences reported in nature. We discuss the implications of our results for the dynamics of male-killing Spiroplasma in natural populations.

Hidden role of mutations in the evolutionary process.

Mutations not only alter allele frequencies in a genetic pool but may also determine the fate of an evolutionary process. Here we study which allele fixes in a one-step, one-way model including the wild type and two adaptive mutations. We study the effect of the four basic evolutionary mechanisms-genetic drift, natural selection, mutation, and gene flow-on mutant fixation and its kinetics. Determining which allele is more likely to fix is not simply a question of comparing fitnesses and mutation rates. For instance, if the allele of interest is less fit than the other, then not only must it have a greater mutation rate, but also its mutation rate must exceed a specific threshold for it to prevail. We find exact expressions for such conditions. Our conclusions are based on the mathematical description of two extreme but important regimes, as well as on simulations.

Molecular phylogeny of the Drosophila tripunctata and closely related species groups (Diptera: Drosophilidae).

We suggest a new phylogenetic hypothesis for the tripunctata radiation based on sequences of mitochondrial genes. Phylogenetic trees were reconstructed by parsimony, maximum likelihood and Bayesian methods. We performed tests for hypotheses of monophyly for taxonomic groups and other specific hypotheses. Results reject the monophyly for the tripunctata group whereas monophyly is not rejected for the tripunctata radiation and other specific groups within the radiation. Although most of the basal nodes were unresolved we were able to identify four clusters within the tripunctata radiation. These results suggest the collection of additional data before a proper taxonomic revision could be proposed.

Decision making and interference in the domestic cat (Felis catus).

Domestic cats (Felis catus) received a task in which an obstacle was introduced at the time they were ready to reach for 1 among 2 or 3 baited targets. The results revealed that when the initially chosen direction (i.e., before the introduction of the obstacle) and the new one were orthogonal, the cats reassessed distance, angular deviation, and visibility once they had turned around the obstacle (Experiments 1, 2, and 3). Then the cats chose a different target than the one that would have been chosen had there been no obstacle. When both the initially chosen and the new direction matched (Experiment 4), the cats kept intact their initial decision. The discussion focuses on the nature of the representational system of the cat. The discussion also emphasizes comparisons with dogs (i.e., cooperative hunters).

Analysis of mitochondrial DNA variability and genetic structure in populations of New World screwworm flies (Diptera: Calliphoridae) from Uruguay.

The New Word screwworm, Cochliomyia hominivorax (Coquerel 1858) (Diptera: Calliphoridae), is one of the most important insect pests of livestock in the Neotropical region. In this work, polymerase chain reaction-restriction fragment length polymorphism of mitochondrial DNA (mtDNA) was used to study the diversity and population structure of seven geographically distinct populations of C. hominivorax from most of the important livestock areas in Uruguay. The control region (A+T/12S) and subunits 1 and 2 of cytochrome oxidase (cox1/cox2) were amplified and digested with restriction endonucleases. Nine haplotypes were observed among the populations sampled. The mean nucleotide diversity and the haplotype diversity indicated high mtDNA variability in this species. The similarity index, average nucleotide divergence, and analysis of molecular variance results showed no evidence of subpopulation differentiation, indicating that the C. hominivorax populations of Uruguay form a single panmitic population. The distribution pattern of the genetic variation in natural populations of C. hominivorax and the implications of these results for establishing control program are discussed.

Breast metastasis as the first clinical manifestation of ileal neuroendocrine tumor. A challenging diagnosis with relevant clinical implications.

Ileal neuroendocrine tumors are slow-growing grade 1 or, more rarely, grade 2 neuroendocrine tumors which, however, are frequently metastatic to regional lymph nodes and the liver. A few cases of ileal neuroendocrine tumors that are metastatic to the breast have also been reported in the medical literature. The knowledge of this uncommon clinical presentation is of great importance because it needs to be differentiated from primary breast carcinomas with neuroendocrine features, which represent completely different entities with a different therapeutic approach. The diagnosis of a breast metastasis from an ileal neuroendocrine tumor and its distinction from a well-differentiated primary neuroendocrine tumor of the breast is a challenging task for clinicians and pathologists. This workup is particularly difficult when the breast lesion is the first sign of malignancy. In the present paper, we describe the clinicopathological features of an ileal neuroendocrine tumor first presenting with a breast metastasis in a 50-year-old woman and we discuss the key diagnostic features for the differential diagnosis with primary well-differentiated neuroendocrine tumor of the breast. Moreover, we have reviewed the medical literature to give the reader a comprehensive overview on this topic.

