Biodiversity of cactophilic microorganisms in western Argentina: community structure and species composition in the necroses of two sympatric cactus hosts

The cactus-yeast-Drosophila system is a model system in evolutionary biology, and the participating saprotrophic microorganisms represent one of the most thoroughly studied microbial communities. However, much of the cactus-dominated regions of South America, home to endemic versions of this classical system, remain understudied. A combined morpho-physiological and molecular approach was employed to identify the fungal members of the cactus-yeast-Drosophila system in western Argentina. We identified twenty one species of saprotrophic organisms in the necroses of Opuntia sulphurea and Trichocereus terscheckii in a region of sympatry, where both cacti are exploited by cactophilic Drosophila. After excluding opportunistic isolates, we determined that the saprobe community of O. sulphurea was composed of eight species (including the first consideration of filamentous fungi as community members), whereas the community of T. terscheckii represented a subgroup of the former. We explain this nested pattern by considering the physiological and ecological attributes of both hosts and vectors involved.     

Yeast communities as descriptors of habitat use by the Drosophila fasciola subgroup (repleta group) in Atlantic rain forests

Yeast communities associated with four species of the Drosophila fasciola subgroup (repleta group) in tropical rain forests were surveyed in an abandoned orchard, and rain forest sites of Rio de Janeiro and Ilha Grande, State of Rio de Janeiro, Brazil. Adult flies of Drosophila carolinae, Drosophila coroica, Drosophila fascioloides and Drosophila onca frequently carried Candida colliculosa, Geotrichum sp, Kloeckera apiculata and a Pichia membranaefaciens-like species. The most frequent yeasts in the crop of flies included Candida collicullosa, C. krusei, Pichia kluyveri and a P. membranaefaciens-like species. The physiological abilities and species composition of these yeast communities differed from those of other forest-inhabiting Drosophila. The narrow feeding niches of the fasciola subgroup suggested the use of only part of the substrates available to the flies as food in the forest environment, as noted previously for cactophilic Drosophila serido (mulleri subgroup of the repleta group) in a sand dune ecosystem. The cactophilic yeasts that were isolated have not been previously found in forests. The fasciola subgroup probably used epiphytic cactus substrates as breeding and feeding sites in the forest. The physiological profile of yeasts associated with the fasciola flies was broader than that of yeasts associated with the cactophilic Drosophila serido, suggesting that the fasciola subgroup represents an older lineage from which the South American repleta species evolved.     

The colonization of organges by the cosmopolitan Drosophila

The guild of cosmopolitan Drosophila coexist almost worldwide and yet the mechanisms that underlie this coexistence are unknown. The larval resource of the guild is decaying fruit and vegetables, but the species show little specialization and can coexist on a single resource, such as oranges. In southern California the guild includes D. simulans (SIM), D. melanogaster (MEL), D. pseudoobscura (OBS), D. immigrans (IMM), D. hydei (HYD) and D. busckii (BUS). These species show consistent differences in their colonization of decaying organges, differences that may promote their coexistence. This study tested whether the colonization pattern of a species is determined primarily by attraction to specific resource types (decayed or fresh organges), by ability to colonize new resource patches, or by dependence on a successional sequence of Drosophila species. The experiments compared oranges that were pre-aged prior to a colonization period and showed that the colonization pattern of each species (except OBS) was driven primarily by its decay-dependent attraction to oranges. While OBS exhibited a pattern of colonization independent of pre-aging, the remaining species all showed some preference for older (7-day pre-aged) over fresh oranges. Their overall pattern of attraction, ordered by high relative abundance on fresher organges, was SIM>MEL=IMM>HYD=BUS. BUS, a specialist on decaying plant material, was the only species that showed a preference for 11-day over 7-day oranges. Pre-aging the oranges under covers, to prevent prior colonization by Drosophila, did not change the interspecific pattern of colonization, indicating that microbial decay was driving the changes in attraction. The patterns of attraction separated two ecologically similar pairs (SIM from MEL; IMM from HYD) and published data on ethanol tolerance show that, in each pair, the earliest colonizer has the lower tolerance. This suggests an important interplay between colonization patterns and physiological optima.     

