Molecular Mechanisms of Insecticide Resistance - American Chemical

Chapter 16 Tribolium as a Model Insect for Study of Resistance Mechanisms 4

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R. W. Beeman1,2, J. J. Stuart , R. E. Denell , W. H. McGaughey , and B. A. Dover Downloaded by NORTH CAROLINA STATE UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: September 22, 1992 | doi: 10.1021/bk-1992-0505.ch016

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1U.S. Grain Marketing Research Laboratory, U.S. Department of Agriculture, Agricultural Research Service, 1515 College Avenue, Manhattan, KS 66502 2Department of Entomology and Division of Biology, Kansas State University, Manhattan, KS 66506 4Department of Entomology, Purdue University, West Lafayette, IN 47905 3

Classical techniques for chromosome manipulation at the organismal level are used routinely by Drosophila biologists to facilitate the study of genetic variation in populations, but such techniques are lacking in other insects. We are attempting to develop the genetic tools needed to carry out such manipulations in the beetle Tribolium castaneum, and to apply them to the study of several types of biological problems, including insecticide resistance. In principle, artificial mutagenesis can be used to induce resistance mutations at a frequency much higher than the spontaneous rate, providing an efficient way to identify genes capable of conferring resistance. Balancer chromosomes, carrying crossover-suppressing rearrangements, lethals, and dominant visible markers, can be used to extract and render homozygous such mutations, whether recessive or dominant. We have demonstrated the feasibility of applying this method to approach the saturation mutagenesis of a portion of the second linkage group, and are now applying it to a search for pathogen resistance mutations. Only a limited number of eukaryotic species are sufficiently tractable as genetic models to qualify as preferred experimental subjects in which a broad range of sophisticated molecular and genetic techniques can be employed for biological research. Any species that would aspire to such preferred status should possess at least a few of the following properties: 1. A short generation time and ease of rearing, handling and making genetic crosses; 2. A genome small in size, well mapped by visible and molecular markers, and containing only minimal amounts of highly dispersed repetitive DNA; 3. A capacity for in vivo chromosome manipulations using deletions, duplications and balancer chromosomes to facilitate genetic mapping, dosage analysis, reversion analysis and chromosome extraction (described in greater detail below); and 4. A capacity for germline transformation and transposon-mediated gene tagging and cloning. Among higher animals, the insect Drosophila melanogaster provides the most powerful experimental system for integrated genetic and molecular studies. This organism was first chosen for property #1 from the above list, but its potential wasn't fully realized until techniques were developed to exploit properties #3 and 4, aided by #2. Today, the utility of Drosophila as an animal model system is rivalled only by the 0097-6156/92/0505-0202506.00/0 © 1992 American Chemical Society

In Molecular Mechanisms of Insecticide Resistance; Mullin, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

Downloaded by NORTH CAROLINA STATE UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: September 22, 1992 | doi: 10.1021/bk-1992-0505.ch016

