Transposon-mediated insertional mutagenesis unmasks recessive insecticide resistance in the aphid Myzus persicae

The evolution of resistance to insecticides threatens the sustainable control of many of the world’s most damaging insect crop pests and disease vectors. To effectively combat resistance, it is important to understand its underlying genetic architecture, including the type and number of genetic variants affecting resistance and their interactions with each other and the environment. While significant progress has been made in characterizing the individual genes or mutations leading to resistance, our understanding of how genetic variants interact to influence its phenotypic expression remains poor. Here, we uncover a mechanism of insecticide resistance resulting from transposon-mediated insertional mutagenesis of a genetically dominant but insecticide-susceptible allele that enables the adaptive potential of a previously unavailable recessive resistance allele to be unlocked. Specifically, we identify clones of the aphid pest Myzus persicae that carry a resistant allele of the essential voltage-gated sodium channel (VGSC) gene with the recessive M918T and L1014F resistance mutations, in combination with an allele lacking these mutations but carrying a Mutator-like element transposon insertion that disrupts the coding sequence of the VGSC. This results in the down-regulation of the dominant susceptible allele and monoallelic expression of the recessive resistant allele, rendering the clones resistant to the insecticide bifenthrin. These findings are a powerful example of how transposable elements can provide a source of evolutionary potential that can be revealed by environmental and genetic perturbation, with applied implications for the control of highly damaging insect pests.

The evolution of insecticide resistance represents a serious threat to the sustainable control of insect crop pests and disease vectors (1). In addition to its applied importance, the study of resistance also offers insights into a range of fundamental evolutionary questions concerning adaptation to environmental change, sources of adaptive variation, and the genetic architecture of novel traits (2).Research into the proximate molecular mechanisms conferring reduced sensitivity to insecticides in a range of insect species has shown that resistance most commonly arises via two main mechanisms termed “metabolic resistance” and “target-site resistance.” Metabolic resistance results from increased activity or production of metabolic enzymes that detoxify or sequester the insecticide (3). In contrast, “target-site” resistance, which we consider in this study, results from structural changes (mutations) in the gene encoding the target protein, reducing its sensitivity to the toxic effect of the insecticide (4).One of the first examples of target-site resistance in insects was the discovery of the molecular basis of knockdown resistance (kdr) to pyrethroid insecticides, a major class of neurotoxic insecticides that have been used extensively to control a wide range of agricultural and human health pests (5). Mutations in the protein target of this insecticide class, the voltage-gated sodium channel (VGSC), were shown to lead to reduced sensitivity of the insect nervous system to these compounds. The first, and most common, mutation identified in pyrethroid-resistant insect strains, referred to as “kdr,” arises from a single-point mutation in the S6 segment of domain II of the VGSC gene, resulting in a leucine to phenylalanine (L1014F) amino acid substitution (5, 6). Subsequently, secondary mutations giving enhanced (super kdr, “skdr”) resistance, such as the M918T substitution within the IIS4-S5 linker of the VGSC, were described (reviewed in ref. 7).The green peach aphid Myzus persicae is a globally distributed, highly damaging crop pest (8). The economic importance of this species is increased by its extremely broad host range of more than 400 plant species, including many important crops, and its efficiency as a vector of more than 100 plant viruses (8). The intensive and widespread use of insecticides against this species has resulted in the evolution of resistance to many of the compounds used for control (9). This includes the pyrethroid insecticides where resistance has been linked to kdr/skdr mutations in the VGSC. Of these, the kdr L1014F and skdr M918T alterations are the most common, with M918T only ever found in combination with L1014F (i.e., on the same allele) (10, 11). More recently, an alternative substitution, M918L, at the skdr locus has been described and has been identified in the absence of L1014F (12, 13). The kdr and skdr mutations are inherited as recessive traits in a range of insect species, with little or no resistance phenotype observed in heterozygotes (5). Intriguingly, this does not appear to hold true in M. persicae, where aphid clones carrying L1014F, M918T, and M918L in the heterozygote state have been reported to display a resistant phenotype to a range of pyrethroids (11, 12), suggesting that kdr/skdr may be dominant in this species. Alternatively, the atypical mode of inheritance of kdr/skdr in M. persicae could be explained by interactions with other genetic elements that modify the phenotypic expression of these resistance alleles. In this regard, while knowledge of the individual genes and mutations that confer insecticide resistance has advanced dramatically over the last three decades, our understanding of the extent to which genetic variants interact to modify resistance phenotypes remains surprisingly poor.In the current study, we investigated whether genetic factors distinct from known kdr/skr mutations modify the pyrethroid-resistance phenotype in M. persicae. We uncover an example of resistance arising from a mutation that silences a dominant insecticide susceptibility allele, resulting in a normally recessive resistance allele being phenotypically expressed.     