Comparative molecular population genetics of the Xdh locus in the cactophilic sibling species Drosophila buzzatii and D. koepferae

The Xdh (rosy) gene is one of the best studied in the Drosophila genus from an evolutionary viewpoint. Here we analyze nucleotide variation in a 1875-bp fragment of the second exon of Xdh in Argentinian populations of the cactophilic D. buzzatii and its sibling D. koepferae. The major electrophoretic alleles of D. buzzatii not only lack diagnostic amino acids in the region studied but also differ on average from each other by four to 13 amino acid changes. Our data also suggest that D. buzzatii populations belonging to different phytogeographic regions are not genetically differentiated, whereas D. koepferae exhibits a significant pattern of population structure. The Xdh region studied is twice as polymorphic in D. buzzatii as in D. koepferae. Differences in historical population size or in recombinational environment between species could account for the differences in the level of nucleotide variation. In both species, the Xdh region exhibits a great number of singletons, which significantly departs from the frequency spectrum expected under neutrality for nonsynonymous sites and also for synonymous sites in D. buzzatii. These excesses of singletons could be the signature of a recent population expansion in D. buzzatii, whereas they may be simply explained as the result of negative selection in D. koepferae.     

A study of wing morphology and fluctuating asymmetry in interspecific hybrids between Drosophila buzzatii and D. koepferae

In this work we investigate the effect of interspecific hybridization on wing morphology using geometric morphometrics in the cactophilic sibling species D. buzzatii and D. koepferae. Wing morphology in F₁ hybrids exhibited an important degree of phenotypic plasticity and differs significantly from both parental species. However, the pattern of morphological variation between hybrids and the parental strains varied between wing size and wing shape, across rearing media, sexes, and crosses, suggesting a complex genetic architecture underlying divergence in wing morphology. Even though there was significant fluctuating asymmetry for both, wing size and shape in F₁ hybrids and both parental species, there was no evidence of an increased degree of fluctuating asymmetry in hybrids as compared to parental species. These results are interpreted in terms of developmental stability as a function of a balance between levels of heterozygosity and the disruption of coadaptation as an indirect consequence of genomic divergence.     

Degradation of benzene compounds by yeasts in acidic soils

Attemps were made to demonstrate the role of yeasts in the degradation of benzene compounds under natural soil conditions. Yeasts were isolated from acidic sandy soil supplied with benzene compounds. For this purpose the slant culture method was used. Growth on the benzene compounds took place on solid growth media at 10°C. Several yeast species were isolated: Leucosporidium scottii, Rhodotorula aurantiaca, Rhodotorula mucilaginosa, Trichosporon dulcitum, Trichosporon moniliiforme and Schizoblastosporion starkeyi-henricii. Cryptococcus humicolus and Cryptococcus laurentii were isolated from liquid enrichment cultures. All these strains assimilated several benzene compounds in pure culture.Cresol removal from contaminated soil was speeded up by inoculation with Rhodotorula aurantiaca G36. It was demonstrated that this yeast utilized this compound in competition with the soil microflora.     

The hernandez and fernandez genes of Drosophila specify eye and antenna

The formation of different structures in Drosophila depends on the combined activities of selector genes and signaling pathways. For instance, the antenna requires the selector gene homothorax, which distinguishes between the leg and the antenna and can specify distal antenna if expressed ectopically. Similarly, the eye is formed by a group of "eye-specifying" genes, among them eyeless, which can direct eye development ectopically. We report here the characterization of the hernandez and fernandez genes, expressed in the antennal and eye primordia of the eye-antenna imaginal disc. The predicted proteins encoded by these two genes have 27% common amino acids and include a Pipsqueak domain. Reduced expression of either hernandez or fernandez mildly affects antenna and eye development, while the inactivation of both genes partially transforms distal antenna into leg. Ectopic expression of either of the two genes results in two different phenotypes: it can form distal antenna, activating genes like homothorax, spineless, and spalt, and it can promote eye development and activates eyeless. Reciprocally, eyeless can induce hernandez and fernandez expression, and homothorax and spineless can activate both hernandez and fernandez when ectopically expressed. The formation of eye by these genes seems to require Notch signaling, since the induction of ectopic eyes and the activation of eyeless by the hernandez gene are suppressed when the Notch function is compromised. Our results show that the hernandez and fernandez genes are required for antennal and eye development and are also able to specify eye or antenna ectopically.     

The genetics of adaptation in Drosophila sechellia

Drosophila sechellia is an island endemic of the Seychelles. After its geographic isolation on these islands, D. sechellia evolved into a host specialist on the fruit of Morinda citrifolia – a fruit often noxious and repulsive to Drosophila. Specialization on M. citrifolia required the evolution of a suite of adaptations, including resistance to and preference for some of the toxins found in this fruit. Several of these adaptive traits have been studied genetically. Here, I summarize what is known about the genetics of these traits and briefly describe the ecological and geographical context that shaped the evolution of these characters. The data from D. sechellia suggest that adaptations are not as genetically complex as historically thought, although almost all of the adaptations of D. sechellia involve several genes.     

