Localizing genes in Drosophila melanogaster polytene chromosomes by fluorescence in situ hybridization

This paper describes a method for the identification of single copy genes in Drosophila melanogaster polytene chromosomes, using fluorescence in situ hybridization (FISH). We demonstrate the detection of white (w) , a gene previously mapped to 1-1.5 region of the linkage map, and to 3C2 region of the cytogenetic map of X chromosome. Squash preparations of polytene chromosomes from salivary glands dissected out from third instar larvae of Drosophila melanogaster were denatured and subjected to hybridization with a digoxigenin labeled probe, corresponding to mini-white gene. The preparations were then washed and incubated with antidigoxigenin-fluorescein antibodies. After removal of the nonspecifically bound antibodies, the polytene chromosomes were counterstained with propidium iodide. Fluorescence microscopy revealed white locus in the X chromosome in a subterminal location, in agreement with the above mentioned maps. The protocol is efficient and adaptable for simultaneously multiple signal detection.     

Drosophila suzukii in Michigan vineyards,and the first report of Zaprionus indianus from this region

Drosophila suzukii is a new invasive pest that in recent years has become established in the Great Lakes region of the United States. Understanding the level of infestation in potentially susceptible crops is an important first step for planning appropriate management responses. This study was conducted in 2010–2012 to determine the infestation potential of this pest in native Vitis labrusca, French hybrid and V. vinifera grape cultivars grown in Michigan vineyards. Drosophila suzukii adults were reared out of collected grape samples in all 3 years, comprising a low proportion of all emerged drosophilids in each of the years. This trend was also found in vacuum sampling, conducted in 2011, with the majority of flies collected being non‐D. suzukii drosophilids. Another recently introduced invasive fly species, Zaprionus indianus, was also reared out of grape samples collected in 2012. While the results of this study indicate no immediate threats to commercial grape production from D. suzukii, further research is needed to elucidate possible secondary effects that this species may have on vineyards, such as the introduction of diseases to the fruit.     

Quantitative in situ hybridization of ribosomal RNA species to polytene chromosomes of Drosophila melanogaster.

In situ hybridization of ¹²⁵I-labelled 5 S and 18 + 28 S ribosomal RNAs to the salivary polytene chromosomes of Drosophila melanogaster was successfully quantitated. Although the precision of the data is low, it is possible to compare the hybridization reaction between an RNA sample and chromosomes in situ with the reaction between the same RNA sample and Drosophila DNA immobilized on nitrocellulose filters. The in situ hybrid dissociates over a narrow temperature range with a midpoint similar to the value expected for the filter hybrid. The kinetics of the in situ hybridization reaction can be fit with a single first-order rate constant that has a value from three to five times smaller than the corresponding filter hybridization reaction. Although the reaction saturates at longer times or higher RNA concentrations, the saturation value does not correspond to an RNA molecule bound to every available DNA sequence. With the acid denaturation procedure most commonly used to preserve cytological quality, only 5 to 10% of the complementary DNA in the chromosomes is available to form hybrids in situ. This hybridization efficiency is a function of how the slides are prepared and the conditions of annealing, but is approximately constant with a given procedure for both 5 S RNA and 18 + 28 S RNA over a number of different cell types with different DNA contents. The results provide further evidence that the formation of RNA-DNA hybrids is the sole basis of in situ hybridization, and show that the properties of the in situ hybrids are remarkably similar to those of filter hybrids. It is also suggested that for reliable chromosomal localization using the in situ hybridization technique, the kinetics of the reaction should be followed to ensure that the correct rate constant is obtained for the major RNA species in the sample and an impurity in the sample is not localized instead.     

Zaprionus tuberculatus: chromosome map and gene mapping by DNA in situ hybridization.

The genus Drosophila has long been used as a model of karyotype evolution, demonstrating change by paracentric inversion and occasional centric fusion of an ancestral karyotype of five rod-shaped and one "dot" chromosome. This study shows, by mapping D. melanogaster probes hybridized to polytene chromosomes of Zaprionus tuberculatus, that this ancestral pattern extends beyond the genus Drosophila. A formal polytene chromosome map of Z. tuberculatus is presented.     