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nematode Caenorhabditis elegans, for which the experimental advantages of inbreeding have been exploited rather than chromosome manipulation. Although the experimental advantages of Drosophila make it the preferred species for detailed examination of the molecular and genetic mechanisms of insecticide resistance, other insect models are clearly needed. In view of the great diversity of insect forms and the unique importance of insects to mankind, much effort is now being devoted to mapping the genomes of several economically important insect species with molecular markers, and to developing new techniques for germline transformation. The absence of methods for chromosome manipulation and other sophisticated genetic techniques in these species places limits on the kinds of information and strategies possible. We have recently shown that the beetle Tribolium castaneum has many of the characteristics that have proven so favorable in Drosophila research. Below, we describe the attributes of Tribolium that recommend it as a genetic model. We further describe our initial efforts to develop the chromosome extraction technique in this insect, and discuss our progress toward incorporating the technique into a genetic analysis of pathogen resistance. Tribolium castaneum (Red Flour Beetle) The red flour beetle (hereafter referred to as RFB) is globally one of the most abundant and widespread pests infesting stored grain, flour and other cereal products. Its ease of laboratory culture has made it a popular system for studies in population dynamics, population genetics and quantitative genetics for over 30 years. It has by far the bestmarked set of genetic linkage maps in the Coleoptera, and one of the best among all insects. It is one of the easiest of all insects to rear and handle in large numbers, can readily be mutagenized, and is amenable to high resolution genetic analysis (/). A compilation of information on the genetics and biology of the genus Tribolium is given in a three volume set (2-4), and in an annual newsletter entitled the Tribolium Information Bulletin, containing stocklists, research results and bibliographies,. RFB is also particularly well suited for molecular genetic study. It has a genome size of 0.21 pg per haploid genome (5, 6), comparable to that of Drosophila melanogaster, making it one of the smaller insect genomes known. The RFB genome, like that of D. melanogaster, has a long-period interspersion pattern of repetitive elements, in which the moderaterly repetitive sequences are well-separated by long tracts of unique-sequence DNA. The RFB genome is organized into a relatively small number of chromosomes: nine pairs of autosomes and one pair of sex chromosomes (n=10). Sex is determined by a simple X X , XY system in which males are the heterogametic sex. This karyotype is typical of many beetle species (7). A diploid chromosome number and meiotic recombination occur in both sexes. RFB has shown great adaptability in developing resistance to all classes of insecticides to which it has been exposed, including pyrethroids (#), organophosphates (9), DDT (10), juvenile hormone analogues (7/) and fumigants (12). The practicality of genetically extracting, purifying and mapping genes for insecticide resistance, including genes for carboxylesterase-related malathion resistance and target insensitivity-type cyclodiene resistance, has also been demonstrated in RFB (13-15). In short, the economic significance of RFB, its favorable biological and genetic attributes and its long history of insecticide resistance strongly recommend this species as a model genetic system for the study of insecticide resistance mechanisms. Our efforts are currently focussed on constructing balancer chromosomes and other rearrangements which will facilitate chromosome manipulations; mapping the genome with visible genetic markers, recessive lethal mutations and restriction fragment length polymorphisms (RFLPs); developing a transposon-mediated gene tagging and transformation system; and inducing and characterizing insecticide and pathogen resistance mutations.


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Chromosome Extraction: General Principles Among the most powerful genetic tools in Drosophila research are multiply-rearranged "balancer" chromosomes used to maintain the integrity of homologs which have been mutagenized or sampled from wild populations. Such balancers typically carry multiple inversions which decrease or totally eliminate the recovery of viable recombinant progeny, as well as a dominant visible marker and a recessive lethal to aid in following the transmission of the balancer and in eliminating homozygous progeny, respectively. Indeed, the utilization of such a balancer for the X chromosome was the key to Muller's Nobel Prize-winning work on the induction of mutations, performed more than six decades ago. Incredibly, this valuable research tool has not been systematically utilized for any higher animal outside of the Drosophilids. Extraction of mutagenized chromosomes is in principle a powerful strategy for discovering previously unknown resistance loci. In Drosophila, this approach has been used to identify methoprene target resistance, previously unknown in any insect species. In this case the discovery had important implication for basic studies on the mode of action of juvenile hormone (JH) analogs and of JH itself (T. G. Wilson, this volume). The availability of crossover-suppressing rearrangements, recessive lethals and dominant visible mutations in the RFB has suggested the possibility of chromosome extraction in this species. The principle by which chromosome extraction operates is illustrated in Figure 1. It is a three-step process involving extracting, amplifying and rendering homozygous the desired chromosome. Two types of marked homologs of the chromosome to be extracted are required. The first is a balancer chromosome carrying a crossover suppressor =C, a lethal =x, and a dominant visible marker =D1. The second type must carry a dominant visible =D2, different from the one on the balancer chromosome, and must have no lethals in common with the balancer chromosome. The crossover suppressor C allows retention of the identity and integrity of the original chromosome during extraction, amplification and generation of homozygotes by preventing recombination (i.e. contamination) with homologous chromosomes during meiosis. The dominant visible marker D2 allows recognition of individuals carrying both the extracted chromosome and the balancer chromosome after the amplification cross, since such individuals possess the Dl phenotype but lack the D2 phenotype. The lethal x prevents the development of D l homozygotes which would be indistinguishable from the desired class. Finally, the Dl marker allows recognition of individuals homozygous for the desired chromosome after the final cross, since these individuals will lack the D l phenotype. By this method all progeny from cross #3 (Figure 1C) that are phenotypically normal with respect to D l and D2 must be homozygous for the extracted chromosome, i.e. the method is 100% efficient. Other regions of the genome can be extracted at reduced efficiency by inbreeding in cross #3, that is by using the same F] male as sire for crosses 2-3 (Figure 1B-C) so that a single Fj male is crossed to his daughters for the final homozygote-generating step. Chromosome Extraction in Tribolium In our initial feasibility study for chromosome extraction in RFB we used the two homologous chromosomes, maxillopedia-Dachs^ (mxpP ^) and Eyeless (Ey). These chromosomes represent the second linkage group (LG2), and emerged from our studies of a cluster of homeotic genes located on this linkage group (I, 16-18). The mxpD h3 chromosome represents the first type (balancer) described above, and contains C, Dl and x traits, while the Ey chromosome corresponds to D2. mxpP 3 is a single, radiation-induced mutation that confers all three necessary properties C, D l cn

