Papers by Frank Shotkoski
Diversity and expression of cytochrome P450 genes in Drosophila melanogaster
International audienc
Expression of an Antisense Dihydrofolate Reductase Transcript in Transfected Mosquito Cells: Effects on Growth and Plating Efficiency
The American Journal of Tropical Medicine and Hygiene, 1994
Novel approaches to control of vector-borne disease include potential use of transgenic insects, ... more Novel approaches to control of vector-borne disease include potential use of transgenic insects, in which molecular mechanisms will be induced to prevent transmission of pathogenic organisms. The infrastructure essential to this technology includes the cloning of essential genes from vector insects, and the development of efficient transformation strategies. In this study, we use a continuous mosquito (Aedes albopictus) cell line and a cloned mosquito dihydrofolate reductase gene to demonstrate a transgenic approach that may be used to select for the presence or absence of particular gene functions in transfected cells. Plasmids containing the dihydrofolate reductase gene in sense and antisense orientation, under the regulation of a temperature-inducible promoter, were expressed in stably transfected mosquito cells. At the normal growth temperature of 28 degrees C, or after mild heat induction at 34 degrees C, expression of the dihydrofolate reductase construct in sense orientation had little effect on cell growth. In contrast, recovery of clones transfected with the antisense construct was reduced, and induction of antisense transcripts at 34 degrees C further compromised cell growth and viability. Clones transfected with the sense construct retained significantly higher copy numbers of foreign DNA than did cells transfected with the antisense construct. These studies provide a basis for use of sense and antisense dihydrofolate reductase constructs to recover transfected mosquito cells with specific desired phenotypes, based on the relative expression of cloned genes of interest.
Agriculture & Food Security, 2017
Background: Environmental heterogeneity, emerging pathogens and limited access to financial and a... more Background: Environmental heterogeneity, emerging pathogens and limited access to financial and agricultural inputs prevent farmers from producing consistent crop yields in many developing countries. Yield instability impedes establishment of processing and export industries, thereby inhibiting economic development. Modern varieties offer significant increases in yield stability. Results: A deeply collaborative, multi-national germplasm trialing network was established in West Africa to identify tomato varieties well adapted to each country and mobilize those varieties into local seed distribution networks alongside an integrated pest management program. Research partners in seven West African countries evaluated over 100 tomato varieties for resistance to tomato leaf curl disease (ToLCD). Using biotechnology, the identity and distribution of the key viruses (begomoviruses) causing ToLCD in these countries were identified, and a vector-independent inoculation method (agroinoculation) was developed. The trials identified a set of high-performing varieties with resistance. Agroinoculation with the three prevalent begomoviruses confirmed resistance. Conclusions: These trial results fulfill the new Economic Community of West African States harmonized seed regulation policy, which requires at least 2 years of national performance trials prior to commercialization of a tomato variety. To compete with a rapidly expanding canned tomato import industry, West African growers need to increase productivity and processing capacity; therefore, we also assessed processing and export trade data for fresh and processed tomatoes from each of the seven countries.
PloS one, 2016
Studies on potential adverse effects of genetically engineered crops are part of an environmental... more Studies on potential adverse effects of genetically engineered crops are part of an environmental risk assessment that is required prior to the commercial release of these crops. Of particular concern are non-target organisms (NTOs) that provide important ecosystem services. Here, we report on studies conducted in the Philippines over three cropping seasons with Bt eggplants expressing Cry1Ac for control of the eggplant fruit and shoot borer (EFSB), Leucinodes orbonalis, to examine potential effects on field abundance, community composition, structure and biodiversity of NTO's, particularly non-target arthropod (NTA) communities. We document that many arthropod taxa are associated with Bt eggplants and their non-Bt comparators and that the number of taxa and their densities varied within season and across trials. However, we found few significant differences in seasonal mean densities of arthropod taxa between Bt and non-Bt eggplants. As expected, a lower abundance of lepidopter...