Cytogenetics of Zaprionus indianus Gupta (Diptera: Drosophilidae): Nucleolar organizer regions,mitotic and polytene chromosomes and inversion polymorphism

Zaprionus indianus, a member of the family Drosophilidae, is one of the commonest and most widespread species in India. It exploits a variety of fermenting fruits in nature. The nucleolar organizer regions (NORs), mitotic and polytene chromosomes were studied. A standard map of the polytene chromosomes has been constructed in order to locate break-points precisely for naturally occurring chromosomal rearrangements. Analysis of population samples of this species from four different geographical areas has revealed the presence of a single paracentric inversion in the second chromosome. Our quantitative data on the above inversion have confirmed the excess of inversion heterozygotes in nature.     

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.     

Spermatogenesis of Zaprionus indianus and Zaprionus sepsoides (Diptera,Drosophilidae): Cytochemical,structural and ultrastructural characterization

Zaprionus indianus is a drosophilid native to the Afrotropical region that has colonized South America and exhibits a wide geographical distribution. In contrast, Z. sepsoides is restricted to certain African regions. The two species differ in the size of their testes, which are larger in Z. indianus than in Z. sepsoides. To better understand the biology and the degree of differentiation of these species, the current study evaluated spermatogenesis in males of different ages by conventional staining techniques and ultrastructural analysis. Spermatogenesis and the ultrastructure of spermatozoa were similar in the two species, and the diploid number was confirmed to be 2n = 12. A greater number of spermatozoa were observed in young Z. indianus (1–3 days old) compared to Z. sepsoides males, which showed a higher frequency of cells at the early stages of spermatogenesis. The head of the sperm was strongly marked by silver staining, lacto-acetic orcein and the Feulgen reaction; the P.A.S. reaction revealed glycogen granules in the testes of both species. Both species presented similar arrangement of microtubules (9+9+2), two mitochondrial derivatives of different size and 64 spermatozoa per bundle. Such similarity within the genus Zaprionus with other species of Drosophila, indicates that these structures are conserved in the family Drosophilidae. The differences observed the number and frequency of sperm cells in the early stages of spermatogenesis, between the young males of Z. indianus and Z. sepsoides, are features that may interfere with reproductive success and be related to the invasive potential of Z. indianus.     

In situ digestion of Drosophila virilis polytene chromosomes by AluI and HaeIII restriction endonucleases

Polytene chromosomes of Drosophila virilis were treated with AluI and HaeIII restriction endonucleases. Both enzymes were capable of extensively digesting chromosomal DNA, with the exception of some regions that contain repetitive DNAs. Moreover, a comparison was made between our data and the data already obtained with the same enzymes in D. melanogaster. On this basis, AluI digestion showed that the 5S RNA genes of D. virilis and D. melanogaster have different base composition, while digestion with HaeIII revealed resistance of the histone genes in D. virilis, contrary to what was previously found in D. melanogaster.     

The localization of tRNA 5 Asn,tRNAHis, and tRNAAla genes from Drosophila melanogaster by in situ hybridization to polytene salivary gland chromosomes

Transfer RNA _5; ^Asn , tRNA _; ^His , and tRNA^Ala were isolated from Drosophila melanogaster by means of Sepharose 4B chromatography and 2-dimensional polyacrylamide gel electrophoresis. The tRNAs were iodinated in vitro with Na¹²⁵I and hybridized in situ to salivary gland chromosomes from Drosophila. Subsequent autoradiography allowed the localization of the genes for tRNA _5; ^Asn in the regions 42A, 59F, 60C, and 84F; for tRNA^His in the regions 48F and 56E; and for tRNA^Ala in the regions 63A and 90C. From these and our previous results it can be concluded that the genes for the Q-base containing tRNAs (tRNA^Asn, tRNA^Asp, and tRNA^His, are not clustered in the Drosophila melanogaster genome.     

