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.

Temporal flexibility of gene regulatory network underlies a novel wing pattern in flies.

Organisms have evolved endless morphological, physiological, and behavioral novel traits during the course of evolution. Novel traits were proposed to evolve mainly by orchestration of preexisting genes. Over the past two decades, biologists have shown that cooption of gene regulatory networks (GRNs) indeed underlies numerous evolutionary novelties. However, very little is known about the actual GRN properties that allow such redeployment. Here we have investigated the generation and evolution of the complex wing pattern of the fly Samoaia leonensis We show that the transcription factor Engrailed is recruited independently from the other players of the anterior-posterior specification network to generate a new wing pattern. We argue that partial cooption is made possible because 1) the anterior-posterior specification GRN is flexible over time in the developing wing and 2) this flexibility results from the fact that every single gene of the GRN possesses its own functional time window. We propose that the temporal flexibility of a GRN is a general prerequisite for its possible cooption during the course of evolution.

Phylogenetic relationships between fungus-associated Neotropical species of the genera Hirtodrosophila, Mycodrosophila and Zygothrica (Diptera, Drosophilidae), with insights into the evolution of breeding sites usage.

The Neotropical region harbors an astonishing diversity of species, but still encompasses the least studied biogeographic region of the world. These properties apply for different taxonomic groups, and can be exemplified by drosophilids. In fact, high levels of cryptic diversity have recently been discovered for Neotropical species of the Zygothrica genus group, but relationships among these species, or them and other Drosophilidae species still remains to be addressed. Therefore, the aim of this study was to evaluate the phylogenetic relationships between fungus-associated Neotropical species of the genera Hirtodrosophila, Mycodrosophila and Zygothrica, which together with Paramycodrosophila and Paraliodrosophila compose the Zygothrica genus group. For this, fragments of the mitochondrial cytochrome oxidase subunits I (COI) and II (COII) genes, and the nuclear alpha methyldopa (Amd) and dopa decarboxylase (Ddc) genes were newly characterized for 43 Neotropical specimens of fungus-associated drosophilids, and analyzed in the context of 51 additional Drosophilinae sequences plus one Steganinae outgroup. Based on the resulting phylogeny, the evolution of breeding sites usage was also evaluated through ancestral character reconstructions. Our results revealed the Zygothrica genus group as a monophyletic lineage of Drosophila that branches after the subgenera Sophophora and Drosophila. Within this lineage, Mycodrosophila species seem to encompass the early offshoot, followed by a grade of Hirtodrosophila species, with derived branches mostly occupied by representatives of Zygothrica. This genus, in particular, was subdivided into five major clades, two of which include species of Hirtodrosophila, whose generic status needs to be reevatuated. According to our results, the use of fungi as breeding sites encompasses a symplesiomorphy for the Zygothrica genus group, since one of the recovered clades is currently specialized in using flowers as breeding sites whereas a sole species presents a reversal to the use of fruits of a plant of Gentianales. So, in general, this study supports the paraphyly of Drosophila in relation to fungus-associated Neotropical species of Drosophilidae, providing the first molecular insights into the phylogenetic patterns related to the evolution of this diverse group of species and some of its characteristic traits.

Survival of Phortica variegata experimentally and naturally infected with Thelazia callipaeda.

Knowledge of the effects that Thelazia callipaeda (Spirurida, Thelaziidae) infection has on the survival of its vector Phortica variegata (Drosophilidae, Steganinae) is scarce. The present study aimed to: (a) assess the rate of infection between experimentally infected (EI) and not experimentally infected (NEI) flies and (b) determine how T. callipaeda infection may affect the survival of P. variegata. In addition, fat composition was evaluated in flies that died during overwintering. Molecular analysis showed that T. callipaeda prevalence in flies that died before experimental infection, plus those from the NEI group, is 0.75% (i.e. 11 out of 1462 individuals). The EI group showed a significantly higher positivity to T. callipaeda (i.e. 51 out of 682 individuals; 7.48%) compared with the NEI group (i.e. 9 out of 750 individuals; 1.2%). Thelazia callipaeda DNA was detected until 147 days after experimental infection. This demonstrates that larvae of this eyeworm may survive in the fly for a significant period of the winter. Fat composition analysis showed that flies produced more unsaturated than saturated fatty acids during diapause, probably because unsaturated fatty acids remain in a liquid state at lower temperatures, providing anti-freeze properties to survive winter.

Thermal analysis of ice and glass transitions in insects that do and do not survive freezing.

