Molecular analysis of Drosophila melanogaster B chromosomes reveals their origin, composition, and structure

ABSTRACTThe number of chromosomes carried by an individual species is one of its defining characteristics. Some species, however, can also carry supernumerary chromosomes referred to as B chromosomes. B chromosomes were recently identified in a laboratory stock of Drosophila melanogaster—an established model organism with a wealth of genetic and genomic resources—enabling us to subject them to extensive molecular analysis. We isolated the B chromosomes by pulsed-field gel electrophoresis and determined their composition through next-generation sequencing. Although these B chromosomes carry no known euchromatic sequence, they are rich in transposable elements and long arrays of short nucleotide repeats, the most abundant being the uncharacterized AAGAT satellite repeat. Fluorescent in-situ hybridization on metaphase chromosome spreads revealed this repeat is located on Chromosome 4, strongly suggesting the origin of the B chromosomes is Chromosome 4. Cytological and quantitative comparisons of signal intensity between Chromosome 4 and the B chromosomes supports the hypothesis that the structure of the B chromosome is an isochromosome. We also report the identification of a new B chromosome variant in a related laboratory stock. This B chromosome has a similar repeat signature as the original but is smaller and much less prevalent. We examined additional stocks with similar genotypes and did not find B chromosomes, but did find these stocks lacked the AAGAT satellite repeat. Our molecular characterization of D. melanogaster B chromosomes is the first step towards understanding how supernumerary chromosomes arise from essential chromosomes and what may be necessary for their stable inheritance.

Larval development and timeliness of pupation in the laboratory of the navel orangeworm, Amyelois transitella (Walker) (Lepidoptera: Phycitidae), on certain diets, under various Ρhοtoperiοd, tempera­ture, aeration and humidity conditions

Development of larvae of the navel orangeworm, Arn.ielois transitella (Walker) (Lepidoptera: Phycitidae), was studied under various photoperiods, temperatures and larval diets, in an effort to induce dormancy. Ready – to – batch eggs or neonate larvae were placed in transparent vials half – full of diet. Fully grown larvae not pupating within 14 days at 26.7oC and L:D 16:8 were considered as being in dormancy. With artificial larval diets containing bran, yeast, vitamins, and fortified or not with a high protein cereal and egg yolk, none of the treatments induced dormancy to a substantial percentage of laboratory stock or wild larvae. With dry walnut meats, larval growth was slower and survival much lower than with the artificial diets. With walnut meats, when eggs were incubated at 32.2oC and L:D 0:24, a certain percentage of grown larvae of the laboratory stock underwent dormancy when grown as larvae under the conditions that follow: 40% at 21oC and L:D 12:12, 17% at 21oC and L:D 0:24, 19% at 26.7oC and L:D 16:8, and 22% at 26.7oC and L:D 0:24 for the first 14 days than at 21oC and L:D 8:16 for the rest of larval life. When both embrya and larvae developed at 21oC and L:D 0:24, 17% of the larvae underwent dormancy. Yet, the relatively small number of grown larvae in the groups fed walnut meats suggests further work for the occurrence of dormancy in this insect to be proven. No larvae developed on straight brewers’ yeast powder. Straight soybean flour or 9:1 and 7:3 mixtures of it with yeast powder allowed the production of grown larvae, pupae and adults of normal appearance. The rate of larval growth on the soy:yeast diets was significantly slower than on a reference diet. At 26.7oC, a L:D 16:8 photoperiod was as good as a 14:10 one. Continuous darkness resulted in significantly reduced yield in adults and rate of larval growth. High relative humidity on the surface of the diet allowed excessive growth of fungi on and in the soy:yeast diets and resulted in a much faster larval growth. Larvae developed well on dry walnut meats and in cracked dry and water-soaked walnuts. Inside the walnuts the rate of larval growth was uneven, some larvae being still fairly small on the 53rd day at 26.7oC, while the majority was fully grown or had already pupated.

Induction of dormancy in Lobesia botrana by long day and high temperature conditions

Lobesia botrana (Denis and Schiffermueller) (Lepidoptera: Tortricidae) is known to enter a facultative autumnohibernal diapause-mediated dormancy in the pupal stage, when the embryonic and early larval stages are exposed to short-day photo-periods. Yet, in a laboratory stock originating from northern Greece and reared for years on an artificial larval diet, dormancy occurred also under a long-day photo-period. When the eggs were incubated at 30oC in the dark or at 26 oC under the natural daylength of August in northern Greece, and the larvae grew at L:D 16:8 and 25-26 oC, but not 20 oC, a substantial percentage of the pupae entered dormancy. This dormancy seems to be diapause-mediated. It is not known whether it is a typical summer diapause.

Multiplexed Metagenomic Deep Sequencing To Analyze the Composition of High-Priority Pathogen Reagents

ABSTRACT Both the integrity and reproducibility of experiments using select agents depend in large part on unbiased validation to ensure the correct identity and purity of the species in question. Metagenomic deep sequencing (MDS) provides the required level of validation by allowing for an unbiased and comprehensive assessment of all the microbes in a laboratory stock. Laboratories studying high-priority pathogens need comprehensive methods to confirm microbial species and strains while also detecting contamination. Metagenomic deep sequencing (MDS) inventories nucleic acids present in laboratory stocks, providing an unbiased assessment of pathogen identity, the extent of genomic variation, and the presence of contaminants. Double-stranded cDNA MDS libraries were constructed from RNA extracted from in vitro-passaged stocks of six viruses (La Crosse virus, Ebola virus, canine distemper virus, measles virus, human respiratory syncytial virus, and vesicular stomatitis virus). Each library was dual indexed and pooled for sequencing. A custom bioinformatics pipeline determined the organisms present in each sample in a blinded fashion. Single nucleotide variant (SNV) analysis identified viral isolates. We confirmed that (i) each sample contained the expected microbe, (ii) dual indexing of the samples minimized false assignments of individual sequences, (iii) multiple viral and bacterial contaminants were present, and (iv) SNV analysis of the viral genomes allowed precise identification of the viral isolates. MDS can be multiplexed to allow simultaneous and unbiased interrogation of mixed microbial cultures and (i) confirm pathogen identity, (ii) characterize the extent of genomic variation, (iii) confirm the cell line used for virus propagation, and (iv) assess for contaminating microbes. These assessments ensure the true composition of these high-priority reagents and generate a comprehensive database of microbial genomes studied in each facility. MDS can serve as an integral part of a pathogen-tracking program which in turn will enhance sample security and increase experimental rigor and precision. IMPORTANCE Both the integrity and reproducibility of experiments using select agents depend in large part on unbiased validation to ensure the correct identity and purity of the species in question. Metagenomic deep sequencing (MDS) provides the required level of validation by allowing for an unbiased and comprehensive assessment of all the microbes in a laboratory stock.