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Adeno-associated virus (AAV) vectors are quickly becoming the vectors of choice for therapeutic gene delivery. To date, hundreds of natural isolates and bioengineered variants have been reported. While factors such as high production titer and low immunoreactivity are important to consider, the ability to deliver the genetic payload (physical transduction) and to drive high transgene expression (functional transduction) remains the most important feature when selecting AAV variants for clinical applications. Reporter expression assays are the most commonly used methods for determining vector fitness. However, such approaches are time consuming and become impractical when evaluating a large number of variants. Limited access to primary human tissues or challenging model systems further complicates vector testing. To address this problem, convenient high-throughput methods based on next-generation sequencing (NGS) are being developed. To this end, we built an AAV Testing Kit that allows inherent flexibility in regard to number and type of AAV variants included, and is compatible with in vitro, ex vivo, and in vivo applications. The Testing Kit presented here consists of a mix of 30 known AAVs where each variant encodes a CMV-eGFP cassette and a unique barcode in the 3'-untranslated region of the eGFP gene, allowing NGS-barcode analysis at both the DNA and RNA/cDNA levels. To validate the AAV Testing Kit, individually packaged barcoded variants were mixed at an equal ratio and used to transduce cells/tissues of interest. DNA and RNA/cDNA were extracted and subsequently analyzed by NGS to determine the physical/functional transduction efficiencies. We were able to assess the transduction efficiencies of immortalized cells, primary cells, and induced pluripotent stem cells in vitro, as well as in vivo transduction in naive mice and a xenograft liver model. Importantly, while our data validated previously reported transduction characteristics of individual capsids, we also identified novel previously unknown tropisms for some AAV variants.

Adeno-associated virus (AAV) vectors are quickly becoming the vectors of choice for therapeutic gene delivery. To date, hundreds of natural isolates and bioengineered variants have been reported. While factors such as high production titer and low immunoreactivity are important to consider, the ability to deliver the genetic payload (physical transduction) and to drive high transgene expression (functional transduction) remains the most important feature when selecting AAV variants for clinical applications. Reporter expression assays are the most commonly used methods for determining vector fitness. However, such approaches are time consuming and become impractical when evaluating a large number of variants. Limited access to primary human tissues or challenging model systems further complicates vector testing. To address this problem, convenient high-throughput methods based on next-generation sequencing (NGS) are being developed. To this end, we built an AAV Testing Kit that allows inherent flexibility in regard to number and type of AAV variants included, and is compatible with in vitro, ex vivo, and in vivo applications. The Testing Kit presented here consists of a mix of 30 known AAVs where each variant encodes a CMV-eGFP cassette and a unique barcode in the 3′-untranslated region of the eGFP gene, allowing NGS-barcode analysis at both the DNA and RNA/cDNA levels. To validate the AAV Testing Kit, individually packaged barcoded variants were mixed at an equal ratio and used to transduce cells/tissues of interest. DNA and RNA/cDNA were extracted and subsequently analyzed by NGS to determine the physical/functional transduction efficiencies. We were able to assess the transduction efficiencies of immortalized cells, primary cells, and induced pluripotent stem cells in vitro, as well as in vivo transduction in naïve mice and a xenograft liver model. Importantly, while our data validated previously reported transduction characteristics of individual capsids, we also identified novel previously unknown tropisms for some AAV variants.

The spotted wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), is an invasive pest of stone fruits and berries currently without effective control management. The sterile insect technique (SIT) is an environmentally friendly and effective pest control method that releases sterile males to mate with wild females resulting in the suppression or eradication of targeted pest populations. New molecular technologies and genome editing methods are paving the way to bring this technology to invasive species like D. suzukii. The CRISPR/Cas technology is emerging as a versatile option for efficient tailored gene manipulation. Components of this system, the guide RNA (gRNA) and Cas9 protein, can be delivered into the organism in the form of DNA, RNA, or protein. We report CRISPR/Cas-mediated site-specific white gene editing using purified Cas9 protein delivered directly into D. suzukii embryos. Mutant flies with 13, 3, and 1 nt deletions within the cutting site of the Cas9 protein were retrieved. Moreover, the two independent G1 deletion events (13 and 3 nt) derived from the same G0 female. Our results show that the recombinant Cas9 protein could be a method of choice for generating heritable gene modifications in D. suzukii.