Some insects rely on the strategy of freeze tolerance for winter survival. During freezing, extracellular body water transitions from the liquid to the solid phase and cells undergo freeze-induced dehydration. Here, we present results of a thermal analysis (from differential scanning calorimetry) of ice fraction dynamics during gradual cooling after inoculative freezing in variously acclimated larvae of two drosophilid flies, Drosophila melanogaster and Chymomyza costata Although the species and variants ranged broadly between 0 and close to 100% survival of freezing, there were relatively small differences in ice fraction dynamics. For instance, the maximum ice fraction (IFmax) ranged between 67.9% and 77.7% total body water (TBW). Chymomyza costata larvae showed statistically significant phenotypic shifts in parameters of ice fraction dynamics (melting point and IFmax) upon entry into diapause, cold acclimation and feeding on a proline-augmented diet. These differences were mostly driven by colligative effects of accumulated proline (ranging between 6 and 487 mmol kg-1 TBW) and other metabolites. Our data suggest that these colligative effects per se do not represent a sufficient mechanistic explanation for high freeze tolerance observed in diapausing, cold-acclimated C. costata larvae. Instead, we hypothesize that accumulated proline exerts its protective role via a combination of mechanisms. Specifically, we found a tight association between proline-induced stimulation of glass transition in partially frozen body liquids (vitrification) and survival of cryopreservation in liquid nitrogen.

Insect fat body cell morphology and response to cold stress is modulated by acclimation.

Mechanistic understanding about the nature of cellular cryoinjury and mechanisms by which some animals survive freezing while others do not is currently lacking. Here, we exploited the broadly manipulable freeze tolerance of larval malt flies (Chymomyza costata) to uncover cell and tissue morphological changes associated with freeze mortality. Diapause induction, cold acclimation and dietary proline supplementation generate malt fly variants ranging from weakly to extremely freeze tolerant. Using confocal microscopy and immunostaining of the fat body, Malpighian tubules and anterior midgut, we described tissue and cytoskeletal (F-actin and α-tubulin) morphologies among these variants after exposure to various cold stresses (from chilling at -5°C to extreme freezing at -196°C), and upon recovery from cold exposure. Fat body tissue appeared to be the most susceptible to cryoinjury: freezing caused coalescence of lipid droplets, loss of α-tubulin structure and apparent aggregation of F-actin. A combination of diapause and cold acclimation substantially lowered the temperature at which these morphological disruptions occurred. Larvae that recovered from a freezing challenge repaired F-actin aggregation but not lipid droplet coalescence or α-tubulin structure. Our observations indicate that lipid coalescence and damage to α-tubulin are non-lethal forms of freeze injury, and suggest that repair or removal (rather than protection) of actin proteins is a potential mechanism of acquired freeze tolerance.

The evolutionary origination of a novel expression pattern through an extreme heterochronic shift.

The evolutionary origins of morphological structures are thought to often depend upon the redeployment of old genes into new developmental settings. Although many examples of cis-regulatory divergence have shown how pre-existing patterns of gene expression have been altered, only a small number of case studies have traced the origins of cis-regulatory elements that drive new expression domains. Here, we elucidate the evolutionary history of a novel expression pattern of the yellow gene within the Zaprionus genus of fruit flies. We observed a unique pattern of yellow transcript accumulation in the wing disc during the third larval instar, a stage that precedes its typical expression pattern associated with cuticular melanization by about a week. The region of the Zaprionus wing disc that expresses yellow subsequently develops into a portion of the thorax, a tissue for which yellow expression has been reported for several fruit fly species. Tests of GFP reporter transgenes containing the Zaprionus yellow regulatory region revealed that the wing disc pattern arose by changes in the cis-regulatory region of yellow. Moreover, the wing disc enhancer activity of yellow depends upon a short conserved sequence with ancestral thoracic functions, suggesting that the pupal thorax regulatory sequence was genetically reprogrammed to drive expression that commences much earlier during development. These results highlight how novel domains of gene expression may arise by extreme shifts in timing during the origins of novel traits.

Fluctuating asymmetry of meristic traits: an isofemale line analysis in an invasive drosophilid, Zaprionus indianus.