Variation at the Est3 locus and adaptability to organophosphorous compounds in Zaprionus indianus populations

Carboxylesterases are enzymes often associated with insect resistance to insecticides. The Est3 locus of Zaprionus indianus Gupta (Diptera: Drosophilidae) harbors four alleles that encode carboxylesterases with potentially detoxifying roles. In this study, we propose a model of resistance to insecticides in Z. indianus based on the adaptability of the Est3 locus inferred from the spread of its alleles over 27 generations in experimental populations, and their frequencies in field populations, which were either exposed or unexposed to the organophosphorous insecticide malathion. The increase in the frequency of this allele in experimental populations, and its high prevalence in field populations exposed to organophosphorous insecticides suggest that natural selection favors individuals with the Est3-3 allele. The low frequency of this allele in unexposed field populations, and the low productivity of Est3-3 homozygotes indicate that an adaptive cost is associated with this allele. The existence of a marker locus for insecticide resistance in Z. indianus makes it possible to use this species as a bioindicator for monitoring the excessive use of organophosphorous products and the emergence of resistance, and to devise strategies for the management of agricultural pests.     

Localization of tRNA genes of Drosophila melanogaster by in situ hybridization

Six purified tRNAs labeled with ¹²⁵I by chemical or enzymatic methods were hybridized to polytene chromosomes of Drosophila melanogaster. The main chromosomal regions of hybridization were: tRNA _GGA ^Gly , 58A, 84C, and 90E; tRNA ₂ ^Leu , 44E, 66B5-8, and 79F; tRNA _2b ^Ser , 86A, 88A9-12, and 94A6-8; tRNA ₃ ^Thr , 47F and 87B; tRNA ₄ ^Thr , 93A1-2; and tRNA ₁ ^Tyr , 19F, 22F-23A, 41, 50C1-4 and 85A. At 50C the hybridization of tRNA ₁ ^Tyr was polymorphic in the giant strains. When the hybridization of three valine isoacceptors studied previously was re-investigated, it was found that only one hybridization site, 90BC, was shared between tRNA _3b ^Val and tRNA ₄ ^Val . tRNA _3a ^Val did not have any sites in common with the other two.     

Chromosomal homologies betweenDrosophila lebanonensis andD. melanogaster determined by in situ hybridization

Twelve biotin-labelled recombinant DNA probes were hybridized to polytene chromosomes ofDrosophila melanogaster andD. lebanonesis. Probes were chosen in order to cover the whole chromosomal complement. Six probes correspond to known genes fromD. melanogaster (RpII215, H3–H4, MHC, hsp28/23, hsp83, hsp70), four probes are clones isolated from aD. subobscura library (Xdh, DsubS3, DsubG3, DsubG4) and the remaining two probes correspond to the Adh gene ofD. lebanonensis and to one sequence (262), not yet characterized, from the same species. The chromosomal homologies obtained from the in situ hybridization results allow us to determine that Muller's C and D chromosomal elements are fused in the karyotype ofD. lebanonensis and constitute the large metacentric chromosome. Single pericentric inversions in theE andB elements have generated the medium and small metacentric chromosomes, respectively. No great changes are detected in Muller'sA element, which remains acrocentric. The changes detected in the karyotypic evolution ofD. lebanonensis are frequently observed inDrosophila evolution, as deduced from chromosomal homologies of severalDrosophila species. The results are also consistent with Muller's proposal that chromosomal elements have been conserved during the evolution ofDrosophila.     