International Journal of Biotechnology, 2016
A few studies have reported some of the costs associated with bringing to market genetically-modi... more A few studies have reported some of the costs associated with bringing to market genetically-modified (GM) crops but no comprehensive studies exist on the real cost of the entire process of developing and releasing one GM variety by a not-for-profit institution in a developing country for sustainable agriculture. Despite the lack of documented studies, it is commonly assumed that such an undertaking is cost prohibitive, based on mere hearsay, and on two private sector cost assessments. The present study assesses the costs and the time expenditures to two not-for-profit programs, one lead by CIP and the other by Cornell University, of developing a late blight resistant (LBr) potato variety for release in one developing country. CIP's costs run to $1.6 million over eight years, while Cornell's costs amount to $1.4 million over nine years. Exogenous disturbances might result in insignificant increases in cost, but can increase time expenditure significantly. A sensitivity analysis revealed that the total cost is markedly influenced by technical parameters determining the production and identification of the pre-commercial LBr transgenic event.
PLOS ONE, 2016
Plants expressing Cry proteins from the bacterium, Bacillus thuringiensis (Bt), have become a maj... more Plants expressing Cry proteins from the bacterium, Bacillus thuringiensis (Bt), have become a major tactic for controlling insect pests in maize and cotton globally. However, there are few Bt vegetable crops. Eggplant (Solanum melongena) is a popular vegetable grown throughout Asia that is heavily treated with insecticides to control the eggplant fruit and shoot borer, Leucinodes orbonalis (EFSB). Herein we provide the first publicly available data on field performance in Asia of eggplant engineered to produce the Cry1Ac protein. Replicated field trials with five Bt eggplant open-pollinated (OP) lines from transformation event EE-1 and their non-Bt comparators were conducted over three cropping seasons in the Philippines from 2010-2012. Field trials documented levels of Cry1Ac protein expressed in plants and evaluated their efficacy against the primary target pest, EFSB. Cry1Ac concentrations ranged from 0.75-24.7 ppm dry weight with the highest in the terminal leaves (or shoots) and the lowest in the roots. Cry1Ac levels significantly increased from the vegetative to the reproductive stage. Bt eggplant lines demonstrated excellent control of EFSB. Pairwise analysis of means detected highly significant differences between Bt eggplant lines and their non-Bt comparators for all field efficacy parameters tested. Bt eggplant lines demonstrated high levels of control of EFSB shoot damage (98.6-100%) and fruit damage (98.1-99.7%) and reduced EFSB larval infestation (95.8-99.3%) under the most severe pest pressure during trial 2. Moths that emerged from larvae collected from Bt plants in the field and reared in their Bt eggplant hosts did not produce viable eggs or offspring. These results demonstrate that Bt eggplant lines containing Cry1Ac event EE-1 provide
Journal of Economic Entomology, 1990
Field studies were done to determine whether continuous exposure to the systemic insecticides ald... more Field studies were done to determine whether continuous exposure to the systemic insecticides aldicarb, carbofuran, disulfoton, and terbufos would induce or select for insecticide-resistant greenbugs, Schizaphis graminum (Rondani). The extent of resistance was determined with a surface residue bioassay for each of the insecticides. In field experiments, greenbug populations exhibited 21-to 104-fold increases in resistance to disulfoton. Greenbugs exposed to the other insecticides did not exhibit significant increases in resistance. Greenhouse studies were done to determine if disulfoton resistance resulted from a change within a genotype (greenbug clone) or was simply the result of selection of resistant genotypes from a genetically diverse population. Greenbugs originating from a single aphid susceptible to insecticides developed 65-to 300-fold increases in resistance. Similar results were obtained when the study was repeated with progeny from a different greenbug. These results indicate that resistance developed within greenbug clones after exposure to disulfoton for 3 wk or longer. When resistant greenbugs were tested for cross-resistanceto carbofuran, dimethoate, malathion, and terbufos, a significant increase in cross-resistance to dimethoate (nine-fold) and terbufos (two-fold) was observed, but cross-resistance to carbofuran and malathion was not significant.