Metric (e.g., body size) and meristic (e.g., bristle number) traits are of general use in quantitative genetic studies, and the phenotypic variance is subdivided into a genetic and a non-genetic environmental component. The non-genetic variance may have two origins: a common garden effect between individuals and a developmental instability within the same individual. Developmental instability may be studied by considering the fluctuating asymmetry (FA) between the two sides of the body. The isofemale line technique is a convenient method for investigating the architecture of natural populations but has been rarely implemented for investigating FA. In this paper, we use this experimental design for analyzing four meristic traits in eight populations of the cosmopolitan Zaprionus indianus. A study of the correlation between left and right side of each line revealed that almost 90% of the variability was due to a developmental noise, while a much higher correlation among the means of the lines from the same population was observed. A slight trend toward a directional asymmetry was observed: more thoracic bristles on the left side. Four kinds of indices, scaled or non-scaled to the mean were used for comparing the different traits. Unscaled values (mean absolute values or standard deviation of each line) revealed a linear increase with the means. Interestingly the results of ovariole number were included in the same regression. With the scaled indices (mean absolute divided by each individual value or stadard deviation devided by the mean), the differences among traits were considerably decreased, but still remained significant. The mean FA of the various traits were not correlated, suggesting that each trait harbors its own developmental stability. The CVs of FA were high with a magnitude similar to those of the trait themselves, slightly less than 10%. Finally, even with the isofemale line design, which is a powerful means for unravelling slight genetic variations, we did not to find any clear indication of a genetic component of FA under the optimal environmental conditions used in this study.

Conserved metallomics in two insect families evolving separately for a hundred million years.

Μetal cofactors are required for enzymatic catalysis and structural stability of many proteins. Physiological metal requirements underpin the evolution of cellular and systemic regulatory mechanisms for metal uptake, storage and excretion. Considering the role of metal biology in animal evolution, this paper asks whether metal content is conserved between different fruit flies. A similar metal homeostasis was previously observed in Drosophilidae flies cultivated on the same larval medium. Each species accumulated in the order of 200 µg iron and zinc and approximately ten-fold less manganese and copper per gram dry weight of the adult insect. In this paper, data on the metal content in fourteen species of Tephritidae, which are major agricultural pests worldwide, are presented. These fruit flies can be polyphagous (e.g., Ceratitis capitata) or strictly monophagous (e.g., Bactrocera oleae) or oligophagous (e.g., Anastrepha grandis) and were maintained in the laboratory on five distinct diets based on olive oil, carrot, wheat bran, zucchini and molasses, respectively. The data indicate that overall metal content and distribution between the Tephritidae and Drosophilidae species was similar. Reduced metal concentration was observed in B. oleae. Feeding the polyphagous C. capitata with the diet of B. oleae resulted in a significant quantitative reduction of all metals. Thus, dietary components affect metal content in some Tephritidae. Nevertheless, although the evidence suggests some fruit fly species evolved preferences in the use or storage of particular metals, no metal concentration varied in order of magnitude between these two families of Diptera that evolved independently for over 100 million years.

POPULATION DYNAMICS OF DROSOPHILA SUZUKII (DIPTERA: DROSOPHILIDAE) IN BELGIUM IN 2013.

The vinegar fly Drosophila suzukii (Matsumura) (Diptera Drosophilidae), spotted wing drosophila, is a new invasive fruit pest that recently became established in Europe. Unlike other fruit flies that typically only infest overripe and rotten fruit, D. suzukii females oviposit in ripe fruit leading to considerable economic losses of fruit during production. In 2011 and 2012 D. suzukii was detected at several places in Belgium. In 2013, a large-scale monitoring in frame of the project "FLY ALERT" (FOD) was executed using traps with liquid attractant (apple cider vinegar) at more than 100 locations across Belgium during the whole fruit growing season. At 16 locations we also compared the efficacy of a 'bottle type' trap with a 'cup type' trap. The results show that D. suzukii has expanded its distribution in Belgium. Remarkably, in 2013 as well as in 2012 the first detections were made only in the second part of the growing season (August) and the populations reached their peak only at the very end of the season (November). In the bottle type trap the first flies were caught 2-3 weeks earlier than in the cup type trap. In addition, also the population peaks were on average 1 week earlier when monitored with the bottle trap compared to when monitored with the cup trap. In 2014, after an exceptional mild winter adult D. suzukii flies were continuously detected throughout the winter and early spring. The implications of these findings for the phenology of D. suzukii in the Northwest climate region of Europe are discussed.

Many ways to make a novel structure: a new mode of sex comb development in Drosophilidae.