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and x. That is, mxpP ^ is a crossover suppressor; has a dominant visible phenotype (=short legs); and is lethal when homozygous. The Ey chromosome has a dominant phenotype (^reduction in the number of ommatidia in the compound eyes) different from that on the mxpP ^ chromosome, and lacks any lethal present on the mxpP ^ chromosome (i.e. mxpP ^ /Ey is a viable genotype), although the Ey chromosome does carry an unrelated lethal that is balanced by mxp^ ^ . Ideally, a balancer should balance an entire chromosome, i.e. it should eliminate all recombination over the length of the chromosome. The extent of the region of LG2 balanced by mxpP ^ is uncertain, but appears to be approximately in the range of 2050 map units (ref. 19 and unpublished observations) which may correspond to one chromosome arm. The total recombinational length of the RFB genome has been estimated at ca. 1000 map units (unpublished observations), or about fourfold greater than that of Drosophila. The karyotype of LG2 is unknown. After ethylmethanesulfonate (EMS) mutagenesis and extraction of LG2, we screened for lethals and for random visible mutations. The results are summarized in Table I. Among 1607 chromosomes screened we found 15 independently-derived lethals representing 7 complementation groups. Many other lethals were found, but because they occurred in the same batches as the aforementioned, they could not be confirmed to be independently-derived. Complementation analysis has not been completed for those in this latter category. In addition, we found 7 independentlyderived visible mutations causing a variety of unique and distinct morphological or behavioral abnormalities. These include the two behavioral abnormalities "tremorous" and "prolapsed genitalia", and the morphological abnormalities "wingless", "short elytra", "broken antennae", "epidermal hypertrophy" and "melanized quinone gland" Detailed genetic analysis of these mutations will be published elsewhere. Assuming that the genome has 5000 genes and that mxp balances 2-5% of the genome, then 100-250 genes are potentially identifiable by mutation, whereas a minimum of 14 genes were in fact identified. The true extent of the balanced region is uncertain. cn