Undesirable consequences of neglecting nonlinearity: response to comments by Liefting et al. (2013) on Rocha & Klaczko (2012).

In response to our previous study, Liefting et al. argue, in defense of their work on latitudinal variation of developmental-rate reaction norms (RNs), that (1) developmental rate (the reciprocal of development time: rate = time(-1) ) is a more biologically relevant variable than development time; (2) the linear RN model is a valid approximation; and (3) three experimental points suffice to estimate RN parameters. Here, we reply to their comments. First, we give evidence that the complexity of actual development challenges the appealing simplicity of developmental rate. Using the same analysis as Liefting et al. to test their hypothesis with development time, instead of rate, reveals a pattern that is the opposite of their conclusion. Second, we show that a quadratic model is consistent with the whole development-time RNs and explains this contradiction. Third, with the quadratic model, we introduce two parameters to study plasticity: the RN shape (the quadratic coefficient) and RN local plasticity (the derivative of the RN function). The first showed a statistically significant correlation with latitude; and the second showed a continuous variation pattern where all localized patterns can be found (positive, negative, or nonsignificant correlations with latitude) but certainly cannot be generalized.

The reaction norm for abdominal pigmentation and its curve in Drosophila mediopunctata depend on the mean phenotypic value.

The idea of a general independence between the phenotypic plasticity and the mean value of a trait is, presently, a consensus. Here, we use the reaction norm of abdominal pigmentation (number of dark spots) of Drosophila mediopunctata in response to temperature, to test this idea. We raised eight strains, bearing two different chromosomal inversions and with varying mean phenotypic values, under 11 temperatures in a thermal gradient to test for predictions concerning mean phenotypic values, chromosomal inversions, and reaction norms. Our results revealed a strong effect of different phenotypic groups and no effect of different karyotypes on reaction norms. Moreover, we found a significant negative correlation between mean phenotypic value and the curvature of the reaction norms, revealing a high dependency of the reaction norm shape on mean phenotypic value. These results clearly reject the idea of genetic independence between mean value and phenotypic plasticity, and may indicate a pattern of correlation, which may include results from other traits and species, with an importance that has not been fully appreciated.

Shape and size variation on the wing of Drosophila mediopunctata: influence of chromosome inversions and genotype-environment interaction.

The second chromosome of Drosophila mediopunctata is highly polymorphic for inversions. Previous work reported a significant interaction between these inversions and collecting date on wing size, suggesting the presence of genotype-environment interaction. We performed experiments in the laboratory to test for the joint effects of temperature and chromosome inversions on size and shape of the wing in D. mediopunctata. Size was measured as the centroid size, and shape was analyzed using the generalized least squares Procrustes superimposition followed by discriminant analysis and canonical variates analysis of partial warps and uniform components scores. Our findings show that wing size and shape are influenced by temperature, sex, and karyotype. We also found evidence suggestive of an interaction between the effects of karyotype and temperature on wing shape, indicating the existence of genotype-environment interaction for this trait in D. mediopunctata. In addition, the association between wing size and chromosome inversions is in agreement with previous results indicating that these inversions might be accumulating alleles adapted to different temperatures. However, no significant interaction between temperature and karyotype for size was found--in spite of the significant presence of temperature-genotype (cross) interaction. We suggest that other ecological factors--such as larval crowding--or seasonal variation of genetic content within inversions may explain the previous results.

Divergent abdominal bristle patterns in two distantly related drosophilids: antero-posterior variations and sexual dimorphism in a modular trait.

