Performing Department
Entomology
Non Technical Summary
Pollinator populations are in decline globally, and existing toolkits of management practices have been inadequate for halting and reversing losses. Recently developed genomic editing technologies can be applied to study the mechanistic basis of diverse stressors on pollinators and develop novel solutions to improve their ability to withstand multiple stressors in the field. Current genomic editing technologies rely upon delivering DNA or endonucleases to preblastoderm embryos via embryonic microinjection. Embryonic microinjection is technically challenging, limited to a small number of arthropod taxa, and is inefficient even in optimized species. A critical need exists to develop methods for arthropod genetic manipulation that are simple, accessible, and compatible for a large variety of arthropod species (including pollinators). During oogenesis, arthropods transfer yolk protein precursors (such as vitellogenin) to developing oocytes by receptor-mediated endocytosis (RME). We have developed a technique (Receptor-Mediated Ovary Transduction of Cargo; ReMOT Control), which exploits RME to transduce cargo into the developing germline for heritable editing of the chromosomal sequence. We will use ReMOT Control to develop easy and efficient genetic manipulation technologies for the native pollinator Bombus impatiens. In Aim One, we will assess the ability for B. impatiens vitellogenin (BiVg) to transduce protein cargo (GFP) into bee oocytes. In Aim Two, we will use BiVg to transduce into the germline transcription activator-like effector nucleases (TALENs) targeting an eye color gene for heritable editing of the genetic sequence. Our experiments will revolutionize studies of managed bee pollinators, and can be applied to other economically important Hymenoptera.
Animal Health Component
50%
Research Effort Categories
Basic
25%
Applied
50%
Developmental
25%
Goals / Objectives
Justification for topical areas: (1) New and emerging innovative ideas, (2) Application of new knowledge or approaches (3) Tools required to have a paradigm shift in the field, (4) Rapid response to natural disasters and similar unanticipated event. Pollinator populations are in decline globally, and our existing toolkit of management practices has been largely inadequate for halting and reversing these losses. Recently developed genomic technologies (including CRISPRs and TALENs for genome editing) can be applied to comprehensively study the mechanistic basis of the effects of diverse stressors on pollinators and possibly develop novel solutions by introducing genomic modifications into pollinators that improve their ability to withstand multiple stressors in the field. In this exploratory proposal, we will develop an easy-to-use toolkit for flexible and rapid genetic engineering of bees, facilitating studies of bee health and the development of novel and innovative solutions to pollinator decline. ?Introduction: Bees are critical pollinators in natural and agricultural landscapes, and important model systems for the study of social behavior (Klein et al. 2007, Ollerton et al. 2011). Genome and transcriptome sequencing of multiple bee species has dramatically expanded understanding of genetic mechanisms underpinning bee health and behavior (Woodard et al. 2011, Kocher et al. 2013, Elsik et al. 2014, Sadd et al. 2015). However, lack of genetic tools to manipulate gene expression has hampered our ability to test hypotheses, characterize gene pathways, and develop transformed strains of bees with improved resistance to pesticides, parasites, pathogens, and traits that increase health, productivity, and pollination services. A recent study (Schulte et al. 2014) demonstrated that honey bees could be genetically transformed. However, as in the case of most arthropod genetic transformation protocols, Schulte et al's methodology involved injecting embryos. Low transformation success rates (< 1%) make this approach laborious and inefficient, and beyond the capability of most laboratories. Genetic modification of arthropods has profoundly altered the molecular entomology research landscape during the last 30 years. Embryonic microinjection (EM) is the main technique used to create arthropod strains with altered genetic characteristics (Boyle et al. 1993, Catteruccia et al. 2000, Degennaro et al. 2013, Aryan et al. 2013). Although EM techniques were developed over 30 years ago, the procedure is still fraught with many limitations and constraints. EM is a highly specialized