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Chromosome Extraction and Pathogen Resistance Genes Having demonstrated that chromosome extraction is feasible in RFB, we can now envision the application of this technique to the search for genes that control resistance to pathogens (or other insecticides) in this species. The mutagenesis and extraction procedure would be unchanged, but instead of screening for visible or lethal mutations, we would screen for variants that can survive discriminating doses of Bacillus thuringiensis var. tenebrionis (BT). An advantage of this approach is that recessive resistance mutations will be detected. Indeed, the first case of field derived pathogen resistance ever reported in an insect involved recessive resistance to BT (20). Under laboratory or field conditions recessive resistance ordinarily has a lower probability of being selected than dominant resistance, even if the two occur with equal frequency. The major limitation to this approach is that only a restricted portion of the genome can be screened with 100% efficiency, namely the region encompassed by the balancer. In the case of RFB, using the mxpP ^ balancer, this may include as much as a single chromosome arm, or approximately 5% of the genome. However, because of the inbreeding scheme (involving father/daughter matings) incorporated into the extraction scheme, the remainder of the genome is also being screened, although at reduced efficiency. Balancers for other regions of the RFB genome are currently being developed in our laboratories. The second possible limitation is inherent in the mutagenesis approach, and does not specifically concern RFB. This limitation stems from the fact that mutagenesis is expected to generate primarily chromosome rearrangements or base cn


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treated extract A. male X C A J 2 L / D 2 >-

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*/ * Figure 1. Generalized chromosome extraction procedure. (A) Extraction of a single mutagenized chromosome from a mutagenized (="treated") male by picking a single Fl adult. (B) Amplification of the mutagenized chromosome by single pair or single harem mating. (C) Homozygosing of the mutagenized chromosome by selecting against the Dl marker. This can be accomplished by self-crossing the appropriate Fl derived from cross (B) or by backcrossing appropriate daughters to the * /C,x,Dl father to facilitate inbreeding. #, mutagenized chromosome; C, crossover suppressor; x lethal mutation; D l , dominant visible mutation; D2, dominant visible mutation different from D l . In each cross the desired class can be phenotypically distinguished, and only this class is shown. v

Table I. Capture of EMS-induced mutations by chromosome extraction using* LG2 (Dch3) balancer type of No. No. o? genes mutation screened found frequency lethal 1607 7 0.004 visible 1607 7 0.004 Total 1607 ^ 14 0.009 1607 mutagenized LG2 chromosomes were screened for both visible and lethal mutations. Data refer only to distinct complementation groups.


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substitutions. Gene amplification-type resistance mechanisms may not arise directly by mutagenesis, although chromosomes carrying such variants can still be extracted from natural or laboratory populations.


BT Susceptibility of Tribolium castaneum As a prelude to attempting to screen for BT resistance mutations after chromosome extraction, we tested the susceptibility of a laboratory strain of RFB to ABG-6263, a beetle-active BT isolate designated BT subsp. tenebrionis (Abbott Laboratories, North Chicago, IL). ABG-6263 (lot #18-092-BR) was a spray-dried powder containing 860 Colorado potato beetle units/mg (determined by Abbott Laboratories). The inert ingredients consisted of fermentation solids, inert clays and various proprietary additives. A formulation blank containing only the inert ingredients was tested as a control. In order to ensure thorough mixing, we blended each formulations into flour as aqueous slurries, then lyophilized the suspension. The results (Table II) show that the threshold for toxicity to first instar larvae is between 0.1% and 1.0% BT in flour, calculated on the basis of total wt. of the BT prep. At toxic doses (1.0%) larvae are stunted and retarded in their development. Newly-hatched first instar larvae are the most susceptible stage, and susceptibility drops rapidly with larval age (unpublished observations). When adults finally do develop they usually show a very specific and localized syndrome suggestive of juvenilization, namely retention of larval urogomphi. Normally these structures are present in larvae and pupae, but are entirely absent in adults. Adult survivors at this dose tend to show reduced fertility. A genetic mutant (termed juvenile urogomphi, or ju) that produces an identical syndrome, has been described and is fertile in both sexes (ref. 4 and unpublished observations). Retention of juvenile urogomphi in the adult is a well-known consequence of threshold doses of the juvenile hormone mimic, methoprene, in Tenebrionid beetles (27). Thus, it is possible that BT intoxication produces an indirect juvenilizing effect on beetle larvae. Such a possibility does not seem to explain the growth retardation seen in BTintoxicated RFB larvae, since this effect of BT is evident throughout larval life.