Embryonic origin and differentiation of the Drosophila heart

We have followed the normal development of the different cell types associated with the Drosophila dorsal vessel, i.e. cardioblasts, pericardial cells, alary muscles, lymph gland and ring gland, by using several tissue-specific markers and transmission electron microscopy. Precursors of pericardial cells and cardioblasts split as two longitudinal rows of cells from the lateral mesoderm of segments T2-A7 (cardiogenic region) during stage 12. The lymph gland and dorsal part of the ring gland (corpus allatum) originate from clusters of lateral mesodermal cells located in T3 and T1/dorsal ridge, respectively. Cardioblast precursors are strictly segmentally organized; each of T2-A6 gives rise to six cardioblasts. While moving dorsally during the stages leading up to dorsal closure, cardioblast precursors become flattened, polarized cells aligned in a regular longitudinal row. At dorsal closure, the leading edges of the cardioblast precursors meet their contralateral counterparts. The lumen of the dorsal vessel is formed when the trailing edges of the cardioblast precursors of either side bend around and contact each other. The amnioserosa invaginates during dorsal closure and is transiently attached to the cardioblasts; however, it does not contribute to the cells associated with the dorsal vessel and degenerates during late embryogenesis. We describe ultrastructural characteristics of cardioblast differentiation and discuss similarities between cardioblast development and capillary differentiation in vertebrates. Correspondence to: V. Hartenstein     

The utilization of naturally occurring yeasts by Drosophila species, using chemically defined substrates

The ability of three Drosophila species; Drosophila obscura, D. subobscura and D. phalerata, to utilize a range of naturally occurring yeasts as food sources was determined. The yeasts used were the dominant species of two commonly used breeding sites, Sorbus aucuparia fruits and the fruiting bodies of the fungus Phallus impudicus. The range of yeast species used was found to reflect the degree ot substrate specialization exhibited by these three species, with D. subobscura being broader niched than the other two species. There was an indication that D. phalerata was adapted to those yeasts normally found on its natural breeding site, Phallus impudicus. Many factors were found to affect the nutritional adequacy of the yeasts, including their growth period, the larval density, the presence of competitors and particularly the chemical composition of the basal growth medium.     

Modification of Drosophila cell surfaces by concanavalin A

Summary The effect of Con A on the surface morphology of cultured cells of Drosophilia melanogaster growing on coverglasses was examined by scanning electron microscopy. With low lectin concentrations (5–10g/ml) surface filaments disappeared and the cells flattened and spread against the glass surface. Cytoplasmic fusion bridges were observed in areas where cells made contact. Concentrations of Con A ranging between 50–500 g/ml caused cell shrinkage and surface distortions without cell flattening and filament loss. These morphologic effects were not apparent if Con A binding sites were blocked by preincubation with -methyl-D-mannopyranoside before application to the cell cultures. However, once the Con A-mediated changes were in effect, the cells failed to show recovery when they were returned to growth medium and a majority of the cells on the coverglasses degenerated. Presumably the cells whose morphology appears unaffected by Con A treatment are the survivors that repopulate cultures returned to growth medium.Supported by Grants CA-12600 and CA 16619 awarded by the National Cancer Institute, DREW and in part by NIH Biomedical Sciences Grant No. RR-07050. CAA's participation in this project was supported by Training Grant No. 5T01-GM-71-17We wish to thank Dr. Imogene Schneider for providing the cell lines     

The actions of the neonicotinoid imidacloprid on cholinergic neurons of Drosophila melanogaster

The neonicotinoid insecticide imidacloprid is an agonist on insect nicotinic acetylcholine receptors (nAChRs). We utilised fura-2-based calcium imaging to investigate the actions of imidacloprid on cultured GFP-tagged cholinergic neurons from the third instar larvae of the genetic model organism Drosophila melanogaster. We demonstrate dose-dependent increases in intracellular calcium ([Ca²⁺]_i) in cholinergic neurons upon application of imidacloprid (10 nM–100 μM) that are blocked by nAChR antagonists mecamylamine (10 μM) and α-bungarotoxin (α-BTX, 1 μM). When compared to other (untagged) neurons, cholinergic neurons respond to lower concentrations of imidacloprid (10–100 nM) and exhibit larger amplitude responses to higher (1–100 μM) concentrations of imidacloprid. Although imidacloprid acts via nAChRs, increases in [Ca²⁺]_i also involve voltage-gated calcium channels (VGCCs) in both groups of neurons. Thus, we demonstrate that cholinergic neurons express nAChRs that are highly sensitive to imidacloprid, and demonstrate a role for VGCCs in amplifying imidacloprid-induced increases in [Ca²⁺]_i.     

