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Experimental investigation of autosomal translocations for insect pest control: Reid, John Arthur Keith 1974

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AN EXPERIMENTAL INVESTIGATION OF AUTOSOMAL TRANSLOCATIONS FOR INSECT PEST CONTROL: FITNESS EFFECTS AND MARKER-FREE ISOLATION TECHNIQUES. BY JOHN ARTHUR KEITH RE.ID B.Sc. # U n i v e r s i t y of B r i t i s h Columbia, 1972 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Zoology We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA AUGUST, 1974 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requ i rement s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Co lumb ia , I ag ree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s tudy . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d tha t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i thout my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Co lumbia Vancouver 8, Canada Date L U ^ i 10 / l j i A B S T R A C T Because of recent advances in genotic insect control theory, i t has become important to investigate the f i t n e s s e f f ects of, and i s o l a t i o n procedures for, homozygous autosomal translocations. I is o l a t e d 57 autosomal translocations in D r o s o D h i l a »elano^aster . Of these 21 were homozygous viable. From data o b t a i n e d during the i s o l a t i o n of these trunslocation.: and from fi t n e s s tests and competition cage experiments, rh • following points can be made; (1) Between one in ten and one in one hundred homozygous viable laboratory produced translocations a r e l i k e l y to be o f value in f i e l d tests of genetic insect control procedures. (2) Translocations which produce high leve l s of unbalanced gametes whan heterozygous do not tend to be lass f i t i n the homozygous state than others. Therefore s c r e e n i n g procedures dependent only on reduced progeny production from translocation heterozygote parents should be satis f a c t o r y for the i s o l a t i o n of useable stocks. ( 3 ) Translocations whose breakpoints are vary near the center of chromosomes tend to produce small progeny reduction in the heterozygous state, making these translocations useless as negative heterotic c a r r i e r s of useful genes. ( 4 ) Those translocations which are the resu l t of multiple break events tend to be less f i t than simple double-break translocations and t h e r e f o r e should be discarded. TABLE OF CONTENTS Abstract i Table Of Contents i i L i s t Of Figures i i i L i s t Of Tables i v Acknowlegements v Introduction 1 Literature Review: 4 Genetic Load Population Suppression: 5 Gene Pool Replacement: 5 Translocation I s o l a t i o n Procedures: 9 Materials And Methods: 10 Fitness Of Translocations: Cage Experiments, Measurement, And Simulation 14 Methodology: .18 Results: ...26 Discussion: 31 Translocation Isolation Procedures: 33 Materials And Methods 33 Results: 34 Discussion 41 Appendix 1: A Cage For Drosophila Population Studies 45 Appendix 2: A Fast, Accurate, Electronic Fly Counter: 48 Appendix 3. Simulation Of Semi-sterile Genetic Systems. .54 Appendix 4: Estimation Of Karyotype Without Markers. 63 i i i LIST OF FIGURES Figure 1 12 Figure 2 18 Figure 3 21 Figure A • 25 Figure 5 • • 36 Figure 6 • 38 Figure 7 . .40 Figure 8 . . . . . . . . . . . . . 47 Figure 9 . . . • 51 Figure 10 . 53 Figure 11 .65 LIST OF TABLES i v 23 Table 1 C D Table 2 . . . 28 29 Table 3 ' Table 4 . . •'. • • • • • • • • • * . " ' ' 3 ° 49 Table 5 . . . 4 * V ACKNOWLEGEMENTS I am indebted to many graduate students and f a c u l t y members f o r the advice and comments that they have o f f e r e d during the course of t h i s study. In p a r t i c u l a r , I would l i k e to thank my r e s e a r c h s u p e r v i s o r , Dr. C. F. Hehrhahn, who has been very generous with h i s time and support. His encouragement and c r i t i c i s m have c o n t r i b u t e d much to my graduate education. For p r o f e s s i o n a l help and advice on g e n e t i c and c y t o l o g i c a l matters I am g r e a t l y indebted t o Mr. A r t h i l l i k e r . Dr. J3. F i t z -E a r l e , Dr. J. Myers, and Dr. D. G. Holm have a l s o provided much u s e f u l a d v i c e . The t e c h n i c a l a s s i s t a n c e of Mr. B i l l Webb and Mr. Ray H i l b o r n are g r e a t l y a p p r e c i a t e d . I thank Mr. S. S t e r n s , Dr. J. Myres, and Dr. C. F. Wehr'hahn fo r t h e i r comments on the t h e s i s . F i n a l l y I would l i k e t o express my extreme g r a t i t u d e to s y l v i a r e i d , L o u i s Giguere, Con Wehrhahn, Judy Myres, A r t H i l l i k e r , and o t h e r s who have provided p e r s o n a l encouragement to my e f f o r t s . 1 IMTBODDCTIOM A number of g e n e t i c i n s e c t c o n t r o l techniques have been proposed i n rec e n t years. S e v e r a l of the t h e o r e t i c a l l y most promising methods i n v o l v e the use of r e c i p r o c a l t r a n s l o c a t i o n s . The main pro p e r t y of t r a n s l o c a t i o n s which makes them p o t e n t i a l l y u s e f u l ( for the c o n t r o l of i n s e c t pests) i s that h y b r i d s a r r i s i n g from c r o s s e s with normal i n s e c t s have reduced f e c u n d i t y s i n c e as many as h a l f of a l l gametes produced by the hybr i d s are g e n e t i c a l l y unbalanced. It should t h e r e f o r e , be p o s s i b l e to use t r a n s l o c a t i o n s to c o n t r o l i n s e c t p e s t s i n s e v e r a l d i f f e r e n t ways. M u l t i p l y t r a n s l o c a t e d l a b o r a t o r y s t o c k s , i f r e l e a s e d i n l a r g e numbers i n t o p o p u l a t i o n s of pest i n s e c t s should reduce the f e c u n d i t y of the p o p u l a t i o n s . Most proposed methods, however, i n v o l v e the use of t r a n s l o c a t i o n s l i n k e d t o genes which, from man's po i n t of view, are p r e f e r a b l e t o t h e i r a l l e l e s i n pest p o p u l a t i o n s . For these methods to work, t r a n s l o c a t i o n homozygotes must have f i t n e s s s u p e r i o r to heterozygotes under f i e l d c o n d i t i o n s . I f t h i s i s the case, and enough t r a n s l o c a t e d i n s e c t s are r e l e a s e d i n t o w i l d p o p u l a t i o n s , negative h e t e r o s i s w i l l ensure that the t r a n s l o c a t i o n w i l l r e p l a c e the w i l d genotype. I t i s f o r t u n a t e that c r o s s i n g over i s s e v e r e l y depressed i n the v i c i n i t y of t r a n s l o c a t i o n p o i n t s . T h i s means t h a t t r a n s l o c a t i o n s can ba used as c a r r i e r s to d r i v e genes i n t o p o p u l a t i o n s . The kind of gene t h a t we might wish to put i n t o a p o p u l a t i o n depends on the s p e c i e s . I f an i n s e c t i s a dis e a s e 2 vector, a gene which renders i t incapable of harboring the disease may be i d e a l , both for man and for the insect. In cases where eradication of a pest i s the ultimate aim, a conditional l e t h a l gene may be best. Good candidates are genes which prevent an insect from going into diapause, at least i n northern l a t i t u d e s . Temperature sensitive l e t h a l s might also be useful. There i s a broad chasm between conceptualizing a genetic insect control program and implementing i t . For many insect pests, a reasonable idea of the cost of rearing enough insects to suppress wild populations can be obtained. But we know very l i t t l e about the time and e f f o r t required to produce the genetic stocks needed to implement any kind of sophisticated genetic insect control program. In fact, we can not, a p r i o r i , be sure that i t i s te c h n i c a l l y possible to engineer the ri g h t kind of stock at a l l . L o g i c a l l y , the best way to f i l l t h i s gap i n our knowledge i s to f i r s t obtain the relevant information by using, as a p i l o t organism, the genetically most tractable insect which has the genetic and l i f e history features of important pest species. That insect i s Droso£hila melanocjastei: . The main unresolved question that I have chosen to ask i s : what proportion of homozygous viable translocations are f i t enough to be useful i n control programs. Many advocates of genetic insect control dogmatically assume