Novel VIP3 Toxins and Methods of Use
Expression and use of novel pesticidal toxins
Integration of Insect-Resistant Genetically Modified Crops within IPM Programs, 2008
Fruits and vegetables are major components of a healthy diet, but are subject to severe pest pres... more Fruits and vegetables are major components of a healthy diet, but are subject to severe pest pressure. Approximately 30% of all insecticides applied worldwide are used to control insects affecting vegetables and fruits. Transgenic, or more commonly referred to as genetically modified (GM), vegetables and fruits offer unique opportunities for controlling insects and the pathogens they transmit. Aphid transmitted viruses have been particularly difficult to manage by tactics aimed at reducing aphid populations and in many cases there has not been virus resistant plant germplasm. Farmers in the USA have benefited from having GM virus resistant squash and papaya available to them as tools in their overall IPM programs. In the USA, Bt sweet corn has proven effective for control of Lepidoptera and continues to be accepted in the fresh market. However, the best opportunities for GM vegetables and fruits are in developing countries where 83% of the world's population lives, the majority of vegetables and fruits are produced and pest problems are most acute.
Acta Horticulturae, 2010
In the more developed countries, genetically engineered crops contribute greatly to agricultural ... more In the more developed countries, genetically engineered crops contribute greatly to agricultural productivity and sustainability. Over the last few years, the largest growth in the adoption of genetically engineered crops has been in developing countries and this trend is expected to continue. The multinational life sciences companies have been leading the way, but they are focusing primarily on a few crop/trait combinations that have high commercial value and occupy large international markets. Because of the costs and complexity of the issues related to crop biotechnology, many crops and traits of importance to subsistence and resource poor farmers around the world have been overlooked. The Agricultural Biotechnology Support Project (ABSPII), a Cornell University-led and USAID funded consortium of public and private sector institutions, provides support for scientists, regulators, extension workers, farmers and the general public in developing countries to make informed decisions about agricultural biotechnology. Organization in Uganda to establish safe and cost effective programs for the development and commercialization of East African highland bananas (Musa spp.), genetically engineered for black sigatoka and nematode resistance. This paper gives a brief description of the work that has been done to date and discuss the ABSPII based strategy that has been adopted to develop and deliver genetically engineered crops for developing countries.
Nature Biotechnology, 2014
An amplified insect dihydrofolate reductase gene contains a single intron
European Journal of Biochemistry, 1991
We have used methotrexate-resistant mosquito (Aedes albopictus) cells as the source of DNA for cl... more We have used methotrexate-resistant mosquito (Aedes albopictus) cells as the source of DNA for cloning an 8.5-kb EcoRI fragment containing an amplified dihydrofolate reductase (DHRF) gene. An estimated 1200 copies of the DHFR gene were represented in nuclear DNA from Mtx-5011-256 cells, which were 3000-fold more resistant to methotrexate than wild-type cells. Southern blot analysis indicated that all of the amplified DHFR genes were contained within a 1.8-kb AccI fragment represented in the cloned DNA. In contrast to mammalian DHFR genes which span approximately 30 kb, the complete amino acid coding sequence of the mosquito DHFR gene spanned 614 nucleotides, including a single 56-nucleotide intron that interrupted a conserved Arg codon at amino acid position 27. Additional introns characteristic of mammalian DHFR genes were absent; conservation of the first intron in the mosquito DHFR gene supports a regulatory role for this intron. The mosquito DHFR gene coded for a 186-amino-acid protein with 43-48% similarity to vertebrate DHFR.