Resistance to all major insecticide classes has developed in numerous and diverse insect field populations. Imidacloprid, the worldwide most used neonicotinoid, has been extensively applied during the last decade for the control of different insect pests. Lately, cases of sporadic resistance also to neonicotinoids, including Imidacloprid, have been reported. Drosophila melanogaster is one of the most popular model organisms in biology and, although not a pest species, a promising model system for insecticide resistance research. In this study, we present a toxicological and karyotypic analysis of a Drosophila mutant (MiT[w-]3R2) resistant to Imidacloprid and cross-resistant to DDT. Karyotype analysis of polytene chromosome of MiT[w-]3R2 flies did not identify any apparent structural change of the polytene chromosome linked with the resistance phenotype. [Projekat Ministarstva nauke Republike Srbije, br. 173012]

We analyzed changes in the genetic structure of two ecologically distinct populations of Drosophila subobscura by assessing inversion polymorphism parameters for over years. The frequencies of gene arrangements on all five chromosomes show variability in each population. Several chromosomal arrangements appeared for the first time in both populations. Significant difference in the overall distribution of gene arrangements between first and last year of study (1998/2006) in beach wood population of D. subobscura is found for chromosome A (a sex chromosome) and autosomes J and E, while in oak wood population the overall frequency of gene arrangements differ for autosomes J, E and O. The present significant changes of the particular gene arrangements are specific for each population and result in significant differences in comparisons at the level of individual chromosomes between years of study. Although overall heterozygosity remained unchanged over the years in beech habitat, it is significantly reduced in oak population in the fourth year. The obtained results indicate that population structure may significantly depend on short-term and long-term microclimatic changes in habitats. Based on the results to date, monitoring of microevolutionary changes using D. subobscura and its relatives seems a promising way to study the effects of global changes. .

As a response to the long-term presence of heavy metals in the environment, populations can evolve resistance. Its maintenance may have detrimental effect on population's fitness, causing a fitness cost. Lead is one of the widely distributed elements in the environment exhibiting high toxicity on organisms. By analyzing developmental stages viability and developmental time, we evaluated fitness cost in Drosophila subobscura flies adapted to low lead concentration and control flies derived from the same wild population, as well as their hybrids. Significant changes in specific developmental stages viability were detected in both lines, as well as their hybrids, suggesting complex response to low lead concentration. The results show that a long-term exposure to low lead concentration may have a significant impact on a population's survival, especially in a changing environment conditions.

Drosophila subobscura is a wild Drosophila species that is spread over almost all of Europe. It possesses an uniquely rich inversion polymorphism on all five long chromosomes. This polymorphism is to a certain degree associated with the variation and dynamics of ecological factors in space and time. We analyzed the changes of inversion polymorphism components of Drosophila subobscura flies maintained on media with different concentrations of lead in laboratory conditions. The effects of lead on inversion polymorphism were observed by cytological analysis of gene arrangements on all of the five acrocentric chromosomes, as well as by cytological analysis of karyotypes on all of the four autosomes. The frequencies of particular gene arrangements on the four autosomes changed significantly in the samples maintained on medium not supplemented with lead. The frequencies of some gene arrangements on all of the five acrocentric chromosomes changed significantly in the flies maintained on media supplemented with lead. The length of exposure to different lead concentrations results in a significant change in the frequency of a few gene arrangements on two autosomes. However, the results show that different concentrations of lead, as well as the length of exposure, do not affect major parameters of inversion polymorphism. The results suggest that some gene arrangements could be linked with adaptive processes in evolving heavy metal resistance.