Tachykinins in the central and peripheral nervous system of the ascidian Ciona intestinalis

Immunochemical studies were carried out on the ascidian Ciona intestinalis to determine the character and distribution of the tachykinins neurokinin A (NKA) and substance P (SP). Antisera specific for the C-terminus of mammalian SP, and for the N-terminus of mammalian SP and NKA, were used to monitor tissue extracts from Ciona. Parallel immunocytochemical studies assessed the distribution of these tachykinins in the central and peripheral nervous systems as well as their occurrence in endocrine cells. HPLC and radioimmunoassay established the presence of both C-terminal SP and NKA-like material in extracts from neural ganglion and body wall/pharynx. Immunocytochemistry revealed the C-SP material to be present in a population of small neuronal cell bodies and fibers in the ganglion as well as in cell bodies and fibers in the periphery. The NKA-like material was restricted to separate and larger neuronal perikarya in the ganglion while in the periphery its distribution reflected that of the C-SP-like material. Endocrine cells in the pharyngeal epithelium were reactive only with the C-terminal SP antiserum. N-terminal SP antisera were unreactive both in radioimmunoassay and immunocytochemistry. These findings are in accord with the idea that the tachykinin family is represented by at least two of its members at the prevertebrate stage of evolution. Interestingly, the SP-like material shows strong C-terminal homology with the mammalian peptide but little N-terminal similarity. Furthermore, the NKA-like peptide is restricted to the nervous system while SP-like molecules may be found in both central and peripheral neurons as well as endocrine cells in the pharynx.     

Self-incompatibility response induced by calcium increase in sperm of the ascidian Ciona intestinalis

Many hermaphroditic organisms possess a self-incompatibility system to avoid self-fertilization. Recently, we identified the genes responsible for self-sterility in a hermaphroditic primitive chordate (ascidian), Ciona intestinalis: sperm-side polycystin 1-like receptors s-Themis-A/B and egg-side fibrinogen-like ligands on the vitelline coat (VC) v-Themis-A/B. Here, we investigated the sperm behavior and intracellular Ca(2+) concentration ([Ca(2+)](i)) in response to self/nonself-recognition. We found that sperm motility markedly decreased within 5 min after attachment to the VC of self-eggs but not after attachment to the VC of nonself-eggs and that the apparent decrease in sperm motility was suppressed in low Ca(2+) seawater. High-speed video analysis revealed that sperm detached from the self-VC or stopped motility within 5 min after binding to the self-VC. Because s-Themis-B contains a cation channel domain in its C terminus, we monitored sperm [Ca(2+)](i) by real-time [Ca(2+)](i) imaging using Fluo-8H-AM (AAT Bioquest, Inc.). Interestingly, we found that sperm [Ca(2+)](i) rapidly and dramatically increased and was maintained at a high level in the head and flagellar regions when sperm interacted with the self-VC but not when the sperm interacted with the nonself-VC. The increase in [Ca(2+)](i) was also suppressed by low-Ca(2+) seawater. These results indicate that the sperm self-recognition signal triggers [Ca(2+)](i) increase and/or Ca(2+) influx, which elicits a self-incompatibility response to reject self-fertilization in C. intestinalis.     