The number of neurosensory bristles on abdominal sternites of Drosophila is a most investigated trait for quantitative genetic studies. However, the developmental pattern expressed on successive segments in both sexes has remained so far a neglected field. We explored three aspects of this general problem with an isofemale line design: comparing two distantly related species, Drosophila melanogaster and Zaprionus indianus, investigating bristle number variation along the antero-posterior axis, and analysing the sexual dimorphism. Antero-posterior variations could be analysed from segment A2 to A7 in females, and A2-A5 in males. In D. melanogaster, males and females showed parallel changes with a consistently lower number in males. In Z. indianus females the number was quite stable along the abdomen, while in males an important antero-posterior increase was found. The sexual dimorphism was further analysed by considering the female-male correlation and the female/male ratio. The results suggest that sternite bristle number is determined by several developmental genetic systems. One is acting along the antero-posterior axis and may be associated to a gradient, since the genetic correlation decreases when more distant segments are compared. Another is acting in the same way on most segments of both sexes, since the female-male genetic correlation is similar between homologous and non-homologous segments. Finally, genes with specific sex effects are acting on A7 in females of both species, and on A5 in Z. indianus males. The overall architecture of female and male abdomen seems to be constrained by the development of reproductive organs. A large difference between species suggests, however, that the sexual dimorphism of abdominal bristle number is not evolutionarily constrained.

Grafting the molecular phylogenetic tree with morphological branches to reconstruct the evolutionary history of the genus Zaprionus (Diptera: Drosophilidae).

A molecular phylogeny for the drosophilid genus Zaprionus was inferred using a mitochondrial (CO-II) and a nuclear (Amyrel) gene using 22 available species. The combined molecular tree does not support the current classification, dubbed phylogenetic, based entirely upon a morphocline of forefemoral ornamentation. For species for which DNA was not available, phylogenetic positioning was only assigned using morphological characters. In order to avoid conflict between DNA and morphology in the combined analyses (supermatrix method), we developed a new method in which few morphological characters were sampled according to an a priori homoplasy assessment on the consensus molecular tree. At each internal node of the tree, a number of synapomorphies was determined, and species with no molecular sequences were grafted thereon. Analogously to tree vocabulary, we called our method 'morphological grafting'. New species groups and complexes were then defined in the light of our findings. Further, divergence times were estimated under a relaxed molecular clock, and historical biogeography was reconstructed under a maximum likelihood model. Zaprionus appears to be of recent origin in the Oriental region during the Late Miocene ( approximately 10 MYA), and colonization of Africa started shortly after ( approximately 7 MYA) via the maritime route of the Indian Ocean Islands. Most of the morphological and ecological diversification took place, later, in Western Africa during the Quaternary cyclic climatic changes. Furthermore, some species became recent invaders, with one, Zaprionus indianus, has successfully invaded South and North America during the last decade.

Evolutionary genetics of Drosophila mediopunctata.

Drosophila mediopunctata belongs to the tripunctata group, which is the second largest Neotropical group of Drosophila with 64 species described. Here I review the work done with this forest dwelling species, and some applications of the methods developed using it as a model organism, to other species. Specifically I look at: the phylogenetic status of the tripunctata group and its relation with other groups in the Hirtodrosophila-immigrans radiation; D. mediopunctata's chromosome inversion polymorphism (altitudinal cline of frequencies and evidences of selection); the morphological variation of the wing and the development and applications of the ellipse method to describe the morphology of the wing; the variation on the number of aristal branches; the genetic basis of the polychromatism present in D. mediopunctata and its association with chromosome inversions; the sex-ratio trait and its use in the demonstration of Fisher's principle; and, finally, the finding of the transposable P-element in this species.

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.

Homage to Professor Charles Thibault. Synthesis of the symposium and conclusions.

"Reflexive biology", using the ambiguities of the term, is altogether a biology that plays with the mirrors of the different levels of organisation, which, endlessly, go from one place to another, organisms in their real environment to the genome via cells in vitro and others, but which is also reflexive because at any moment biology reflects the finalities and the worry of what it should serve.