technique, requiring extensive training, expertise and a substantial financial outlay for the microinjection equipment. EM is only suitable for a small minority of insects, most of which are model species. This methodology is especially challenging in the case of social insects such as bees where only a few individuals are reproductive (requiring special rearing conditions to produce queens) and reproductive individuals require a full colony to survive and produce offspring (Winston 1991, Amsalem et al. 2015). Development of novel easy-to-use methods for arthropod genetic manipulation that can be utilized in a diverse array of model and non-model organisms will dramatically change the field of arthropod molecular biology, bringing genetic modification technology within the reach of everyday researchers working on non-model organisms.Receptor-mediated endocytosis (RME) is a phenomenon whereby molecules bind to specific membrane-bound receptors, triggering invagination of the plasma membrane and uptake of the molecules into the cell within plasma membrane-derived vesicles (Figure 1). RME has been exploited to deliver molecules of interest to specific cell types, such as tumor cells, by fusing drugs to a protein ligand that specifically binds to and is taken up by the cell type of interest (Sato et al. 2000, Fahrer et al. 2013). Insects transfer yolk protein precursors (YPPs) to their eggs during oogenesis (egg development) by RME, providing a resource for the embryo and the resultant offspring (Sappington et al. 1998). The primary YPP molecules are vitellogenins (Vgs), yolk proteins (YPs) and lipophorin (Lp), which are synthesized in the fat body and transferred to the developing ovarian oocytes by RME (Cheon et al. 2001). Once bound, the cell wall invaginates, creating vesicles filled with the YPPs that are processed, transported to the developing oocytes and deposited in the developing embryo (Snigirevskaya et al. 1997).Here, we demonstrate that recombinant Drosophila melanogaster yolk protein 1 (DmYP1) can be used to shuttle protein cargo (GFP) into Drosophila and Anopheles ovaries by RME during vitellogenesis (see below). We hypothesize that DmYP1 or other YPP proteins can be exploited to deliver cargo to developing insect oocytes while still in utero. We refer to this strategy as Receptor-Mediated Ovary Transduction of Cargo, or "ReMOT Control". In principle, ReMOT Control can be used to transduce an almost unlimited variety of cargo to the developing insect germline, ranging from DNA expression constructs, transposons, RNA, or DNA editing proteins such as zinc-finger nucleases, TALENS or CRISPRs. In this proposal, we will develop ReMOT control into a generalizable technique to easily genetically modify hymenopterans by injecting adult reproductive female insects rather than preblastoderm embryos. Once optimized, ReMOT Control will dramatically change the landscape of molecular entomology, allowing the easy genetic manipulation of a wide variety of vector arthropods and non-model species.Our model system: We will test our system hypotheses using Bombus impatiens (the common eastern bumble bee) as a model system. Bumble bees are second only to honey bees as pollinators in agroecosysems, serving as the primary or sole pollinator for economically important fruit and vegetable crops (McGregor 1976, Delaplane and Mayer 2000, Velthuis and van Doorn 2006). Several bumble bee species are native to North America, but are also tractable for commercial production and management, and thus function as both wild and managed pollinators in many crop systems, including commercial greenhouses (note that honey bees are not suitable pollinators for commercial greenhouse crops). However, many bumble bee species are in decline in the US (Cameron, Lozier et al. 2011, Cordes, Huang et al. 2012), threatening production of these economically important crops. With a relatively small colony size, year-round availability from commercial breeding operations, and its ability to complete its entire colony cycle indoors and thus be quarantined, B. impatiens bumble bees are a very tractable model system for these studies. It is important to note that the approach and results of our studies can easily be applied to honey bees and other managed and native pollinator bee species. Indeed, there are more than 4000 native bee species in the US (National Research Council 2007), and it is imperative that we develop a comparative framework to improve our conservation and management of these critical pollinators.