A novel biological role of tachykinins as an up-regulator of oocyte growth: identification of an evolutionary origin of tachykininergic functions in the ovary of the ascidian, Ciona intestinalis

Tachykinins (TKs) and their receptors have been shown to be expressed in the mammalian ovary. However, the biological roles of ovarian TKs have yet to be verified. Ci-TK-I and Ci-TK-R, characterized from the protochordate (ascidian), Ciona intestinalis, are prototypes of vertebrate TKs and their receptors. In the present study, we show a novel biological function of TKs as an inducible factor for oocyte growth using C. intestinalis as a model organism. Immunostaining demonstrated the specific expression of Ci-TK-R in test cells residing in oocytes at the vitellogenic stage. DNA microarray and real-time PCR revealed that Ci-TK-I induced gene expression of several proteases, including cathepsin D, chymotrypsin, and carboxy-peptidase B1, in the ovary. The enzymatic activities of these proteases in the ovary were also shown to be enhanced by Ci-TK-I. Of particular significance is that the treatment of Ciona oocytes with Ci-TK-I resulted in progression of growth from the vitellogenic stage to the post-vitellogenic stage. The Ci-TK-I-induced oocyte growth was blocked by a TK antagonist or by protease inhibitors. These results led to the conclusion that Ci-TK-I enhances growth of the vitellogenic oocytes via up-regulation of gene expression and enzymatic activities of the proteases. This is the first clarification of the biological roles of TKs in the ovary and the underlying essential molecular mechanism. Furthermore, considering the phylogenetic position of ascidians as basal chordates, we suggest that the novel TK-regulated oocyte growth is an "evolutionary origin" of the tachykininergic functions in the ovary.     

Molecular and functional characterization of cionin receptors in the ascidian, Ciona intestinalis: the evolutionary origin of the vertebrate cholecystokinin/gastrin family

Cholecystokinin (CCK) and gastrin are vertebrate brain-gut peptides featured by a sulfated tyrosine residue and a C-terminally amidated tetrapeptide consensus sequence. Cionin, identified in the ascidian, Ciona intestinalis, the closest species to vertebrates, harbors two sulfated tyrosines and the CCK/gastrin consensus tetrapeptide sequence. While a putative cionin receptor, cior, was cloned, the ligand-receptor relationship between cionin and CioR remains unidentified. Here, we identify two cionin receptors, CioR1 and CioR2, which are the aforementioned putative cionin receptor and its novel paralog respectively. Phylogenetic analysis revealed that CioRs are homologous to vertebrate CCK receptors (CCKRs) and diverged from a common ancestor in the Ciona-specific lineage. Cionin activates intracellular calcium mobilization in cultured cells expressing CioR1 or CioR2. Monosulfated and nonsulfated cionin exhibited less potent or no activity, indicating that CioRs possess pharmacological features similar to the vertebrate CCK-specific receptor CCK1R, rather than its subtype CCK2R, given that a sulfated tyrosine in CCK is required for binding to CCK1R, but not to CCK2R. Collectively, the present data reveal that CioRs share a common ancestor with vertebrate CCKRs and indicate that CCK and CCK1R form the ancestral ligand-receptor pair in the vertebrate CCK/gastrin system. Cionin is expressed in the neural complex, digestive organs, oral siphon and atrial siphons, whereas the expression of ciors was detected mainly in these tissues and the ovary. Furthermore, cioninergic neurons innervate both of the siphons. These results suggest that cionin is involved in the regulation of siphonal functions.     