that the proportion i s high! However, p r a c t i c a l experience suggests that the assumption i s wrong. Robertson and Curtis (1973), in an experiment involving one Drosophila translocation, found that 3 their translocation could not compete successfully with wild f l i e s . Foster (pers. Comm.) found that only one of t h i r t y induced translocations in the sheep blowfly, L u c i l i a cuDrina, was homozygous viable and f e r t i l e . I t , however, had a low fecundity. Another major objective of my research was to examine, in d e t a i l , the p r a c t i c a b i l i t y of a simple procedure for i s o l a t i n g useful translocations. This method would be applicable in pest species where methods involving v i s i b l e genetic markers, crossover-suppressors and polytene chromosome squashes can not be used. 4 LITERATURE REVIER: There are two categories of genetic insect control techniques. These w i l l be termed genetic load techniques and. the Emulation replacement technigues^ The objective of genetic load techniques i s to eradicate or lower to commercially tolerable lev e l s pest populations through the introduction of translocated or otherwise genetically aberrant i n d i v i d u a l s : introduced individuals, by mating with members of a wild population, lower the average f i t n e s s of the population. Such techniques were f i r s t suggested by Serebrovskii (1940). The best known procedure of t h i s sort i s the s t e r i l e insect technique, which was used to eradicate, at least temporarly, the screw-worm f l y from the South Western U.S.A. Gene pool replacement of pest populations by s p e c i a l l y taylored laboratory st r a i n s was suggested by Curtis (1968). The nature of an introduced genetic stock must be such that i t w i l l tend to replace the o r i g i n a l wild-type stock. The key point i s that the introduced 2§netic material must i n i t i a l l y confer a f i t n e s s advantage so that individuals carrying i t are'favored in competition with wild stocks^ The more sophisticated variants of both categories of genetic control procedures, load and replacement, re l y on a single p r i n c i p l e of population genetics. This p r i n c i p l e i s termed negative heterosis^ Negative heterosis implies that the heterozygote between two strains i s less f i t than either of the 5 two homozygotes. T h i s r e s u l t s i n an o v e r a l l p o p u l a t i o n f i t n e s s d e p r e s s i o n and i n an unstable e q u i l i b r i u m between wild-type and t r a n s l o c a t e d s t o c k s . T h i s i n t u r n , r e s u l t s i n frequency dependent s e l e c t i o n i n f a v o r of the genotype which i s present i n excess of the unstable e q u i l i b r i u m l e v e l . G enetic Load P o p u l a t i o n Suppression^ Moderately s u c c e s s f u l experimental g e n e t i c manipulations of pest p o p u l a t i o n s have been r e p o r t e d . Wagoner et a h (1973), using sex-autosome t r a n s l o c a t i o n s , produced a s i g n i f i c a n t d e p r e s s i o n i n a wild house f l y p o p u l a t i o n ( f i e l d t e s t ) . Laven (1969), a l s o using sex-autosome t r a n s l o c a t i o n s , e r a d i c a t e d s t r u c t u r a l l y w i l d - t y p e Culex E i E i e n s (large cage t e s t ) . These examples encourage f u r t h e r development of g e n e t i c i n s e c t c o n t r o l methods. Gene Pool Replacement^ The development of replacement techniques was f i r s t suggested by C u r t i s (1968). His idea was to use s i n g l e t r a n s l o c a t i o n s to cause an u n s t a b l e e q u i l i b r i u m c o n d i t i o n which c o u l d be e x p l o i t e d t o i n t r o d u c e c l o s e l y l i n k e d d e s i r a b l e genes i n t o i n s e c t p o p u l a t i o n s . In such a case, the t r a n s l o c a t i o n i s termed a c a r r i e r mechanism as i t can be used to t r a n s p o r t a d e s i r a b l e t r a i t i n t o wild p o p u l a t i o n s . D e s i r a b l e t r a i t s i n c l u d e high or low temperature l e t h a l i t y and i n s e c t i c i d e s u s c e p t i b i l i t y as w e l l as the i n a b i l i t y to be a disease v e c t o r . 6 The theory was l a t e r expanded by C u r t i s and Robinson(1972) nho examined the use of double t r a n s l o c a t i o n s f o r pest c o n t r o l . Whitten (1971a) f u r t h e r extended the theory by c o n s i d e r i n g the use of m u l t i p l y t r a n s l o c a t e d s t r a i n s which would c a r r y d e s i r a b l e c h a r a c t e r s . He developed a simple d i s c r e t e g e n e r a t i o n computer s i m u l a t i o n model. The r e s u l t s of t h i s model suggested to Whitten t h a t f o r a t r a n s l o c a t e d s t r a i n with a f i t n e s s as low as .7, replacement of the wild p o p u l a t i o n would be v i r t u a l l y complete i n seven g e n e r a t i o n s . Whitten (1971a) s u b s c r i b e d to two myths t h a t pervade the l i t e r a t u r e of g e n e t i c i n s e c t c o n t r o l . The f i r s t i s t y p i f i e d by the f o l l o w i n g quote: "Once a set of s u i t a b l e s t r a i n s has been developed ... and the system t e s t e d . . . i t s permanence i s i n s u r e d s i n c e n a t u r a l s e l e c t i o n cannot oppose i t ; r a t h e r n a t u r a l s e l e c t i o n i s an e s s e n t i a l i n g r e d i e n t of the program." (Whitten 1971a, page 684.) This may be paraphrased as "God i s i n the l a b o r a t o r y and mother nature i s no match f o r him". The second myth i s t y p i f i e d by the f o l l o w i n g quote: "Although t h i s m u l t i p l e t r a n s l o c a t i o n s t r a i n , (developed i n the lab) can be expected to equal the base s t r a i n i n f i t n e s s . . . " (Whitten 1971a, page 682) This second myth may have a r i s e n from a statement i n C u r t i s (1968), "...But t r a n s l o c a t i o n homozygotes (T/T) are u s u a l l y 7 f u l l y f e r t i l e . " S e r e b r o v s k i i (1940) on the other hand had suggested that the g r e a t e s t problem, even f o r genetic load techniques, would be development of h i g h l y f i t l a b o r a t o r y s t r a i n s . A l a r g e p o r t i o n of the t h e o r e t i c a l and experimental l i t e r a t u r e i s based on the second myth, t h a t t r a n s l o c a t e d s t r a i n s with extremely high f i t n e s s can e a s i l y be c o n s t r u c t e d . F o s t e r e t a l . . (1972) note, as d i d C u r t i s (1963) , the problem of i n s e p a r a b l y l i n k i n g d e s i r e d c h a r a c t e r s to the g e n e t i c a l l y rearranged s t r a i n . They pointed out t h a t i f a d e s i r a b l e a l l e l e i s g e n e t i c a l l y s e p a r a b l e from the t r a n s p o r t i n g mechanism, then the d e s i r e d a l l e l e (for temperature s e n s i t i v i t y etc.) could be a c c i d e n t l y l o s t v i a g e n e t i c c r o s s i n g - o v e r i n the heterozygote during gene-pool replacement. Thus the net e f f e c t of such a replacement program would be t h a t one wild-type s t r a i n would be r e p l a c e d by another at g r e a t expense. F o s t e r et al._ (1972) a l s o c o n s i d e r e d the use of compound chromosomes (a s p e c i a l type of t r a n s l o c a t e d chromosome where the l e f t and r i g h t arms are attached to the same centromere) as a t r a n s p o r t i n g mechanism. Compound chromosomes seem very a t t r a c t i v e as c a r r i e r s s i n c e the d e s i r e d a l l e l e s cannot be removed by c r o s s i n g over i n heterozygotes. T h i s i s so because compound by wild-type c r o s s e s produce no v i a b l e heterozygotes. Foster e t a L (1972) r e p o r t e d on d i s c r e t e generation experiments showing d e n s i t y dependent s e l e c t i o n i n competition between a compound stock and a s t r u c t u r a l l y wild-type stock. 