Certain xenobiotics have the capacity to induce the expression of genes involved in various biological phenomena, including insecticide resistance. The induction potential of different chemicals, among them different insecticides, has been documented for a number of insect species. In this study, we have analyzed the induction potential of Imidacloprid, a widely used member of the neonicotinoid insecticide family. Genes Cyp6g1 and Cyp6a2, known to be involved in the resistance of mutant Drosophila melanogaster line MiT[W⁻]3R2 to Imidacloprid and DDT were included in the analyzed sample. We find that Imidacloprid does not induce expression of the analyzed genes.

We analyzed the developmental time, egg-to-adult viability, and developmental stability (fluctuating wing size asymmetry) in Drosophila subobscura, maintained for six generations on different concentrations of lead. Development time is significantly affected by generation and lead concentration, but interaction of these factors is not a significant source of variability for this fitness component. Generation and the interaction generation × concentration of lead significantly affect egg-to-adult viability. Levene’s test of heterogeneity of variance showed that variability of FA is not significant in any of the samples. Within both lead concentrations females showed significantly higher FA indices for the wing width than males. Within sexes, a significantly higher FA was found only in females for wing width FA between the control and the lower concentration of lead. The results show that if strong relationship between FA and the studied fitness components exists, it results in a stronger selection of unstable genotypes under lead as a stress factor and, consequently, FA needs to be used with caution as a biomarker in natural populations under environmental stress.

We analyzed changes in the genetic structure and effective population size of two ecologically distinct populations of Drosophila subobscura over several years. Population sizes of D. subobscura in beech and oak wood habitats for a period of 6 years were estimated by the capture-mark-release-recapture method. Inversion polymorphism parameters were also assessed in the same populations for a period of 3 years. Significant differences in the numbers of individuals were observed between sexes. This affected the effective population sizes between particular years. The ratio of the effective size over the cenzus dropped significantly in beech wood in 2 years. Although overall heterozygosity remained unchanged during the years in both habitats, frequencies of gene arrangements on five chromosomes show variability. After the bottleneck, some complex chromosomal arrangements appeared for the first time in both populations. Standard gene arrangements of chromosome A increased in frequency over the years in each habitat, while the complex arrangements remain rather stable and specific for each population. The results obtained indicate that the population structure may significantly change if the effective size of D. subobscura population is reduced, which is mostly related to microclimatic changes in habitats. Based on the results to date, monitoring of microevolutionary changes by using D. subobscura and its relatives seems a promising way to study the effects of global climate changes.

Insecticide resistance is a worldwide problem with major impact on agriculture and human health. Understanding the underlying molecular mechanisms is crucial for the management of the phenomenon; however, this information often comes late with respect to the implementation of efficient counter-measures, particularly in the case of metabolism-based resistance mechanisms. We employed a genome-wide insertional mutagenesis screen to Drosophila melanogaster, using a Minos-based construct, and retrieved a line (MiT[w(-)]3R2) resistant to the neonicotinoid insecticide Imidacloprid. Biochemical and bioassay data indicated that resistance was due to increased P450 detoxification. Deep sequencing transcriptomic analysis revealed substantial over- and under-representation of 357 transcripts in the resistant line, including statistically significant changes in mixed function oxidases, peptidases and cuticular proteins. Three P450 genes (Cyp4p2, Cyp6a2 and Cyp6g1) located on the 2R chromosome, are highly up-regulated in mutant flies compared to susceptible Drosophila. One of them (Cyp6g1) has been already described as a major factor for Imidacloprid resistance, which validated the approach. Elevated expression of the Cyp4p2 was not previously documented in Drosophila lines resistant to neonicotinoids. In silico analysis using the Drosophila reference genome failed to detect transcription binding factors or microRNAs associated with the over-expressed Cyp genes. The resistant line did not contain a Minos insertion in its chromosomes, suggesting a hit-and-run event, i.e. an insertion of the transposable element, followed by an excision which caused the mutation. Genetic mapping placed the resistance locus to the right arm of the second chromosome, within a ∼1 Mb region, where the highly up-regulated Cyp6g1 gene is located. The nature of the unknown mutation that causes resistance is discussed on the basis of these results.