Large-scale, saturating insertional mutagenesis of the mouse genome

We describe the construction of a large-scale, orderly assembly of mutant ES cells, generated with retroviral insertions and having mutational coverage in >90% of mouse genes. We also describe a method for isolating ES cell clones with mutations in specific genes of interest from this library. This approach, which combines saturating random mutagenesis with targeted selection of mutations in the genes of interest, was successfully applied to the gene families of G protein-coupled receptors (GPCRs) and nuclear receptors. Mutant mouse strains in 60 different GPCRs were generated. Applicability of the technique for the GPCR genes, which on average represent fairly small targets for insertional mutagenesis, indicates the general utility of our approach for the rest of the genome. The method also allows for increased scale and automation for the large-scale production of mutant mice, which could substantially expedite the functional characterization of the mouse genome.     

Germ-line transgenesis of the Tc1/mariner superfamily transposon Minos in Ciona intestinalis

The tadpole larva of the basal chordate Ciona intestinalis has the most simplified, basic body-plan of chordates. Because it has a compact genome with a complete draft sequence, a large quantity of EST/cDNA information, and a short generation time, Ciona is a suitable model for future genetics. We establish here a transgenic technique in Ciona that uses the Tc1/mariner superfamily transposon Minos. Minos was integrated efficiently into the genome of germ cells and transmitted stably to subsequent generations. In addition, an enhancer-trap line was obtained. This is a demonstration of efficient, Minos-mediated transgenesis in marine invertebrates.     

piggyBac-based insertional mutagenesis in the presence of stably integrated P elements in Drosophila

P element-mediated mutagenesis has been used to disrupt an estimated 25% of genes essential for Drosophila adult viability. Mutation of all genes in the fly genome, however, poses a problem, because P elements show significant hotspots of integration. In addition, advanced screening scenarios often require the use of P element-based tools like the generation of germ-line mosaics using FLP recombinase-mediated recombination or gene misexpression using the UAS/Gal4 system. These techniques are P element-based and can therefore not be combined with the use of P elements as mutagenic agents. To circumvent these limitations, we have developed an insertional mutagenesis system using non-P element transposons. An enhanced yellow fluorescent protein-marked piggyBac-based mutator element was mobilized by a piggyBac specific transposase source expressed from a Hermes-based jump-starter transposon marked with enhanced cyan fluorescent protein. In a pilot screen, we have generated 798 piggyBac insertions on FRT bearing third chromosomes of which 9% have sustained a putatively piggyBac-related lethal hit. The FRTs present on the target chromosome remained stably integrated during the screen and could subsequently be used to generate germ-line clones associated with maternal and zygotic phenotypes. PCR-based analysis of insertion loci shows that 57% of the insertions are in genes for which no P element insertions have been reported. Our data demonstrate the potential of this technique to facilitate the quest for saturation mutagenesis of the Drosophila genome. The system is Drosophila nonspecific and potentially applicable in a broad spectrum of nonmodel organisms.     

On the evolutionary significance and metal-binding characteristics of a monolobal transferrin from Ciona intestinalis

Transferrins are a family of proteins that bind and transport Fe(III). Modern transferrins are typically bilobal and are believed to have evolved from an ancient gene duplication of a monolobal form. A novel monolobal transferrin, nicatransferrin (nicaTf), was identified in the primitive ascidian species Ciona intestinalis that possesses the characteristic features of the proposed ancestral Tf protein. In this work, nicaTf was expressed in Pichia pastoris. Extensive solution studies were performed on nicaTf, including UV-vis, fluorescence, CD, EPR and NMR spectroscopies, and electrospray time-of-flight mass spectrometry. The expressed protein is nonglycosylated, unlike the protein isolated from the organism. This property does not affect its ability to bind Fe(III). However, Fe(III)-bound nicaTf displays important spectral differences from other Fe(III)-bound transferrins, which are likely the consequence of differences in metal coordination. Coordination differences could also account for the weaker affinity of nicaTf for Fe(III) (log K = 18.5) compared with bilobal human serum transferrin (HsTf) (log K = 22.5 and 21.4). The Fe-nicaTf complex is not labile, as indicated by slow metal removal kinetics by the high-affinity chelator tiron at pH 7.4. The protein alternatively binds up to one equivalent of Ti(IV) or V(V), which suggests that it may transport nonferric metals. These solution studies provide insight into the structure and function of the primitive monolobal transferrin of C. intestinalis for comparison with higher order bilobal transferrins. They suggest that a major advantage for the evolution of modern transferrins, dominantly of bilobal form, is stronger Fe(III) affinity because of cooperativity.     