8-The main problem with r e p l a c i n g pest s p e c i e s by r e l e a s i n g s t r a i n s with compound chromosomes i s t h a t compound chromosomes are very d i f f i c u l t to c o n s t r u c t i n pest s p e c i e s which are not g e n e t i c a l l y w e l l c h a r a c t e r i z e d . Smith and von B o r s t e l (1972) a l s o noted the c r o s s i n g over problem. They coined the term "Time Bomb Technique" f o r the replacement of i n s e c t p o p u l a t i o n s by c o n d i t i o n a l l e t h a l s attached to a c a r r i e r . wehrhahn and Klassen (197 1) suggested the use of c o n d i t i o n a l l e t h a l s i n s e p a r a b l y l i n k e d to genes f o r i n s e c t i c i d e r e s i s t a n c e . Among other t h i n g s , they f a v o r the use of i n s e c t i c i d e s to promote replacement of w i l d p o p u l a t i o n s with c o n d i t i o n a l l e t h a l s . T i g h t l i n k a g e between r e s i s t a n c e and c o n d i t i o n a l l e t h a l genes i s more c r i t i c a l to the u s e f u l n e s s of t h e i r method than any s m a l l decrement i n f i t n e s s would be. Wehrhahn (1973) noted that s p e c i e s with low powers of d i s p e r s a l would be l e a s t amenable to c o n t r o l because "hot spots" of wild-type i n s e c t s , not e a s i l y swamped by i n t r o d u c e d genotypes, would develop a f t e r a few generations of a c o n t r o l program. F i t z - E a r l e at al._ (1974), using compound chromosomes of D r o s o p h i l a l e l a n o g a s t e r , achieved t o t a l replacement of the wild-type stock from which the compound was d e r i v e d . T h i s was the f i r s t time negative h e t e r o s i s was used to achieve t r u e w i l d -type (as opposed to s t r u c t u r a l l y wild-type) population replacement. 9 l £ S O.slocation I s o l a t i o n Procedures^ A l l the work mentioned to t h i s p o i n t has been done i n i n s e c t s which have p r e v i o u s l y been g e n e t i c a l l y very w e l l c h a r a c t e r i z e d . The development of v i s i b l e g e n e t i c markers and c r o s s - o v e r suppressors would cause a g e n e t i c i n s e c t c o n t r o l p r o j e c t i n most pests t o be delayed by s e v e r a l years. Because of such time c o n s i d e r a t i o n s , the work repo r t e d by C u r t i s (1971), with t s e t s e f l i e s , and Wijnands-Stab and Van Heemert (1974), with the onion root-maggot f l y , i s of s p e c i a l i n t e r e s t . C u r t i s (1971) i s o l a t e d t r a n s l o c a t i o n homozygotes i n t s e t s e f l i e s without r e s o r t i n g to the use of g e n e t i c markers. Kijnands-Stab and Van Heemert (1974) i s o l a t e d t r a n s l o c a t i o n s by i r r a d i a t i n g sperm and checking f o r r e d u c t i o n i n progeny numbers i n the F2 g e n e r a t i o n . They were ab l e to confirm t h e i r f i n d i n g s with the s i m p l e s t of c y t o g e n e t i c methods, somatic chromosome p r e p a r a t i o n s . 10 MATERIALS AND METHODS^ Dros o p h i l a p o p u l a t i o n s were kept i n cages (see Appendix 1) s i m i l a r to those f i r s t developed by Bennett (1956). These cages are d e s c r i b e d i n Appendix 1. Each cage contained e i g h t food v i a l s . One food v i a l was r e p l a c e d by a f r e s h one every t h r e e days. During experiments, cages were kept o p e r a t i o n a l f o r from one to two months. In a l l cases standard Oregon-r c o n t r o l p o p u l a t i o n s were i n i t i a t e d at the same time as the experimental p o p u l a t i o n s to a i d i n the d e t e c t i o n of any change i n c o n d i t i o n s t h a t , otherwise, might be construed to be an experimental e f f e c t . H v d r o s o p h i l a S S i ^ n o g a s t e r s t o c k s were obtained from t h r e e s o u r c e s . T r a n s l o c a t e d s t r a i n s , g l a s s y , smudge, and h a i r y , came from Dr. P. T. Ives' l a b o r a t o r y i n Amherst Massachusetts. (The smudge stock i s the same as t h a t used by Robinson and C u r t i s (1974)). A l l other s t o c k s except those r e f e r r e d to as TVR , were provided by Dr. D. Holn^s l a b o r a t o r y at U.B.C. Those s t o c k s l a b e l l e d TVR1 through TVR57 are the t r a n s l o c a t e d s t o c k s which I d e r i v e d from Dr. H o l i e s Oregon-r, wild-type, s t o c k . I i s o l a t e d TVR1 through TVR57 by the standard Drosophila S®I§£23§§i§£ marker system o u t l i n e d i n Figure 1a. These t r a n s l o c a t i o n s were checked f o r homozygous v i a b i l i t y by a s i b l i n g c r o s s between males and females heterozygous f o r the t r a n s l o c a t i o n and a m u l t i p l e - b r e a k , homozygous l e t h a l , c r o s s i n g -Figure //la. A Drosophila melano^aster translocation i d e n t i f i c a t i o n procedure which u t i l i z e s the two recessive eye colour markers, brown (bw), and scarlet ( s t \ These interact to produce the phenotype white eye. When the sperm contributes a 2-3-translocated haploid genome, one half of the progeny never become adults. Figure #lb. A Drosophila melanogaster p u r i f i c a t i o n scheme which u t i l i z e s the "balancer" chromosome, 2L2R Cy bw (dominant phenotype Curly) . When a homozygous viable translocation i s present, unmarked progeny w i l l occur i n the F A generation. F l • F2 Phenotype A l i v e (A) or Dead (D) 2-3-T NON-2-3-T (sperm) + ; + \ bw; s t / ^ bw st w i l d - t y p e A A 1 + + W bw st /' +. s t //\ bw'st s c a r l e t D A I — ; 0 fbw'st 1 I X bw ;st \ Ibw'st • brown D A (ovum) 1 r i i i . |bw.st 1bw'st white A A Pheno type A l i v e (A) or Dead (D) HOMOZYGOUS HOMOZY GOUS LETHAL VIABLE Df(2R) bw In(2L2R)Cy bw'+ + In(2L2R) Cy bw'+ I + + ! +;+ + + J In(2L2R)Cy bw'-lln(2L2R)Cy bw + 1 + ' + + + jIn(2L2R)Cy bw.+ In(2L2R) Cy bw'+ Jln(2L2R)Cy bw'+ w i l d - t y p e C u r l y wing Cur l y wing A 13 over suppressing, i n v e r s i o n , C u r l y - S M I . T h i s i s a "missing c l a s s system" i n which homozygous v i a b l e t r a n s l o c a t e d i n d i v i d u a l s are the on l y progeny produced. (see F i g u r e 1b.) 14 FITNESS OF TRANSLOCATIONCAGE EXPERIMENTS, MEASUREMENT, AND SIMULATION As I have mentioned p r e v i o u s l y , ( L i t e r a t u r e review page 7) many workers i n the f i e l d of g e n e t i c i n s e c t c o n t r o l have assumed that most t r a n s l o c a t i o n s w i l l be f i t enough to be u s e f u l . There i s reason to suspect that t h i s assumption may be f a l s e . The main o b j e c t i v e of my r e s e a r c h i s to determine what p r o p o r t i o n of l a b o r a t o r y i s o l a t e d t r a n s l o c a t i o n s are s u f f i c i e n t l y f i t to ba u s e f u l i n g e n e t i c i n s e c t c o n t r o l . In order to achieve t h i s o b j e c t i v e , three t h i n g s must be done. The f i r s t i s t o dev i s e f i t n e s s measures and to i n c o r p o r a t e them i n t o a s i m u l a t i o n model. Such a model can be used t o p r e d i c t the a c c e p t a b i l i t y or u n a c c e p t a b i l i t y of any t r a n s l o c a t i o n . The second t h i n g that must be done i s to t e s t the r e l i a b i l i t y of the model and of the f i t n e s s measures upon which i t i s based. F i n a l l y i t i s necessary to generate a sample of new unmarked t r a n s l o c a t i o n s and then, using the f i t n e s s measures and the model, determine the pr o p o r t i o n of t r a n s l o c a t i o n s which might be u s e f u l i n g e n e t i c i n s e c t c o n t r o l . Previous Work^ Robinson and C u r t i s (1973) performed f i t n e s s t e s t s on a s i n g l e D r o s o p h i l a melanogaster homozygous v i a b l e t r a n s l o c a t e d s t r a i n . They obtained t h i s s t r a i n , smudge, from Dr. P. T. Ives of Amherst C o l l e g e , Amherst Massachusetts. Robinson and C u r t i s devised two measures of f i t n e s s , egg-production and egg-15 hatch a b i l i t y . U t i l i z i n g these measures of f i t n e s s they constructed a simulation model of competition between the translocated s t r a i n and wild-type f l i e s . Because of discrepancies between the predictions from th e i r discrete-generation computer simulation model and r e s u l t s from caged populations, their measures of f i t n e s s must be regarded as inadequate. The apparent reason for the discrepencies i s that th e i r measures give a gross over-estimate of the true f i t n e s s of the s t r a i n under study. Fitness^. Measures And Models^ I chose to use more complete measures of f i t n e s s . These are (1)carrying capacity in a population cage and (2)maximum production of adult offspring by single females. My basic simulation model (Appendix 3) has four main features. The f i r s t i s that i t i t e r a t e s from one three-day, simulation period to the next. At the end of each i t e r a t i o n a l l adult individuals mate panmictically. These two u n r e a l i s t i c assumptions, 3-day mating period and panmixis, affect the model only i n that they increase the speed of a l l e l e frequency changes occuring in the populations. I t i s unlikely that these assumptions w i l l a l t e r the d i r e c t i o n of selection, and i t i s the d i r e c t i o n rather than the speed with which I am concerned. The second feature i s the simulation of replacement a c t i v i t y over many three-day i t e r a t i o n s . The t h i r d feature determines the carrying capacity which w i l l produce equilibrium 16 gene f r e q u e n c i e s a t a g i v e n t r a n s l o c a t i o n ' s maximum p r o d u c t i o n . F i n a l l y , by p e r f o r m i n g many b i n a r y s e a r c h e s the model can d e s c r i b e an e q u i l i b r i u m l i n e on the f i t n e s s space .above" which the t r a n s l o c a t i o n w i l l r e p l a c e t h e w i l d - t y p e , and below which i t w i l l be e l i m i n a t e d . ( F i g . 