Population genetics and recent colonization history of the invasive drosophilid Zaprionus indianus in Mexico and Central America

Zaprionus indianus, also known as the African fig fly, is an invasive pest of a variety of commercial and native fruit. The species was first reported in Brazil in 1999, but has established itself in much of the New World within the last 10–15 years. We used nucleotide sequences from a segment of the mitochondrial cytochrome c oxidase subunit I (COI) gene to examine haplotype relationships, population structure, and infer the colonization history of Z. indianus in Mexico and Panama. Construction of a haplotype network showed that six COI haplotypes, obtained from flies collected at six localities in Mexico and one in Panama, clustered into three distinct clades. Clade composition was generally consistent in flies from Panama to northwestern Mexico, and analysis of molecular variance indicated no significant structure among populations. Three of the six haplotypes from Mexico and Panama were identical to previously reported haplotypes from Brazil. None of the six haplotypes, however, were shared with previously reported haplotypes from potential source populations in the Old World. The results of our genetic analysis suggest that the invasion of Z. indianus into Central America and Mexico most probably includes a northward migration of individuals from Brazil, with the possibility of at least one additional introduction of Z. indianus to the New World. Additional sequence data from potential source populations in the Old World will be required to confidently determine the number of introductions of Z. indianus into the New World, and to identify the geographic source.     

Multicolour whole-mount in situ hybridization to Drosophila embryos

 We report an extended whole-mount in situ hybridization procedure for Drosophila embryos. By using probes labelled with digoxigenin, fluorescein and biotin, respectively, this protocol allows the detection in three colours of RNAs derived from three different genes. Hybridized probes are detected by consecutive staining with appropriate alkaline phosphatase conjugates using different chromogenic substrate combinations, and serial removal of the antibody conjugates by low pH washes. Received: 7 May 1996/Accepted: 7 July 1996     

Ecophysiological interaction between nitrifying bacteria and heterotrophic bacteria in autotrophic nitrifying biofilms as determined by microautoradiography-fluorescence in situ hybridization

Ecophysiological interactions between the community members (i.e., nitrifiers and heterotrophic bacteria) in a carbon-limited autotrophic nitrifying biofilm fed only NH(4)(+) as an energy source were investigated by using a full-cycle 16S rRNA approach followed by microautoradiography (MAR)-fluorescence in situ hybridization (FISH). Phylogenetic differentiation (identification) of heterotrophic bacteria was performed by 16S rRNA gene sequence analysis, and FISH probes were designed to determine the community structure and the spatial organization (i.e., niche differentiation) in the biofilm. FISH analysis showed that this autotrophic nitrifying biofilm was composed of 50% nitrifying bacteria (ammonia-oxidizing bacteria [AOB] and nitrite-oxidizing bacteria [NOB]) and 50% heterotrophic bacteria, and the distribution was as follows: members of the alpha subclass of the class Proteobacteria (alpha-Proteobacteria), 23%; gamma-Proteobacteria, 13%; green nonsulfur bacteria (GNSB), 9%; Cytophaga-Flavobacterium-Bacteroides (CFB) division, 2%; and unidentified (organisms that could not be hybridized with any probe except EUB338), 3%. These results indicated that a pair of nitrifiers (AOB and NOB) supported a heterotrophic bacterium via production of soluble microbial products (SMP). MAR-FISH revealed that the heterotrophic bacterial community was composed of bacteria that were phylogenetically and metabolically diverse and to some extent metabolically redundant, which ensured the stability of the ecosystem as a biofilm. alpha- and gamma-Proteobacteria dominated the utilization of [(14)C]acetic acid and (14)C-amino acids in this biofilm. Despite their low abundance (ca. 2%) in the biofilm community, members of the CFB cluster accounted for the largest fraction (ca. 64%) of the bacterial community consuming N-acetyl-D-[1-(14)C]glucosamine (NAG). The GNSB accounted for 9% of the (14)C-amino acid-consuming bacteria and 27% of the [(14)C]NAG-consuming bacteria but did not utilize [(14)C]acetic acid. Bacteria classified in the unidentified group accounted for 6% of the total heterotrophic bacteria and could utilize all organic substrates, including NAG. This showed that there was an efficient food web (carbon metabolism) in the autotrophic nitrifying biofilm community, which ensured maximum utilization of SMP produced by nitrifiers and prevented buildup of metabolites or waste materials of nitrifiers to significant levels.