Synthesis of fucosyl-containing glycoproteins of the vitelline coat in oocytes of Ciona intestinalis (Ascidia).

The sperm receptors of the ascidian oocyte are located at the outer surface of the vitelline coat (formerly called the chorion). The fucose residues are the receptor's most important components for sperm recognition and binding. We asked whether the fucosyl-containing glycoproteins of the vitelline coat are a product of the oocyte, the follicle cells, or the test cells. Ovaries of Ciona intestinalis were injected with L-[3H]fucose and the progress of its incorporation was followed by using autoradiography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the injected gonads and of the isolated vitelline coats. We found that incorporation of fucose begins within the vitellogenic oocytes, and fucose slowly accumulates in the differentiating vitelline coat. At no time could fucose incorporation be detected in the follicle cells or in the test cells. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of vitelline coats prepared from the injected ovaries showed fucose incorporation into the same three glycoproteins present in vitelline coats from mature oocytes and identified by their affinity for 125I-labeled fucose-binding protein [Pinto, M. R., De Santis, R., D'Alessio, G. & Rosati, F. (1981) Exp. Cell Res. 132, 289-295]. A radioactive band not found in the mature oocyte was also present.     

Induction of gamete release by gonadotropin-releasing hormone in a protochordate, Ciona intestinalis.

Gonadotropin-releasing hormone (GnRH) of vertebrates is now believed to have multiple functions in addition to its role as a hypophysiotropic hormone, as originally defined. Recently, it has been shown that GnRH occurs also in the ascidians, which are considered ancestral chordates. Here the author shows that GnRH induces spawning of gametes from mature individuals of Ciona intestinalis. Ciona accumulates mature gametes in the gonoducts and maintains them until spawning is triggered by a photoperiodic cue(s). Injection of synthetic tunicate GnRH-I or -II into various sites of mature individuals effectively induced gamete release (spawning), although the former was more potent. Gamete release often occurred on a larger scale than in spontaneous spawning. However, moderate gamete release, similar to spontaneous spawning, was often triggered by exogenous tunicate GnRH. GnRH in vivo apparently is released from the GnRH-containing neurons that are distributed from the region of the cerebral ganglion to the proximal part of the ovary along the dorsal strand within the blood sinus; this indicates that both forms of tunicate GnRH may be the actual inducers of spawning. It is suggested that, in the ancestral chordate, GnRH neurons release GnRH prior to the spawning and the released GnRH acts directly on the epithelium of gonoducts or functions as a neuromodulator of other neurons innervating the gonoducts to induce spawning.     

Mammalian and chicken I forms of gonadotropin-releasing hormone in the gonads of a protochordate, Ciona intestinalis

Two forms of gonadotropin-releasing hormone (GnRH) were isolated from the gonads of the tunicate, Ciona intestinalis. The primary structure of the purified peptides was determined by MS and chemical sequence analysis. Both GnRH forms have blocked NH(2) and COOH termini, and their primary structures are identical to mammalian (mGnRH) and chicken I (cGnRH-I) forms reported previously in vertebrates. A total of 1.2 mg of purified cGnRH-I and 0.98 mg of mGnRH was obtained from 100 g of Ciona gonads. The physiological effects of native GnRHs included the induction of synthesis and secretion of sex steroids from ciona gonads and the secretion of luteinizing hormone from rat pituitary. These results suggest that the primary structure and functional roles of mGnRH and cGnRH-I have been highly conserved throughout evolution of chordates.     