2.) M e t h o d o l o g y D e t e r m i n a t i o n Of F i t n e s s Measures^ To e s t i m a t e t h e c a r r y i n g c a p a c i t y of a s t r a i n i n v o l v e s cage e x p e r i m e n t s l a s t i n g about two months. Hence, r e p l i c a t i o n i s time consuming and I l i m i t e d m y s e l f t o t h r e e r e p l i c a t e s per s t r a i n . I n d i v i d u a l r e p l i c a t e s are shown i n F i g u r e 2 as s i n g l e s m a l l d o t s . Smudge" i s u n r e p l i c a t e d . E x p e r i m e n t s t o measure maximum p r o d u c t i o n a r e , however, easy t o perform and c o n s e q u e n t l y were r e p l i c a t e d 9 or more t i m e s . The mean v a l u e s shown i n f i g u r e 2 a r e t h u s based on 9 or more r e p l i c a t e s . E x p e r i m e n t s I n v o l v i n g Marked T r a n s l o c a t i o n s Using the f o u r a v a i l a b l e marked t r a n s l o c a t i o n s , r e p l i c a t e d f i t n e s s measures were determined. Cage p o p u l a t i o n s were i n i t i a t e d w i t h v a r i o u s i n i t i a l f r e q u e n c i e s o f Oregon-r and t r a n s l o c a t e d f l i e s . The e x p e r i m e n t a l cages were i n c u b a t e d f o r one months a t 25 c. At the end o f t h i s p e r i o d t h e cage e x p e r i m e n t s ware t e r m i n a t e d and samples of a d u l t s taken to d e t e r m i n e t r a n s l o c a t i o n f r e q u e n c i e s . FIGURE #2. Mean progeny production from single females of five genotypes: SM-smudge, H-hairy, GL-glassey, TVR5-brilliant, and ORR-oregon-r, versus their cage carrying populations. Both axes are expressed as a proportion of wild-type, ORR, performance. 3 replicates of the cage populations are separate dots except in SM where 2 cages were terminated because of mold. The line expresses the modelled prediction above which replacement of a wild population w i l l occur via negative heterosis. 1.0. ORR TVR 5 o o ZD o o ct: Q_ 0.5 J .. . \GL SM . H 0.0 0.0 0e5 CAGE POPULATION 19 Experiments I n v o l v i n g 0nraark§d T r a n s l o c a t i o n s ^ T o create new translocations, I t r e a t e d males with gamma radiation ( 1 0 0 0 rd.) and collected progeny from the f i f t h and sixth days of exhaustive mating. I detected new translocations by the procedure outlined in F i g u r e 1 a . T h o s e translocations which were homozygous viable, (as d e t e c t e d by using the system out-lined in F i g u r e 2 b . ) were sorted on the basis of two preliminary measures of fitness. ( S e e F i g u r e 3 . ) T h e f i r s t measure of fitness i s the number of progeny produced when a wild-type female is mated t o the translocation heterozygote. T h e number of progeny produced should be inversely related to the usefulness of the translocation. T h e second measure of fitness i s the ratio'of translocation homozygote to wild-type progeny from a sib-cross of translocation heterozygotes ( F i g . 3 ) . T h e heterozygote contains a homozygous lethal multiple-break-inversion and thus the expected proportions of translocation homozygotes to heterozygotes is 1 : 2 . S o the expected position for a f u l l y viable translocation i s . 5 on the "y" axis of F i g u r e 3 . I consider this to be an indirect measure of translocation homozygote v i a b i l i t y . I took a sample of five suitable homozygous viable translocations, as judged by two preliminary fitness c r i t e r i a : ( 1 ) proportion of translocation homozygotes recovered from translocation heterozygote sib crosses, and ( 2 ) the number of progeny obtained from crosses of wild females with male Figure #3. Locations of the homozygous v i a b l e t r a n s l o c a t i o n s on the plane formed by two p r e l i m i n a r y f i t n e s s measures. The two measures are: (X-axis) number of o f f s p r i n g produced by a s i n g l e w i l d karyotype female when mated to a T/+ male (no r e p l i c a t e s ) . , (Y-axis) r a t i o of T/T to T/+ progeny from a T/+ by.T/+ c r o s s , where + c a r r i e s a r e c e s s i v e l e t h a l (0 - § r e p l i c a t e s ) . . PRODUCTION OF T/T .FROM T/+ X.TA MATINGS o o o cn X ~0 o o c o CO o CD O 0 CO CO a CD « cn co 9 o co CO \1 a CO CO a CO 4 ^ s CO K> CO co 22 t r a n s l o c a t i o n h a t e r o z y g o t e s . These homozygous v i a b l e t r a n s l o c a t i o n s were chosen i n such a way t h a t they would cover a range of the two primary f i t n e s s measures. The' f i v e t r a n s l o c a t i o n s , TVR's 10, 19, 26, 34, and 39 were t e s t e d using the more comprehensive f i t n e s s measures o u t l i n e d i n F i t n e s s : Measures and Models. I compared these r e s u l t s with r e s u l t s from w i l d - t y p e c o n t r o l s and checked f o r s i g n i f i c a n t d i f f e r e n c e s . A l l f i v e homozygous t r a n s l o c a t i o n s chosen, were t e s t e d f o r t h e i r a b i l i t y to compete with the wild-type Oregon-r stock from which they had been d e r i v e d . To t h i s end, cages were i n i t i a t e d with 13 p a i r s of t r a n s l o c a t i o n homozygotes and two p a i r s of normals. Cages i n i t i a t e d with the reverse r a t i o were a l s o maintained. At the end of a one month i n c u b a t i o n p e r i o d , males from a l l experimental cages were sampled to determine the t r a n s l o c a t i o n f r e q u e n c i e s . The sampling procedure was i d e n t i c a l to the procedure f o r the recovery of t r a n s l o c a t i o n s ( F i g . 1), except t h a t f i v e progeny from each male were c o l l e c t e d and checked f o r karyotype. In t h i s way the o r i g i n a l male's genotype could be i n f e r r e d (Table 1) . 23 TABLE 2 i Number of sampled progeny of v a r i o u s genotypes and r e s u l t i n g c o n c l u s i o n s as to male parent's genotype. O r i g i n a l B a l e r s Kar^otjpe-Kary.otv.Ee 2-3 zT Non-2-3 zT T/T 5 0 +/+ 0 5 +/T 1 to 4 1 to 4 A p r e l i m i n a r y estimate of the frequency of the two karyotypes i n the o r i g i n a l p o p u l a t i o n can be obtained before the above procedure has been done. Because of the reduced number of progeny produced when one parent i s a t r a n s l o c a t i o n heterozygote ( F i g . 5), production of progeny from the sample-male X wild-karyotype-female c r o s s together with c y t o g e n e t i c subsamples (Fi g . 