On macroevolutionary time scales, the same genes can regulate the development of homologous structures through strikingly different cellular processes. Comparing the development of similar morphological traits in closely related species may help elucidate the evolutionary dissociation between pattern formation and morphogenesis. We address this question by focusing on the interspecific differences in sex comb development in Drosophilids. The sex comb is a recently evolved, male-specific structure composed of modified bristles. Previous work in the obscura and melanogaster species groups (Old World Sophophora) has identified two distinct cellular mechanisms that give rise to nearly identical adult morphologies. Here, we describe sex comb development in a species from a more distantly related lineage, the genus Lordiphosa. Although the expression of key regulatory genes is largely conserved in both clades, the cell behaviors responsible for sex comb formation show major differences between Old World Sophophora and Lordiphosa. We suggest that the many-to-one mapping between development and adult phenotype increases the potential for evolutionary innovations.

Contrasting levels of genotype by environment interaction for life history and morphological traits in invasive populations of Zaprionus indianus (Diptera: Drosophilidae).

It has been demonstrated that phenotypic plasticity and genotype by environment interaction are important for coping with new and heterogeneous environments during invasions. Zaprionus indianus Gupta (Diptera: Drosophilidae) is an Afrotropical invasive fly species introduced to the South American continent in 1999. This species is generalist and polyphagous, since it develops and feeds in several different fruit species. These characteristics of Z. indianus suggest that phenotypic plasticity and genotype by environment interaction may be important in this species invasion process. In this sense, our aim was to investigate the role of genetic variation for phenotypic plasticity (genotype by environment interaction) in Z. indianus invasion of the South American continent. Specifically, we quantified quantitative genetic variation and genotype by environment interactions of morphological and life history traits in different developmental environments, that is, host fruits. This was done in different populations in the invasive range of Z. indianus in Argentina. Results showed that Z. indianus populations have considerable amounts of quantitative genetic variation. Also, genotype by environment interactions was detected for the different traits analyzed in response to the different developmental environments. Interestingly, the amounts and patterns of these parameters differed between populations. We interpreted these results as the existence of differences in evolutionary potential between populations that have an important role in the short- and long-term success of the Z. indianus invasion process.

Genome size diversity in the family Drosophilidae.

Flies in the genus Drosophila have been the dominant model organisms in genetics for over a century and, with a dozen complete sequences now available, continue as such in modern comparative genomics. Surprisingly, estimates of genome size for this genus have been relatively sparse, covering less than 2% of species. Here, best practice flow cytometric genome size estimates are reported for both male and female flies from 67 species from six genera in the family Drosophilidae, including 55 species from the genus Drosophila. Direct and phylogenetically corrected correlation analyses indicate that genome size is positively correlated with temperature-controlled duration of development in Drosophila, and there is indication that genome size may be positively related to body size and sperm length in this genus. These findings may provide some explanation for the streamlined genomes found in these insects, and complement recent work demonstrating possible selective constraints on further deletion of noncoding DNA.

Adaptation to different climates results in divergent phenotypic plasticity of wing size and shape in an invasive drosophilid.

The phenotypic plasticity of wing size and wing shape of Zaprionus indianus was investigated in relation to growth temperature (17 degrees C to 31 degrees C) in two natural populations living under different climates, equatorial and subtropical. The two populations were clearly distinguished not only by their wing size (the populations from the colder climate being bigger in size), but also by the shape of the response curves to growth temperature i.e., their reaction norms. In this respect, the temperature at which the size of the wing was maximum was about 3 degrees C higher in the equatorial population. Such a difference in size plasticity is already found in two other nonclosely related species, might be a general evolutionary pattern in drosophilids. Wing shape was investigated by calculating an ellipse included into the wing blade, then by considering the ratio of the two axes, and also by analysing the angular position of 10 wing-vein landmarks. For an overall shape index (ratio of the two axes of the ellipse), a regular and almost linear increase was observed with increasing temperature i.e., a more round shape at high temperatures. Wing shape was also analysed by considering the variations of the various angles according to temperature. A diversity of response curves was observed, revealing either a monotonous increase or decrease with increasing temperature, and sometimes a bell shape curve. An interesting conclusion is that, in most cases, a significant difference was observed between the two populations, and the difference was more pronounced at low temperatures. These angular variations are difficult to interpret in an evolutionary context. More comparative studies should be undertaken before reaching some general conclusions.

A new genus of anthophilous drosophilids, Impatiophila (Diptera, Drosophilidae): morphology, DNA barcoding and molecular phylogeny, with descriptions of thirty-nine new species.