Stem cell gene therapy, position effects and chromatin insulators
Stem Cells, 1997
Low efficiency of gene transfer is the main obstacle for a clinically effective gene therapy at t... more Low efficiency of gene transfer is the main obstacle for a clinically effective gene therapy at the level of the pluripotent hematopoietic stem cell. Another important aspect of stem cell gene therapy, the actual expression of the transduced genes, has only been investigated adequately in very few studies, mainly for globin genes. Transcriptional silencing and position effects due to negative effects of surrounding chromatin on the expression of randomly integrated vector sequences may seriously jeopardize the success of current gene therapy strategies, even if transduction efficiency can be significantly improved. We propose the incorporation of chromatin insulators in the design of gene therapy vectors to overcome the problem of position effects. Chromatin insulators are protein-binding DNA elements that lack intrinsic promoter/enhancer activity but shelter genes from transcriptional influence of surrounding chromatin. The best characterized insulators are from Drosophila. We hypothesize that the important cellular function of chromatin organization is evolutionarily conserved and that human homologs to Drosophila insulator binding proteins such as the suppressor of Hairy-wing exist and can be cloned. Using these putative proteins, it should be possible to identify corresponding minimal binding sites with insulator activity. The design and incorporation of effective chromatin insulator sequences in the next generation of gene therapy vectors should lead to improved and more predictable expression of therapeutic transgenes and constitute an important step toward clinically effective gene therapy.
Polymerase chain reaction diagnostic for cyclodiene insecticide resistance in the mosquito Aedes aegypti
Medical and Veterinary Entomology, 1994
Cyclodiene insecticide resistance in Aedes aegypti (L.) is associated with the same amino acid su... more Cyclodiene insecticide resistance in Aedes aegypti (L.) is associated with the same amino acid substitution (Thompson et ul., 1993) as that proved to cause cyclodiene resistance in Drosophila melanogaster Meigen (ffrenchConstant et al., 1993a, b). An alanine residue in the second membrane-spanning domain of the GABA receptor/ chloride ion channel gene Rdl (Resistant to dieldrin) is replaced by a serine. This substitution results in the addition of only a single hydroxyl group to the putative lining of the chloride ion channel. The precise mechanism whereby the interaction of cyclodienes (putative channel blockers) with the receptor is hindered is currently under investigation in our laboratory. However, the identification of the single base pair mutation associated with resistance in Aedes has enabled us t o design a rapid molecular diagnostic based on the amplification of specific alleles in the polymerase chain reaction (PCR). The cloning of the Rdl homologue from Aedes using the Drosophila cDNA and the resistance-associated mutation has been described elsewhere (Thompson et al., 1993). In order to achieve specific amplification of the resistance allele we used PCR primers with the resistance-associated mutation placed at their 3' end. Under particular reaction conditions, specific amplification of the matching resistance allele can be achieved whilst the susceptible allele (or another resistance allele) fails to amplify due to mismatch between the DNA template and the primer. Two allelespecific primers were made, both forward and reverse, as previous experiments with Drosophila have often shown that optimal resolution between alleles can only be achieved using one of the two primers (Steichen & ffrenchConstant, 1993). PCR was performed between the allele-specific primers and flanking primers known to be nested within exon 7 of Rdl (ffrench-Constant & Rocheleau, 1992) which contains the resistance associated mutation (Fig. 1). This allows for DNA amplification from a simple genomic DNA preparation (Steichen & ffrench-Constant, 1993). For PCR,
Functional expression of insecticide‐resistant GABA receptors from the mosquito Aedes aegypti
Insect Molecular Biology, 1994
We are interested in cloning insecticide resistance genes from vector mosquitos for use as select... more We are interested in cloning insecticide resistance genes from vector mosquitos for use as selectable markers in their genetic transformation. As a first step towards this goal, we here report the functional homo‐muitimeric expression of a γ‐aminobutyric acid (GABA) receptor subunit gene, Resistance to dieldrin (Rdl), from the yellow fever mosquito Aedes aegypti in baculovirus‐infected insect cell lines. Replacement of alanine296 with a serine leads to approximately 100‐fold insensitivity to picrotoxin as previously observed in Drosophila. This shows not only that the mosquito GABA receptor cDNA is functional but also that it can be simply mutated to resistance. Strategies for incorporation of this cDNA into a minigene for the genetic transformation of mosquitoes are discussed.