4) of l a r v a e from v i a l s with v a r i o u s numbers of progeny, can g i v e us such an estimate (Data of t h i s type i s presented i n Table 4.). (The sampling procedure i s o u t l i n e d i n Appendix 4.) Figure if A. An example polytene chromosome pre p a r a t i o n . This shows c l e a r l y that both break p o i n t s are i n euchromatin, though one i s near to the heterochromatin. " 3 -26 R e s u l t s : l xES£il§Sis I n v o l v i n g Marked T r a n s l o c a t i o n s ^ F i g u r e 2 i s a s c a t t e r diagram of mean progeny production versus c a r r y i n g c a p a c i t y f o r each of fo u r marked t r a n s l o c a t i o n s t r a i n s and Oregon-r„ My s i m u l a t i o n model p r e d i c t s that t r a n s l o c a t i o n s which f a l l below the curve i n Figure 2, are in c a p a b l e of r e p l a c i n g w i l d - t y p e p o p u l a t i o n s . Thus the p r e d i c t i o n i s that smudge, h a i r y , and T V R 5 ( b r i l l i a n t ) w i l l be unable to out-compete the w i l d - t y p e . Glassy f a l l s c l o s e to the border l i n e but i s a l s o p r e d i c t e d to be unable to out-compete Oregon-r. The r e s u l t s of my competition cage experiments c l e a r l y v a l i d a t e the model, s i n c e at the end of the competition cage experiments i n a l l cases not one t r a n s l o c a t i o n homozygote appeared i n cage samples of between 50 and 100 f l i e s . These r e s u l t s were found even i n cages where the t r a n s l o c a t i o n was i n t r o d u c e d a t a frequency of 48 p a i r s to 2 wild-type p a i r s . Experiments I n v o l v i n g Unmarked Tr a n s l o c a t i o n s j _ F i g u r e 3 shows t h a t there i s no high c o r r e l a t i o n between the two p r e l i m i n a r y measures of f i t n e s s . Two (TVR19 and TVR9) of the s i x t e e n t r a n s l o c a t i o n s f o r which I have data seem to be at the upper t h e o r e t i c a l l i m i t (.5) of the v i a b i l i t y measure of f i t n e s s . Using my two comprehensive measures of f i t n e s s , production 27 (Table 2) and c a r r y i n g c a p a c i t y (Table 3), there are two s i g n i f i c a n t d e v i a t i o n s between the f i t n e s s of the unmarked t r a n s l o c a t i o n s and the Oregon-r wild-type stock. TVR10 shows a neg a t i v e d e v i a t i o n i n c a r r y i n g c a p a c i t y , while TVR.19 shows a p o s i t i v e d e v i a t i o n i n progeny pr o d u c t i o n . The r e s u l t s of the cage experiments with TVR26 i n d i c a t e d that only about one i n twenty t r a n s l o c a t e d chromosomes remained at the end of a one and one-half month experiment s t a r t e d with an i n i t i a l p o p u l a t i o n t h a t was 90% t r a n s l o c a t e d . P r e l i m i n a r y r e s u l t s of cage experiments i n v o l v i n g the other f o u r t r a n s l o c a t i o n s are presented i n t a b l e 4. These p r e l i m i n a r y r e s u l t s i n d i c a t e that a l l t r a n s l o c a t i o n s except f o r TVR19, are poor competitors with the Oregon-r stock from which they were d e r i v e d . TVR 19, TVR34, TVR39, TVR10, and TVS26 have both of t h e i r b r e a k p o i n t s i n euchromatin. An example of such a break i s shown i n F i g u r e 4. The f i r s t three of these t r a n s l o c a t i o n s are simple two-breakpoint t r a n s l o c a t i o n s , and the l a s t two are m u l t i p l e breakpoint t r a n s l o c a t i o n s . Table 2. Number of progeny produced by s i n g l e females of Oregon-r and f i v e t r a n s l o c a t e d s t o c k s ^ _Oregon =r TVRIC) TVR19 TVR32 TVR34 ^_TVR39 . 42 62 52 37 37 20 32 43 48 16' 38 39 37 54 51 35 56 . 46 48 55 48 38 38 41 45 44 36 37 63 36 38 '. 45 36 30 38 47 35 41 . 54 29 46 44 38 37 52 26 37 57 28 27 . 54 17 38 46 21 62 36 45 44 53 20 MEAN 37.6 45.4 49.3* 29.5*' 42.8 41.2 S i g n i f i c a n t l y d i f f e r e n t from Oregon-r. Table 3. Number per po p u l a t i o n cage f o r Oregon-r and four t r a n s l o c a t e d s t o c k s . Twenty p a i r s of f l i e s were used t o ' i n i t i a t e each population. The populations were censused a f t e r i n c u b a t i o n f o r one month at 25^ C. Oregon-r TVR10 TVR19 TVR34 TVR39 1406 1315 1046 1203 1-EAN 1242.5 850 1380 1155 1119 1086 900 757 I860 707 993 1089 1082 " 1257 1389 1012 . S i g n i f i c a n t l y d i f f e r e n t from Oregon-r Table 4. Number of i n d i v i d u a l s of each of three p o s s i b l e karyotypes i n samples from population cages i n i t i a t e d w i t h 18 p a i r s of the t r a n s l o c a t i o n and.2 p a i r s w i l d - t y p e . Populations were incubated f o r one month at 25 C. T r a n s l o c a t i o n TVR 10 TVR 19 TVR34 TVR39 -T/T 16 28 30 25 Karyotype T/+ 4 3 6 8 +/+ 6 2 5 15 Number of 16 7 16 38 w i l d a l l e l s T o t a l a l l e l s sampled 52 66 80 96 Frequency of w i l d a l l e l s .31 .11 .2 .26 31 D i s c u s s i o n ^ Since the f o u r marked t r a n s l o c a t i o n s and TVR10 were shown to be s i g n i f i c a n t l y l e s s f i t than Oregon-r and s i n c e these f i v e t r a n s l o c a t i o n s have been shown to be poor competitors, I conclude that my f i t n e s s measures are an improvement over the p r e v i o u s l y used measures; egg-production and h a t c h a b i l i t y (Robinson and C u r t i s 1974). (TVR26 was not s u b j e c t e d to these two f i t n e s s measures because s c a r l e t markers were i n a d v e r t a n t l y i n c o r p o r a t e d i n t o i t d u r i n g the i n i t i a l i s o l a t i o n phase, l i k e l y by male somatic c r o s s i n g - o v e r ) . However TVR34 and TVS39 a l s o appear to be at a competitive disadvantage i n competition with Oregon-r even though my f i t n e s s t e s t s i n d i c a t e d t hat n e i t h e r one i s of s i g n i f i c a n t l y lower f i t n e s s than oregon-r. In these two cases the p r e l i m i n a r y f i t n e s s measures appear t o be more r e l i a b l e . I t i s i n t e r e s t i n g t h a t , of the 57 t r a n s l o c a t i o n s i s o l a t e d , only 21 were v i a b l e i n the homozygous s t a t e . Of these, only one was shown to be a good competitor with the Oregon-r type. I t may be t h a t TVR9 would have done as well as TVR19 had TVR9 been as i n t e n s i v e l y t e s t e d . Thus the r e s u l t s of my cage experiments suggest that only about two i n twenty homozygous v i a b l e t r a n s l o c a t i o n s are worth t e s t i n g i n a f i e l d s i t u a t i o n . I t i s not s u r p r i s i n g t h a t the f i t n e s s measures used by Robinson and C u r t i s (1974) gave over-estimates of the true f i t n e s s of s t r a i n s . One would expect egg p r o d u c t i o n and h a t c h a b i l i t y to overestimate f i t n e s s s i n c e these measures are 32 affactad by only a small portion of the insect's l i f e - c y c l e . It should be mentioned at t h i s juncture that, in cage experiments la s t i n g one month one cannot expect negative heterosis to have a strong e f f e c t , since only two f u l l generations of incubation elapse before termination of the experiments and heterozygotes are not produced u n t i l the end of the second generation. Thus heterozygotes, upon which negative hetarosis acts, are not produced u n t i l very near the end of each experiment. 33 TRANSLOCATION ISOLATION PROCEDURES! The use of t r a n s l o c a t i o n procedures has a tremendous advantage over other g e n e t i c i n s e c t c o n t r o l methods i n that the needed g e n e t i c rearrangements can be i s o l a t e d without the use of v i s i b l e markers, c r o s s - o v e r - s u p p r e s s o r s or e x t e n s i v e c y t o g e n e t i c s (Wijnands-Stab and Van Heemert (1971) and C u r t i s (1971) ) . The technique used i s to i r r a d i a t e males and mate them to untreated v i r g i n females. The progeny are then crossed to untreated f l i e s and c r o s s e s i n which there i s lew progeny pr o d u c t i o n are suspected of c a r r y i n g t r a n s l o c a t i o n s . The suspected l i n e s are then examined c y t o l o g i c a l l y , { Wijnands-Stab and Van Heemert (1974) or t e s t c r o s s e d , ( C u r t i s 1971), to determine i f a t r a n s l o c a t i o n i s indeed present. M a t e r i a l s And Methods I t r e a t e d Oregon-r Droso£hila melanoc[aster males with 1000 r d . of gamma r a d i a t i o n and then mated them to k a r y o t y p i c a l l y wild-type females homozygous f o r the r e c e s s i v e markers brown (chromosome 2) and scarlet(chromosome 3). Then I back-crossed the progeny to the maternal s t r a i n . Any 2-3 t r a n s l o c a t e d s t o c k s can be i d e n t i f i e d by an i n s e p a r a b l e l i n k a g e of the brown and s c a r l e t markers. By t h i s procedure a l l 2-3 t r a n s l o c a t i o n s produced are picked up i n the heterozygous s t a t e . T h i s should account f o r about f o u r - f i f t h s of a l l t r a n s l o c a t i o n s produced. 