Nucleotide divergence of the rp49 gene region between Drosophila melanogaster and two species of the Obscura group of Drosophila

Summary A 2.1-kb SStI fragment including the rp49 gene and the 3 end of the -serendipity gene has been cloned and sequenced in Drosophila pseudoobscura. rp49 maps at region 62 on the tip of chromosome II of this species. Both the coding and flanking regions have been aligned and compared with those of D. subobscura. There is no evidence for heterogeneity in the rate of silent substitution between the rp49 coding region and the rate of substitutions in flanking regions, the overall silent divergence per site being 0.19. Noncoding regions also differ between both species by different insertions/deletions, some of which are related to repeated sequences. The rp49 region of D. pseudoobscura shows a strong codon bias similar to those of D. subobscura and D. melanogaster. Comparison of the rates of silent (K _S ) and nonsilent (K _a ) substitutions of the rp49 gene and other genes completely sequenced in D. pseudoobscura and D. melanogaster confirms previous results indicating that rp49 is evolving slowly both at silent and nonsilent sites. According to the data for the rp49 region, D. pseudoobscura and D. subobscura lineages would have diverged some 9 Myr ago, if one assumes a divergence time of 30 Myr for the melanogaster and obscura groups.Offprint requests to: C. Segarra     

The association between malathion resistance and acetylcholinesterase in Drosophila melanogaster

The relationship between the 50% survival time for flies feeding on a malathion-containing medium and the activity of acetylcholinesterase (AChE) was determined for 15 isofemale lines of Drosophila melanogaster. A significant correlation was found (r=0.28, P<0.05), with more resistant lines tending to have a lower level of AChE activity. An association between AChE and malathion resistance was also observed in a selection experiment. The AChE activity decreased in two of two populations selected for malathion resistance. AChE from these populations was altered in kinetic parameters (measured in crude head extracts) and electrophoretic mobility. Although the resistant AChE had a lower activity (V _m) on either a per milligram protein or a per individual basis, its apparent K _m for acetylthiocholine was lower than that of susceptible AChE. Recombination mapping of both low activity and fast electrophoretic mobility localized these traits to the region of the structural locus (Ace) on the third chromosome. The AChE activity of flies heterozygous for a variety of Ace lesions (kindly provided by Dr. W. M. Gelbart) was consistent with this location. The changes in AChE were suggested to have been caused by selection of alleles at the Ace locus.This work was supported by NSERC Grants A5857, G0183, and A0629.     

Notch signalling in Drosophila: three ways to use a pathway

Cell-cell interactions mediated by Notch are critical at multiple stages of development. Our current understanding of the Notch signalling pathway suggests a comparatively simple transduction mechanism. However, this core pathway can be deployed in three different types of developmental process: lateral inhibition, lineage decisions and boundary formation. These illustrate how the activity of the pathway can be modulated both at the cell surface, through availability and effectiveness of ligand interactions, and inside the cell, through effects on the transduction pathway and the responsiveness of target genes.     

Modulation of ionizing radiation-induced apoptosis by bantam microRNA in Drosophila

In Drosophila, heterozygosity in the pro-apoptotic gene hid significantly reduces apoptosis that is induced by ionizing radiation (IR). Therefore, mechanisms that regulate Hid levels can potentially contribute to life-or-death decision of an irradiated cell. 3′UTR of hid mRNA contains 5 potential binding sites for bantam microRNA. Ectopic expression of ban attenuated apoptosis that results from ectopic expression of hid but the significance of this regulation under physiological conditions remained to be investigated. We report here that ban is needed to limit IR-induced apoptosis in larval imaginal discs. Using tubulin–EGFP ban sensors with ban consensus sequences in the 3′UTR, we find that EGFP decreases following IR, indicating that IR activates ban. Likewise, a tubulin–EGFP reporter with hid-3′UTR is repressed in irradiated discs and this repression requires ban consensus sites in the hid 3′UTR. ban mutant larvae show increased sensitivity to killing by IR, which is suppressed by a mutation in hid. These results can fit into a model in which IR activates ban and ban represses hid to limit IR-induced apoptosis. miRNAs have been shown previously to be induced by radiation but this is the first report that a miRNA is functionally important for radiation responses.     

The cell biology of Drosophila wing metamorphosis in vitro

Summary We have examined the metamorphosis of the wing imaginal disc of Drosophila during culture in vitro in the continuous presence of 20-hydroxy ecdysone (0.1 g/ ml). We find that the sequence of cellular changes in the wing blade during culture closely match those occurring in situ, involving two periods at which the dorsal and ventral surfaces are joined only by cell processes containing trans-alar microtubule arrays. Good pupal and imaginal cuticle secretion is found in this system.