An amplified mosquito dihydrofolate reductase gene: ampiicon size and chromosomal distribution
Insect Molecular Biology, 1993
The dihydrofolate reductase amplicon in methotrexate-resistant mosquito cells provides an amplifi... more The dihydrofolate reductase amplicon in methotrexate-resistant mosquito cells provides an amplified gene in insects that can be compared directly to the corresponding amplified locus in mammalian cells. A cloned Aedes albopictus dihydrofolate reductase gene was used as a probe to examine the structure of dihydrofolate reductase alleles in sensitive and resistant cells. In wild type cells, two distinct alleles could be distinguished by restriction fragment length polymorphisms, one of which was amplified in methotrexate-resistant cells. Subsequent to amplification, an additional polymorphism at a ten base-pair XmnI recognition site indicated that the amplified mosquito gene is subject to genetic changes similar to those that have been described in amplified genes from mammalian cells. Pulsed-field gel electrophoresis was used to determine that the minimum size of the mosquito dihydrofolate reductase amplicon was 140 kilobases; ethidium bromide staining patterns suggested a size of at least 233 kilobases. Dihydrofolate reductase probes hybridized to distinct locations in five of the thirteen chromosomes in Mtx-5011-128 cells, indicating that the amplified DNA probably occurs as tandem direct or inverted repeats.
Differential induction of antibacterial transcripts in Drosophila susceptible and resistant to parasitism by Leptopilina boulardi
Insect Molecular Biology, 1996
Two well-described elements of the immune response of insects include encapsulation of metazoan p... more Two well-described elements of the immune response of insects include encapsulation of metazoan parasites (blood-cell-mediated) and the production of antibacterial peptides (humoral and/or cellular). However, the possible functional interrelationship between cellular encapsulation and antibacterial responses, and the extent to which the two components may be co-regulated, are poorly understood. We used a novel approach involving strains of Drosophila resistant (R) or susceptible (S) to the wasp parasitoid Leptopilina boulardi to study the expression of three genes involved in the antibacterial response: Dorsal-related immunity factor (Dif), Cecropin (CecA1) and Diptericin (Dip). Both S and R strains produced high levels of all antibacterial transcripts upon bacterial injection. However, when parasitized the R strain showed no induction whilst the S strain did. This lack of antibacterial transcript induction in the parasitized R strain not only clarifies the separation of these two types of immune response but also raises the fascinating possibility of a link in their genetic regulation.
Insect Biochemistry and Molecular Biology, 1993
An Aedes albopictus dihydrofolate reductase gene was used to construct two chimeric DNA vectors t... more An Aedes albopictus dihydrofolate reductase gene was used to construct two chimeric DNA vectors that functioned as dominant selectable markers in transfected, wild type mosquito cells. Stably transformed clones were recovered after 10-15 days in the presence of selective medium containing 1/iM methotrexate. The transformed clones contained an estimated 100-500 copies of transfected DNA per nucleus. Combined data from Southern blots and in situ hybridization to metaphase chromosomes indicated that transfected DNA was likely integrated into chromosomes both as repeated structures and as randomly integrated single copy molecules, with minimal rearrangement of coding sequences. Transfected DNA was stably maintained under selective conditions, hut in some cases was lost when cells were maintained for prolonged periods in the absence of methotrexate. These observations provide a general framework for further development of stable gene transfer systems for mosquito cells in culture.
Functional analysis of a mosquito γ-aminobutyric acid receptor gene promoter
Gene, 1996
A single point mutation in the insect γ-aminobutyric acid receptor (GABAR)-encoding gene (Rdl) co... more A single point mutation in the insect γ-aminobutyric acid receptor (GABAR)-encoding gene (Rdl) confers high levels of resistance to cyclodienes in Drosophila and other insects. We were interested in studying the promoter of this gene for two reasons. Firstly, to define the elements underlying Rdl expression. Secondly, to identify the minimum set of regulatory elements necessary for construction of a
Uploads
Papers by Frank Shotkoski