34 Progeny p r o d u c t i o n of the t r a n s l o c a t i o n s was e s t i m a t e d at the time they were p i c k e d up and thus we can t e s t the u s e f u l n e s s of a progeny r e d u c t i o n scheme of t r a n s l o c a t i o n i d e n t i f i c a t i o n . R e s u l t s ^ There i s a marked r e d u c t i o n i n progeny numbers when t r a n s l o c a t i o n h e t e r o z y g o t e male p a r e n t s are mated to wi ld k a r y o t y p e females ( F i g . 5 ) . The mean p r o d u c t i o n per mating f o r n o n - t r a n s l o c a t e d males mated to w i l d females was 55 while the mean p r o d u c t i o n of a d u l t s f o r 2-3 t r a n s l o c a t e d males was 35. Two q u e s t i o n s r e m a i n . what p r o p o r t i o n of the t r a n s l o c a t i o n s produced c o u l d be d e t e c t e d by n o t i n g a r e d u c t i o n i n progeny numbers? Secondly how can we maximize the number or q u a l i t y of t r a n s l o c a t i o n s produced per u n i t e f f o r t ? F i g u r e 5 demonstrates t h a t t h e r e i s a c o n s i d e r a b l e d i f f e r e n c e i n the number of progeny p r o d u c e d , between t r a n s l o c a t i o n h e t e r o z y g o t e males and normal males . U n f o r t u n a t e l y t r a n s l o c a t i o n s made up o n l y a maximum of 4 p e r c e n t of the genomes t r e a t e d . (It i s wise not to attempt t o i n c r e a s e the p r o p o r t i o n of t r a n s l o c a t i o n s by i n c r e a s i n g the gamma r a y dose because l a r g e numbers of r e c e s s i v e l e t h a l s are p r o d u c e d . ) Thus i f F i g u r e 5 i s m o d i f i e d to account f o r t h i s and the t r a n s l o c a t e d progeny are added to the n o r m a l i z e d v e r s i o n of the h i s t o g r a m , those c r o s s e s i n d i c a t i n g the presence of a t r a n s l o c a t i o n become much harder to s e p a r a t e , F i g . 6. F i g u r e 7, d e r i v e d from F i g u r e 6, g i v e s the r e l a t i o n s h i p FIGURE #5. Histograms of frequency versus .the progeny produced per k a r y o t y p i c a l l y w i l d female mated to males e i t h e r , non-2-3-translocated ( c o n t r o l , t o p graph), or 2 - 3 - t r a n s l o c a t i o n heterozygotes (experimental, bottom graph). Data represents 47 t r a n s l o c a t i o n s and t h e i r c o n t r o l s . 3 6 N U M B E R O B S E R V E D J M13 E R O B S E R V E D O t -CD ~'J .. i. J i \ • , 1 ' ' I <• i ' i t:_) J J ' i i ' i n r rn CD rn - i I'M U r i r-1 -/ C ) o - I r n :< "D f" ' f n i j i UJ i") i " " o T) I ) in : n r" 111 LO K ~V> — l M I o o o n rn T) r" i n (./•) r n i • i FIGURE #6. Histogram of frequency versus number of progeny produced'per k a r y o t y p i c a l l y w i l d female mated to non-2-3-translocated males (data expanded from f i g u r e 5 £ to account f o r sample s i z e ) , and mated to 2 - 3 - t r a n s l o c a t i o n heterozygote males (shaded area). NUMBER OBSERVED o o o I N J CD ' (ZD-CD CD O O LO CD CD X> r _ r~ rn m ui to D —i r" -< o m X) —i rn O FIGURE ill. ' Graph of p r o p o r t i o n of t r a n s l o c a t i o n s that w i l l be i d e n t i f i e d versus the proportion of treated genomes that the i n v e s t i g a t o r examines. PROPORTION OF TRRNSLOCRTIONS IDENTIFIED f l between the c u m u l a t i v e p r o p o r t i o n of a l l t r a n s l o c a t i o n s which would be d e t e c t e d and the cummulative p r o p o r t i o n of the t r e a t e d genomes examined.. From t h i s r e l a t i o n s h i p i t i s apparent t h a t most t r a n s l o c a t i o n s can be i s o l a t e d from those matinys y i e l d i n g f e w e s t progeny. The b e s t s t r a t e g y f o r i s o l a t i n g t r a n s l o c a t i o n s when s u i t a b l e markers are not a v a i l a b l e i s dependent on two f e a t u r e s of t h e pest i n q u e s t i o n . These are the e f f o r t r e q u i r e d t o r a i s e s i n g l e p a i r o f f - s p r i n g and the e f f o r t t o examine the o f f - s p r i n g , e i t h e r c y t o l o g i c a l l y or w i t h s i n g l e p a i r mating e x p e r i m e n t s . I f much e f f o r t i s r e q u i r e d t o make s i n g l e p a i r ma t i n g s then the best s t r a t e g y i s t o c y t o l o y i c a l i y examine a l a r g o p r o p o r t i o n of those matinys y i e l d i n g f e w e st progeny. A l t e r n a t i v e l y , i f the s i n g l e p a i r mating i s easy r e l a t i v e t o the e f f o r t of k a r y o t y p i n g tha progeny, than o n l y genomes from the--most g r e a t l y depressed p r o g e n i e s s h o u l d be examined. I n these rur;pects I agree w i t h the approach taken by w i j u a n d s - S t a b and Van Heeinert (1974) and C u r t i s (1971). S i n c e t h e r e appears to be no c o r r e l a t i o n between my two p r e l i m i n a r y measures of f i t n e s s , i t i s q u i t e p o s s i b l e t h a t soma vary f i t t r a n s l o c a t i o n s may come from the extreme l e f t t a i l of the d i s t r i b u t i o n F i g u r e 6. Wijnands-St-ib and Van Heeinert (1974) and C u r t i s (19/1) .chose to i g n o r e the extreme d e v i a n t s and p r o b a b l y missed p r o m i s i n g t r a n s l o c a t i o n s . 42 BIBLIOGRAPHY: Bennett, J.(1965) Inexpensive P o p u l a t i o n Cages. D. I.S., V o l . 30, Page 159. C u r t i s , C. F. (1968), P o s s i b l e Use Of T r a n s l o c a t i o n s To Fix D e s i r a b l e Genes In I n s e c t P o p u l a t i o n s . Nature, V o l . 218, Page 368. C u r t i s , C. F. (1971) Experiments On Breeding T r a n s l o c a t i o n Homozygotes In Ts e t s e F l i e s . In S t e r i l i t y P r i n c i p _ l e _ f o r I n s e c t C o n t r o l _ o r E r a d i c a t i o n IAEA Page C u r t i s , C. F. and H i l l , W. G. (1971) T h e o r e t i c a l S t u d i e s On The Use Of T r a n s l o c a t i o n s For The C o n t r o l Of Tsetse F l i e s And Other Disease V e c t o r s . T h e o r e t i c a l P o p u l a t i o n B i o l o g y , V o l . 2, Page 71. C u r t i s , C. F. and Robinson, A. S. (1972) Computer S i m u l a t i o n Of The Use Of Double T r a n s l o c a t i o n s F o r Pest C o n t r o l , Genetics, V o l . 69, Page 97, September /72. F i t z - E a r l e , M., Holm, D. G., and Suzuki, D. T. (1974) Genetic C o n t r o l Of I n s e c t P o p u l a t i o n s , G e n e t i c s , V o l . 74, Page 461. F o s t e r , G. G. , Whitten, M. J . , Prout, T., G i l l , R. (1972) Chromosome Rearrangements For The C o n t r o l Of Insect Pests, Science V o l . 137, Page 875, May 72. F o s t e r , G. G. and Whitten, M. J . (1974) The Development Of Genetic Methods Of C o n t r o l i n g The A u s t r a l i a n Sheep Blo w f l y , L u c i l i a cujgrina In The Use Of Gen e t i c s In Insec t Control.. Ed. by R. Pal and M. J . Whitten. Frydenberg, 0. (1962) E s t i m a t i o n Of Some G e n e t i c a l And V i t a l S t a t i s t i c s Parameters Of Bennett Po p u l a t i o n s Of i^rosop_hila £elanogaster, Hereditas, V o l . 48, Page 83. King, C. E. and Anderson, W. W. (1971) A g e - S p e c i f i c S e l e c t i o n . I I . The I n t e r a c t i o n Between r And k During P o p u l a t i o n Growth, American N a t u r a l i s t , V o l . 105, Page 137. Laven, H. , J o s t , E. , Meyer, H., and S e l i n g e r , R. (1971) S e m i s t e r i l i t y For I n s e c t C o n t r o l . In S t e r i l i t y , P r i n c i p _ l e _ f o r I n s e c t C o n t r o l _ o r E r a d i c a t i o n IAEA Page. 4?57 Laven H. (1969) E r a d i c a t i n g Mosquitoes Using T r a n s l o c a t i o n s . Nature, Vol.221, Page 958. McDonald, I. C. and Overland, D. E. (1973a) Use Of An I n v e r s i o n To F a c i l i t a t e The Recovery Of T r a n s l o c a t i o n 43 Homozygotes And To Reduce Genetic Recombination On T r a n s l o c a t e d T h i r d Chromosomes. J o u r n a l Of Heredity, Vol.64, Page 247. HcDonald, I. C. and Overland, D. E.(1973b) I s o l a t i o n Of A Heat-S e n s i t i v e T r a n s l o c a t i o n Homozygote. J o u r n a l Of Heredity, V o l . 64, Page 253. Hoordink, J . Ph. W. (1972) I r r a d i a t i o n , Competitiveness And The Use Of R a d i o i s o t o p e s In S t e r i l e - H a l e S t u d i e s With The Onion F l y , Hylenry_a Antigua ( Meigen) , IAEA-SM-133/62, Page 323. P a l , R. and LaChance, L. E. (1974) The O p e r a t i o n a l F e a s a b i l i t y Of Genetic Methods For The C o n t r o l Of I n s e c t s Of Medical And V e t e r i n a r y Importance. Annual Review Of Entomology, V o l . 