Project Methods
Aim 1. Assess ability for B. impatiens vitellogenin (BiVg) fragments to transduce protein cargo (GFP) into developing bee oocytes. GFP will be fused to fragments of BiVg, expressed using our developed in vitro system and assessed for entry into vitellogenic bee oocytes by fluorescence microscopy and western blot. The smallest effective BiVg fragment will be used for transduction of TALEN cargo in Aim 2.B. impatiens rearing: Young colonies of B. impatiens that provide adult bees will be obtained from Koppert Biological Systems (Howell, Michigan). Colonies will be maintained in nest boxes at a constant temperature of 28-30°C and 40-50% humidity, and supplied ad libitum with 60% sugar and honey bee-collected pollen. Workers of approximately the same size and age will be collected and caged in pairs in small plastic boxes for under the same conditions. Upon separation from the queen, workers become vitellogenic within 5 days and lay eggs within 7-9 days. Because these workers will not be mated, oviposited eggs will produce haploid males, allowing for the easy screening of recessive null mutants in Aim 2.Construction and expression of BiVg fusion proteins: We have already isolated and cloned the complete vitellogenin gene from B. impatiens. This gene is approximately 5.4 kb in length. In order in increase the amount of cargo that can be fused to the ligand, we will use deletion mapping (as described above for DmYP1) to localize the binding site of BiVg to the vitellogenin receptor (VgR). The BiVg gene will be split into ten fragments of approximately 500-600 bp each, and each fragment fused to GFP as described above. Transgenic cell lines will be made for each BiVg fragment-GFP fusion.Screening BiVg-GFP fusion proteins for ReMOT Control potential: Secreted fusion proteins from each cell line will be collected and concentrated using Amicon Ultra centrifugal filters (Millipore) as described above and injected into the thorax of bees currently undergoing vitellogenesis using a Nanojet microinjector. 24-48 hours post-injection, ovaries will be dissected and examined for GFP by epifluorescence microscopy using an Olympus BX-41 epifluorescent microscope. GFP per ovary will be quantified by quantitative Western Blot. The fragment giving best results will be used as the basis for TALEN transducing constructs in Aim Two.Aim 2. Use ReMOT Control to transduce transcription activator-like effector nucleases (TALENs) to the B. impatiens oocyte for specific gene deletion. TALENs are engineered restriction enzymes that are generated by fusing a sequence-specific TAL effector DNA binding domain to a DNA cleavage domain. TALENs targeting the B. impatiens kmo eye color gene will be fused to the BiVg fragment identified in Aim One and injected into unmated adult female bees that are currently undergoing vitellogenesis. Specific gene knockout will be detected by the production of haploid white-eyed male offspring and will be confirmed by sequencing.Construction and expression of BiVg -TALEN fusion proteins: For these experiments, we will create TALENs targeting the B. impatiens kynurenine 3-monooxygenase (kmo) gene (GenBank #: XM_003491181.1). The kmo gene is involved in the ommochrome eye pigment pathway, where it catalyzes the conversion of kynurinine to 3-hydroxykynurinine (Rasgon and Scott 2004). Null mutations in this gene lead to a lack of ommochrome eye pigments and phenotypically white eyes in a wide range of insects, including bees (Lopatina and Dolotovskaya 1987, Rasgon and Scott 2004). In addition, the kmo gene has previously been used as a marker for TALEN mutagenesis in mosquitoes (Aryan et al 2013). We will commercially synthesize (Cellectis Bioresearch, Paris, France) validated TALENs targeting the fifth exon of the kmo gene, which has been shown to result in null mutants in previous studies (Aryan et al. 2013).We will create 2 new stable transgenic S2 cell lines that will secrete fusion proteins consisting of the selected BiVg fragment identified in Aim One and the left or right Bi-kmo TALENs. The fusion proteins will be collected and concentrated from the culture medium and co-injected into bees as previously described. Eggs will be reared and the haploid males will emerge approximately 26 days post-oviposition, which will be screened for white eyes. In white-eye males, the kmo TALEN locus will be PCR amplified and sequenced to characterize the kmo modification induced by the TALEN. Efficiency per unit effort will be quantified as the number of knockout individuals obtained per 100 females injected.