Breeding habits of essential dependence on flowers for larval food resources have evolved repeatedly in separate lineages of the Drosophilidae. However, flowers of Impatiens L. have never been recognized as hosts for drosophilid flies until recently: two Hirtodrosophila species, H. actinia (Okada) and H. yapingi Gao, were found feeding and breeding on Impatiens flowers. During our recent field surveys in central and southern China, a great number of drosophilid flies morphologically resembling the two species were collected, almost exclusively from flowers of Impatiens (family Balsaminaceae) and the family Gesneriaceae. In the present study, these specimens were identified on the basis of morphological characters and/or partial DNA sequences of the mitochondrial COI (cytochrome c oxidase subunit I gene, used as a barcoding marker). As a result, 39 new species were recognized. We then reconstructed the phylogenetic relationships among most of them, based on concatenated DNA sequences (3047 nucleotide sites) of two mitochondrial (COI and COII, i.e., cytochrome c oxidase subunits I and II, respectively) and three nuclear genes (ATPsyn-alpha, alphaTub84B and Hsc70cb, i.e., ATP synthase alpha, alpha-Tubulin at 84B and Hsc70Cb isoform H, respectively). In the resulting Bayesian and ML (maximum likelihood) trees, three well-supported clades were recognized, with a few species having remained uncertain for their phylogenetic positions. We also conducted a cladistic analysis with data of adult morphological characters to investigate the phylogenetic positions of a few species of which DNA sequence data were not available, and to investigate the classification of species groups with definition of their diagnoses. In consequence, we established a new genus, Impatiophila, for the species visiting flowers of Impatiens and Gesneriaceae, described all the new species, and revised the taxonomy of some known species.

Four new species of the Stegana ornatipes species group (Diptera: Drosophilidae) from Yunnan, China, with DNA barcoding information.

Fore new species of Stegana (Steganina) ornatipes species group are found from Yunnan, China: S. (S.) angustifoliacea sp. nov., S. (S.) crinata sp. nov., S. (S.) nigripes sp. nov. and S. (S.) polysphyra sp. nov. The DNA sequences of the mitochondrial COI gene with BOLD Process ID and GenBank accession numbers are provided for the Chinese species. 

Differences in larval nutritional requirements and female oviposition preference reflect the order of fruit colonization of Zaprionus indianus and Drosophila simulans.

Species coexist using the same nutritional resource by partitioning it either in space or time, but few studies explore how species-specific nutritional requirements allow partitioning. Zaprionus indianus and Drosophila simulans co-exist in figs by invading the fruit at different stages; Z. indianus colonizes ripe figs, whereas D. simulans oviposits in decaying fruit. Larvae feed on yeast growing on the fruit, which serves as their primary protein source. Because yeast populations increase as fruit decays, we find that ripe fruit has lower protein content than rotting fruit. Therefore, we hypothesized that Z. indianus and D. simulans larvae differ in their dietary requirements for protein. We used nutritional geometry to assess the effects of protein and carbohydrate concentration in the larval diet on life history characters in both species. Survival, development time, and ovariole number respond differently to the composition of the larval diet, with Z. indianus generally performing better across a wider range of protein concentrations. Correspondingly, we found that Z. indianus females preferred to lay eggs on low protein foods, while D. simulans females chose higher protein foods for oviposition when competing with Z. indianus. We propose the different nutritional requirements and oviposition preference of these two species allows them to temporally partition their habitat.

The New World genus Rhinoleucophenga (Diptera: Drosophilidae): new species and notes on occurrence records.

The genus Rhinoleucophenga Hendel comprises 26 nominal species with New World distribution. In the present study, two new species are described from samples in the Pampa and Caatinga biomes in Brazil, R. punctuloides sp. nov. and R. trivisualis sp. nov., respectively. Rhinoleucophenga punctuloides sp. nov. is a sibling species of R. punctulata Duda. Furthermore, two females of R. joaquina Schmitz, Gottschalk & Valente were found for the first time and a description is presented. A taxonomic dichotomous key with pictures is given for the Rhinoleucophenga species recorded in the Caatinga and Pampa biomes. The Neotropical open environments are areas of high diversity for Rhinoleucophenga. The description of new species and review of some older descriptions can change the area of species distribution and improve the faunistic knowledge of other localities in which previous studies have shown unidentified or misidentified Rhinoleucophenga species.

The genera Luzonimyia and Pararhinoleucophenga from China (Diptera: Drosophilidae), with DNA barcoding information.

Four new species are described from Yunnan, China, which belong to two different genera within the subfamily Steganinae: Luzonimyia hirsutina sp. nov., Luzonimyia setocauda sp. nov., Pararhinoleucophenga amnicola sp. nov. and Pararhinoleucophenga sylvatica sp. nov. The DNA sequences and GenBank accession numbers of the mitochondrial COI gene among Chinese species are provided.