19. Page 269. Ray, K. S. And McDonald, P. T. Chromosomal T r a n s l o c a t i o n s And Genetic C o n t r o l Of Ades a e g y p t i . In S t e r i l i t y EEi2£iP.I e_f° r I n s e c t C o n t r o l _ o r E r a d i c a t i o n IAEA Page 437._ Robinson, A. S. And C u r t i s , C. F. ( 1973) C o n t r o l e d Crosses And Cage Experiments With A T r a n s l o c a t i o n In Drosophila. Genetica, V o l . 44, Page 591 . S e r e b r o v s k i i , A. S. (1940) On The P o s s i b i l i t y Of A New Method For The C o n t r o l Of I n s e c t Pests, Z o o l o g i c h e s k i i Zhurnal 19(4) Page 618, T r a n s l a t e d By C. F. C u r t i s . Smith, R. H. and von B o r s t e l , R. C. (1972) Genetic C o n t r o l Of In s e c t P o p u l a t i o n s , S c i e n c e , V o l . 178, Page 1164. Wagoner, D. E. , Morgan, P. B., LaBrecgue, G. C , and Johnson, 0. A. (1973) Genetic Manipulation Used Against A F i e l d P o p u l a t i o n Of House F l i e s . 1. Males Bearing A Heterozygous T r a n s l o c a t i o n , Envionmental Entomology, Vo l . 2, Page 128. Wagoner, D. E., McDonald, I. C., and C h i l d r e s s , D. (1974) The Present Status Of Genetic C o n t r o l In The House F l y , Musca domestica L. i n The Use Of Genetics In Insect C o n t r o l Id. by R. P a l and M. J . Whitten. ~ Wagoner, D. E., N i c k e l , A., and Johnson, A . (1969) Chromosomal T r a n s l o c a t i o n Heterozygotes In The House F l y , J o u r n a l Of Heredity, V o l . 60, Page 301. Wehrhahn, C. F. (1973) An Approach To Modelling S p a t i a l l y Heterogeneus P o p u l a t i o n s And The S i m u l a t i o n Of P o p u l a t i o n s Subject To I n s e c t Release Programs, i n Computer Models And A p p l i c a t i o n Of The S t e r i l e Male Technigue^ IAEA-PL-466/3, Page 45. Wehrhahn, C. F. and K l a s s e n , W. (1971) Genetic Insect C o n t r o l 44 Methods I n v o l v i n g The Release Of R e l a t i v e l y Few L a b o r a t o r y - R e a r e d I n s e c t s . The Canadian E n t o m o l o g i s t , V o l . 103, Page 1387. W h i t t e n , M. S. And P a l , R. (1974) I n t r o d u c t i o n To The Use Of G e n e t i c s In I n s e c t C o n t r o l E d . by R. P a l and M. J . W h i t t e n . W h i t t e n , M. J . (1974) G e n e t i c s Of P e s t s In T h e i r Management, i n Concepts Of P e s t Management Ed . R. Rabb And F. E. G u t h r i e . W h i t t e n , M. S. (1b971) I n s e c t C o n t r o l By G e n e t i c M a n i p u l a t i o n Of N a t u r a l P o p u l a t i o n s . S c i e n c e , V o l . 171, Page 682. W h i t t e n , M. S. (1971b) Use Of Chromosome Rearrangements F o r M o s q u i t o C o n t r o l . In S t e r i l i t y P r i n c i . £ l e _ f o r I n s e c t C o n t r o l _ o r E r a d i c a t i o n IAEA Page 399.. W i j n a n d s - S t a b , K. J . A. and Van Heemert, C . (1974) R a d i a t i o n Induced S e m i - S t e r i l i t y F o r G e n e t i c C o n t r o l Purposes In The Onion F l y Hylemya _anticjua ( Meigen) T h e o r e t i c a l And A p p l i e d G e n e t i c s ? V o l . 44? Page 111. 45 APPENDIX Jk A CAGE FOR DROSOFHILA POPULATION STUDIES Bennett (1956) in t r o d u c e d an in e x p e n s i v e B r o s o p h i l a p o p u l a t i o n cage which c o n s i s t e d of a s m a l l square p l a s t i c f r e e z e r c o n t a i n e r with a removable l i d . T h i s was modified by i n s e r t i n g f o u r holes on each of two opp o s i t e s i d e s along with two a i r ho l e s . T h i s cage, though a gre a t advance over previous wooden models, has two drawbacks. F i r s t l y , removal of a d u l t f l i e s i s an i n e f f i c i e n t and l a b o r i o u s t a s k ; and secondly, i n my experience, the cage w a l l s i n v a r i a b l y develop c r a c k s around the food v i a l h o l e s , a l l o w i n g f l i e s to escape. I have developed a new cage ( F i g . 8) which s o l v e s both of these problems, and i s cheaper than the p l e x i g l a s s cages used by F i t z - E a r l e t al... (1974). Each cage c o n s i s t s of a 2 qt. D a i r y l a n d p l a s t i c milk b o t t l e . E i g h t h o l e s are placed i n two rows on one s i d e and at l e a s t two a i r holes covered i n f i n e mesh are bored i n the adjacent s i d e s . As with Bennett cages, one food v i a l i s removed once every two to f o u r days. My cages support s i m i l a r a d u l t p o p u l a t i o n s to those observed by Frydenberg (1962), eg. 1000 to 2000 a d u l t s . The a d u l t s can be as e a s i l y removed by banging the open cage i n t o an e t h e r i z e r . Twenty-five of these cages have been i n use f o r the past two years, but only one has cracked around the v i a l openings. Figure ;'/8. A s m a l l , economical Drosophila population m i l k - j u g w i t h eight holes, s u i t a b l y s i z e d for two a i r holes- are bored i n the adjacent sides cage. I t c o n s i s t s of a 2qt. p l a s t i c food v i a l s , bored i n one s i d e . At l e a s t and covered w i t h f i n e f l y - p r o o f mesh. 48 APPENDIX 21 A FAST, ACCURATE, ELECTRONIC FLY COUNTER:_ T h i s c o u n t e r was developed t o s o l v e the problem of c o u n t i n g l a r g e numbers of l i v e e t h e r i s e d D r o s o p h i l a where c o u n t i n g by hand r e q u i r e s such a l o n g time p e r i o d t h a t heavy m o r t a l i t y almost always r e s u l t s . The a p p a r a t u s was c o n s t r u c t e d and p e r f e c t e d (with the g r e a t a s s i s t a n c e of A. Reid) over the c o u r s e o f the two year s t u d y a t a c o s t o f a p p r o x i m a t e l y two hundred d o l l a r s . I t c o n s i s t s o f t h r e e b a s i c p a r t s ( F i g . 9 ) : (1) a photo-t r a n s i s t o r and l i g h t beam through which t h e f l i e s a r e passed v i a a vacuum a p p a r a t u s , (2) a p r e - a m p l i f i e r which i n c r e a s e s the d i f f e r e n c e i n r e s i s t a n c e caused by f l u c t u a t i o n s i n beam i n t e n s i t y d e t e c t e d by the photo t r a n s i s t o r , and (3) an e l e c t r o n i c beam i n t e r r u p t i o n c o u n t e r . The a c c u r a c y a c h i e v e d by the c o u n t e r i s dependent on the u n i f o r m i t y i n s i z e of t h e i n s e c t s b e i n g counted, as w e l l as the l i k e l i h o o d o f t h e i r s t i c k i n g t o g e t h e r . A t e s t o f a c c u r a c y i s r e p o r t e d i n T a b l e 5. As can be seen from t h e s e r e s u l t s , the c o u n t e r never o v e r - e s t i m a t e s the number of i n s e c t s passed through the l i g h t beam but o f t e n w i l l s l i g h t l y u n d e r - e s t i m a t e t h e c o r r e c t count. The e x t e n t of t h i s e f f e c t v a r i e s w i t h the s i z e v a r i a b i l i t y and s t i c k y n e s s o f the i n s e c t s b e i n g counted. The e f f e c t can l a r g e l y be removed by d o i n g a s m a l l sample hand count and machine count to e s t a b l i s h a c o n v e r s i o n f a c t o r . (See T a b l e 2.) S T A B L E 5. COMPARISON OF EXACT HAND COUNTS OF F L I E S AND COUNTS I N D I C A T E D BY MX .ELECTRONIC BEAM-INT ERUPT ION COUNTER. EXACT E L E C T R O N I C . D**2 CORRECTED D2**2 HAND COUNT E L E C T R O N I C COUNT . . COUNT 37 3 5 4. 00 3 6 . 16 0.70 1 6 16 0.00 1 6 . 5 3 0.28 3 5 35 0.00 3 6 . 16 1.35 38 36 4.00 3 7 . 19 0.65 3 7 37 0. 00 3 8 . 2 3 1.51 3 0 3 0 0. 00 3 1 . 0 0 0.99 29 27 4.00 2 7 . 9 0 1 .22 26 2 5 1 . 00 2 5 . 83 0.0 3' 17 17 0.00 17. 56 0. 32 62 61 1 .00 6 3 . 0 2 1.05 43 4 0 9. 00 4 1.33 2 .80 54 51 9.00 5 2 . 6 9 1.71 5 5 5 1 16. 00 5 2 . 69 5.33 44 44 0.00 45 . 4 6 2. 13 45 42 9. 00 4 3 . 39 2.58 4 1 3 9 4 . 00 4 0 . 2 9 0.50 27 26 1.00 2 6 . 86 0.02 34 34 0.00 3 5 . 13 1.27 3 8 36 4. 00 3 7 . 19 0.65 56 54 4 .00 5 5 . 7 9 0.04 38 38 0. 00 39 . 2 6 1 .59 63 62 1.00 6 4 . 0 6 1.12 38 38 ' . 0.00 3 9 . 26 1 .59 TOTALS 9 0 3 . 8 7 4 7 1 . 0 0 9 0 3 . 0 0 2 9 . 4 2 5 8 . 6 % o f s q u a r e d d e v i a t i o n s a c c o u n t e d f o r by c o r r e c t e d c o u n t . 53 51 4. 00 5 2 . ,69 0. 09 73 7 1 4. 00 73. , 3 6 0. 13 6 7 67 0. 00 6 9 . ,22 4. 94 5 5 , 5 2 9. 00 53. ,73 1 . 62 7 9 7 8 1. 00 8 0 . , 59 2. 52 97 9 1 3 6 . 00 94. .02 8. 88 9 9 9 5 1 6 . 00 9 8 . , 15 0. 72 86 8 1 2 5 . 00 83. . 69 5. 35 6 1 60 1. 00 61 , , 99 0. 98 94 90 16. 00 92. . 99 1 . 03 10 1 1 0 0 1 . 00 1 0 3 . ,32 5. 37 3 8 38 0. 00 39. , 26 1 . 59 T OTALS 9 0 3 8 7 4 1 1 3 . 