Induction of leukemia in chicken by bovine leukemia virus due to insertional mutagenesis

Bovine leukemia virus (BLV) was inoculated into one-day-old chickens. In a small part of inoculated chickens leukemia developed during observation period of one year. Out of 88 birds inoculated, only 4 developed histopathologically verified leukemia. The induced leukemia was characterized by enlarged liver and spleen. The organs were infiltrated with leukemic cells. The DNAs of body organs of inoculated chickens were analysed by Southern blot hybridization for the presence of BLV specific sequences. Out of 9 suspicious chickens tested in 6 birds the BLV was found to be integrated into host DNA either as a complete viral genome or as a part corresponding to its 3'-end. The leukemic cells were monoclonal as regard to the integration site of the BLV provirus. Neither the expression of BLV provirus in chicken leukemic cells nor the antibody response to BLV antigens in inoculated birds was detected. The rearrangements and amplification of erb-B and myb loci of protooncogenes in leukemic cells was detected. There were no changes in loci of following protooncogenes: myc, sis, fes, fps, erb A, src and yes. All obtained data taken together suggest that the BLV induced leukemia in chickens is caused by insertional mutagenesis.     

Application of the mini-Mu-phage for target-sequence-specific insertional mutagenesis of the herpes simplex virus genome.

An earlier technique for insertional mutagenesis of large viral genomes involved the insertion of the thymidine kinase (TK) gene at a specific target site, cotransfection of the fragment carrying the insertion with the intact viral genome, and selection of the progeny for viral recombinants expressing the TK gene. The inserted TK gene could then be replaced by cotransfection of the recombinant DNA with fragments carrying a foreign sequence or a deletion in the target sequence. To enable the probing of larger target domains and facilitate insertional mutagenesis, we extended this technique by insertion of a 2.2-kilobase-pair (kbp) herpes simplex virus 1 (HSV-1) chimeric alpha TK gene into the 7.5-kbp mini-Mu-phage (alpha TK-mini-Mu) and lysogenized Escherichia coli with the helper Mu phage and the alpha TK-mini-Mu. Induction of phage multiplication of the lysogenized E. coli after transformation with plasmids carrying HSV-1 DNA and subsequent infection of E. coli RecA+ lysogenized with Mu phage yielded plasmid populations carrying randomly inserted alpha TK-mini-Mu DNA. Application of this procedure for insertional mutagenesis of the BamHI B fragment, which spans the junction between the unique and reiterated sequences of the L component of viral DNA, yielded two types of recombinants. Viral recombinant designated RBMu1 contained the intact alpha TK-mini-Mu inserted into unique sequences of BamHI B fragment. In recombinant RBMu2, the alpha TK-mini-Mu was inserted at or in the repeated sequences, but approximately equal to 14 kbp comprising most of the internal reiterations of the viral genome in the prototype arrangement were deleted.     

Unique risk factors for insertional mutagenesis in a mouse model of XSCID gene therapy

Although gene therapy can cure patients with severe combined immunodeficiency (SCID) syndromes, the clinical occurrence of T cell malignancies due to insertional mutagenesis has raised concerns about the safety of gene therapy. Several key questions have remained unanswered: (i) are there unique risk factors for X-linked SCID (XSCID) gene therapy that increase the risk of insertional mutagenesis; (ii) what other genetic lesions may contribute to transformation; and (iii) what systems can be used to test different vectors for their relative safety? To address these questions, we have developed an XSCID mouse model in which both the Arf tumor-suppressor gene and the gammac gene were ablated. Gene therapy in this animal model recapitulates the high incidence of integration-dependent, T cell tumors that was seen in the clinical trial. Ligation-mediated PCR analysis showed integration sites near or within established protooncogenes (Chd9, Slamf6, Tde1, Camk2b, and Ly6e), demonstrating that T cell transformation was associated with targeting of oncogene loci; however, no integrations within the Lmo2 locus were identified. The X-SCID background in transplanted cells was required for high rate transformation and was associated with expansion of primitive hematopoietic cells that may serve tumor precursors. This model should be useful for testing safety-modified vectors and for further exploring the risk factors leading to insertional mutagenesis in gene therapy trials.