Table II. Effect of BT on Tribolium exposed from the egg stage dose (%>) adults pupae larvae Fl 0 50 0 0 >100 10C 31 0 0 51 .1BT 28 0 0 0 .3BT 6* 0 0 0 1BT 2* 0 0 0 3BT 0 0 0 0 10BT 0 0 0 0 Each datum (given as # of individuals) is total of 4 independent replicates. Two females were allowed to oviposit in each vial for three days. Vials were scored at day 60 for all developmental stages. C=control (formulation blank). BT=Bacillus thuringiensis formulation. Fl refers to progeny of adults recovered at day 60. •Retention of juvenile urogomphi in adults.


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Future Prospects The widespread utility of chromosome extraction in RFB in the future will depend on the availability of new balancer chromosomes that cover larger regions of the genome, such as entire chromosomes. Pseudolinkage of nonhomologous balancer chromosomes by rearrangement might produce more complex balancers that could be used to simultaneously extract two or more chromosomes. Sets of recessive visible point mutations needed to detect and recover such rearrangements are available and are now being used in balancer screens. Downloaded by NORTH CAROLINA STATE UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: September 22, 1992 | doi: 10.1021/bk-1992-0505.ch016

Literature Cited 1. Beeman, R. W.; J. J. Stuart; M. S. Haas; R. E. Denell. Devel. Biol. 1989, 133, 196-209. 2. Sokoloff, A. The Biology of Tribolium; Oxford Press: London, 1972; Vol. 1. 3. Sokoloff, A. The Biology of Tribolium; Oxford Press: London, 1974; Vol. 2. 4. Sokoloff, A. The Biology of Tribolium; Oxford Press: London, 1977; Vol. 3. 5. Brown, S. J.; Henry, J. K.; Black, W. C., III; Denell, R. E. Insect Biochem. 1990, 20, 185-193. 6. Juan, C.; Petitpierre, E. Genome 1991, 34, 169-173. 7. Smith, S. G.; Virkki, N. In Animal Cytogenetics; John, B., Ed.; Berlin/Stuttgart, 1978, Vol. 3, No. 5 (Coleoptera). 8. Collins, P. J. Pestic. Sci. 1990, 28, 101-115. 9. Halliday, W. R.; Arthur, F. H.; Zettler, J. L. J. Econ. Entomol. 1988, 81, 7477. 10. Dyte, C. E.; Blackman, D. G. J. Stored Prod. Res. 1967, 2, 211-228. 11. Dyte, C. E. Nature 1972, 238, 48-49. 12. Zettler, J. L.; Halliday, W. R.; Arthur, F. H. J. Econ. Entomol. 1989, 82, 15081511. 13. Beeman, R. W. J. Econ. Entomol. 1983, 76, 737-740. 14. Beeman, R. W.; Nanis, S. M. J. Econ. Entomol. 1986, 79, 580-587. 15. Beeman, R. W.; Stuart, J. J. J. Econ. Entomol. 1990, 83, 1745-1751. 16. Beeman, R. W. Nature 1987, 327, 247-249. 17. Beeman, R. W.; Brown, S. J.; Stuart, J. J.; Denell, R. E. In: Molecular Insect Science; Hagedorn, H.; Hildebrand, J.; Kidwell, M.; Law, J., Eds.; Proc. Int'l. Symp. Molec. Insect Sci.; Plenum Press: NY, NY, 1990; pp. 21-29. 18. Stuart, J. J.; Brown, S.J.; Beeman, R.W.; Denell, R.E. Nature 1991, 350, 7274. 19. Beeman, R W.; Johnson, T. R.; Nanis, S. M. J. Hered. 1986, 77, 451-456. 20. McGaughey, W. H. Science 1985, 229, 193-195. 21. Edwards, J. P.; Short, J. E.; Rowlands, D. G. J. Stored Prod. Res. 1988, 24, 165-172. R E C E I V E D December 9, 1991


Molecular Mechanisms of Insecticide Resistance - American Chemical

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