00 9 0 3 . . 00 3 3 . 23 10% o f s q u a r e d d e v i a t i o n s a c c o u n t e d f o r b y c o r r e c t e d c o u n t Figure 119. A block diagram of a l i g h t beam-interruption i n s e c t counter. Insects move through the beam from the l i g h t source i n an a i r stream, causing v a r r i a t i o n i n the l i g h t i n t e n s i t y reaching the photo sensor. This variarion.produces a v a r r i a t i o n i n the r e s i s t a n c e of the photo sensor which i s a m p l i f i e d and f i n a l l y counted as a beam i n t e r r u p t i o n . BEAM INTERRUPTION INSECT COUNTER A-L I G H T I N T E N S I T Y C O N T R O L LIGHT S O U R C E A-B^ C* REGULATED POWER SUPPLY 3> - - i!ZE3 P H O T O S E N S O R VIM AMPLIFIER r -B— C — COUNTER Figure #10. . • Photo of l i g h t beam-interruption i n s e c t counter. Insects move through the system from r i g h t to l e f t i n t e r r u p t i n g the l i g h t beam between the l i g h t source and the photo sensor, the i n s e c t s then.pass i n t o the c o l l e c t i n g b o t t l e at l e f t . L I G H T S O U R C E 54 APPENDIX 3. SIMULATION OF SEMI-STERILE GENETIC SYSTEMS. B a s i c Algorithm R e a l i s t i c s i m u l a t i o n models of semi-incompatible g e n e t i c systems, such as wild p o p u l a t i o n s under a t r a n s l o c a t i o n r e l e a s e program, q u i c k l y become completely i n t r a c t a b l e . Therefore I have developed a new modelling system s p e c i f i c a l l y designed to handle such problems. The b a s i s of t h i s system i s a Markovian t i m e - i t e r a t i o n procedure which u t i l i z e s an INCOMPATIBILITY MATRIX, a type of t r a n s i t i o n matrix formed by the s e r i a l a d d i t i o n of I n c o m p a t i b i l i t y V e c t o r s , Also r e q u i r e d i s simple, f o r t r a n d e f i n e d f u n c t i o n t o convert the r e s u l t s i n t o new animals or nonsense (incomplete) animals. As an example, the I n c o m p a t i b i l i t y Matrix f o r the simple case of one wild-type genome and one homozygous v i a b l e t r a n s l o c a t i o n i s c o n s t r u c t e d as f o l l o w s : IH£21£§.£ikiiity Vectors f o r one homozygous v i a b l e t r a n s l o c a t i o n ^ +/+ [ 0, 0, 0, 0 ] T/+ [ 0,-3, 4, 1] T/T [ 1, 1, 1, 1 ] The i d e n t i t y numbers of the +/+, T/+, and T/T genotypes are 55 0, 1, and 2 r e s p e c t i v e l y . The f o r t r a n f u n c t i o n i s simply d e f i n e d and has the f o l l o w i n g a c t i o n . FUHCTI0H= F(IR) = F( 1 to 2= same, - i n f i n i t y to -1 = 3, and 3 to i n f i n i t y = 3) Thus a T/+ by T/+ mating g i v e s the f o l l o w i n g INCOMPATIBILITY MATRIX. 0, -3, 4, 1 0, 3, 3, 1 -3,-6,1,-2 3 , 3 , 1 , 3 4, 1, 8, 5 WHICH THE FUNCTION 3, 1, 3, 3 1, -2, 5, 2 CONVERTS INTO 1 , 3 , 3 , 2 A l l 3's appearing i n the matrix i n d i c a t e nonsense or incomplete animals. We can e a s i l y c a l c u l a t e the p r o p o r t i o n s of the three p o s s i b l e genotypes produced by a t r a n s l o c a t i o n heterozygote by t r a n s l o c a t i o n heterozygote mating. These are T/T= 1/16, T/+= 4/16, +/+= 1/16, and l e t h a l = 10/16. Th i s modelling system was used e x t e n s i v e l y to a s s i s t i n choosing those t r a n s l o c a t i o n s most l i k e l y to compete f a v o r a b l y with the wild-type genome. 56 NAME:. CPD TRANS.SIM AUTHOR: K e i t h Reid D a t e : JAN 1, 1974 CPU TRANS.SIM - I t e r a t i v e Homozygous T r a n s l o c a t i o n F i t n e s s Phase-space C o n s t r u c t i o n P r o c e d u r e . I . TYPE OF ROUTINE: M a i n - l i n e program w r i t t e n i n F o r t r a n I I . PURPOSE: To c o n s t r u c t a p h a s e - s p a c e of two f i t n e s s measures, c a r r y i n g c a p a c i t y and maximum progeny p r o d u c t i o n (which can be o b t a i n e d from v a r i o u s g e n e t i c i n s e c t s t r a i n s 9 . The phase space g i v e s t r a n s l o c a t i o n f i t n e s s l e v e l s which w i l l produce u n s t a b l e e q u i l i b r i a between a t r a n s l o c a t e d s t r a i n and the w i l d - t y p e . I I I . RESTRICTIONS: Must be c o n c a t i n a t e d with l i n k : p l o t or the PDP 11 system r o u t i n e s , KGA, KLINE, and KLABL (see l i s t i n g s i n c l u d e d ) . A l s o s h o u l d be run o n ' s y n - 6 (see " o u t p u t s s e c t i o n " ) . I V . VIRTUAL MEMORY: F i t s on the c u r r e n t PDP 11 V. METHOD: I t e r a t i v e s e a r c h over the phase -space to l o c a t e the a r e a s o f u n s t a b l e e q u i l i b r i a . At each Y - f i t n e s s l e v e l a b i n a r y s e a r c h i s used t o l o c a t e the r e l e v a n t X - a x i s l e v e l f o r e q u i l i b r i u m . V I . HOW TO USE: 35RUN *FTN PAR=S=SIM.S L=-L $RUN - L + LINK:PLOT 5= DATA 6=0UTPUT 57 CPU TRANS. S I M "5=data" the data i s to be s t o r e d i n D A T A " 6 = o u t p u t » t h i s i s where the p r i n t e d (as opposed to p l o t t e d ) o u t - p u t goes . 1) I n p u t s : B i r t h r a t e s (min and max - - t r e a t e d d e n s i t y -d e p e n d e n t l y ) , death r a t e s s t a r t i n g p o p u l a t i o n s , c a r r y i n g c a p a c i t i e s , and maximum b i r t h s per i t e r a t i o n . 2) O u t p u t s : A l l o u t p u t , p l o t t e d goes to the s y n - 6 s c r e e n , but i f paper p l o t s are r e q u i r e d , then use "HCOPY" o or c o n c a t i n a t e the run command with w e b b : l i b i n s t e a d of l i n k : p l o t and run p l o t : q par=-plot# a f t e r the f i r s t run i s complete . V I I . ACKNOWLEDGEMENT: No thanks would be too much f o r my good f r i e n d s and b u d d i e s who l i k e to dragg me o f f to the " p i t " . A l s o I would l i k e to thank B i l l Webb and R. Yourk f o r t h e i r h e l p . \ 58 S R U N * F T N P A R = S = M 0 D ( 4 ) L = H O D . O $ R U N W E B B : T R A P + M O D . 0 + P L O T . O + L I N K : P L O T 6 = -SP 5=MOD ( 2 0 0 ) $ E N D D I M E N S I O N T ( 3 , 4 ) D I M E N S I O N T l ( 3 , 4 ) D I M E N S I O N X Y ( 2 , 1) , X Y B ( 2 , 1 5 ) , X Y T ( 2 , 15) C T H E A B O V E I S F O R P L O T P U R P O S E S O N L Y C O MMON D ( 3 ) ,B ( 3 ) C T H E A B O V E A R E D E A T H A N D B I R T H R A T E S F O R T T T P AND P P COMMON DM ( 3 ) C O MMON P O P C C T H E A B O V E A R E T H E S T A T E M A T R I X ( T E N E T I C S T A T E , A G E ) , A ND P T COMMON T K ( 3 ) ,TMA X C A B O V E A R E G E N E T I C S P E C I F I C C A R R Y I N G C A P A C I T I E S , A N D M AX GROW COMMON F I N T C A B O V E I S N U M B E R O F F L I E S I N T R O D U C E D P E R S T E M C T H E A B O V E I S T H E N U M B E R O F T HO M O S I N T R O D U C E D P E R T I M E P E R I O D C O M M O N Y , Y 1 , Y S Y 1 N Y S K = 1 1 2 R E A D ( 5 , 1 0 0 ) B , D, DM , ( ( T ( I , J ) , 1= 1, 3) , J = 1 , 4 ) , T K , T M A X , F I N T W R I T E ( 6 , 1 0 0 ) B , D , D H , ( ( T ( I , J ) ,1 = 1 , 3 ) , J = 1 , 4 ) , T K , T M A X , F I N T 1 0 0 F O R M A T ( 3 F 1 0 . 2 ) I F ( T M A X . E Q . 0 ) GO T O 9 9 9 YM= B ( 1 ) X M = T K ( 1 ) C A L L K G A ( X M , Y M ) X Y ( 1 , 1) = 0 . XY ( 2 , 1) = Y M * 1. 0 5 C A L L K L I N E ( X Y , 1 , - 1 , 1 ) W R I T E ( 7 , 1 0 4 ) D , T M A X , F I N T C A L L P F L A G 1 0 4 F O R M A T ( * D E A T H R T S . D T T = » , F 5 . 3 , ' D T P = ' , F 5 . 3 , ' D P P = « 2 , F 5 , 3 , 1 • M A X YO U N G / I T T . = *, F6,0 , ' T T I N T R O D U C T I O N ^ ' , F 6 . 0 ) C A L L K L A B L ( X M , Y M , 2 ) W R I T E ( 7 , 1 0 7 ) C A L L P F L A G 1 0 7 F O R M A T (* B I R T H R A T E O F T T » ) C A L L K L A B L ( X M , Y M , 1 ) W R I T E ( 7 , 1 0 8 ) C A L L P F L A G 1 0 8 F O R M A T ( ' C A R R Y I N G C A P . O F T T ' ) C A L L F C H A R ( 0 . , Y M , . 1 0 , . 1 5 , 0 . 0 ) C O U T E R M O S T L O O P O V E R T H E I N I T A L S T A R T I N G V A L U E S DO 8 I R = 1 , 4 Q *************** S E T / R E S E T B ( 1 ) B ( 1 ) = B ( 3 ) c *** ****** Z E R O OUT P L O T F I L E S DO 1 1 J = 1 , 1 5 DO 1 1 1 = 1 , 2 X Y B ( I , J ) = 0 . 59 11 XYT(I,J)=0. C BINARY SEARCH OF REAGON BETWEEN TK (3) AND C AND BETWEEN B(3) AND 10. K=0 7 CONTINUE K=K+1 WRITE (6,100)B,TK Q ************** SET RESET TK(1)=TK(3) TT=TK (3) TB=0 . TK (1) =TT C SET UP FLIP-FLOP FF=1 DO 5 1=1,10 C WEE WILL MAKE THE ABOVE NO OF CUTS ON THE BINARY SEARCH DO 1 11=1,3 DO 14 JJ=1,3 14 T1 (II,JJ)=0. 1 T 1 ( 1 1 , 4 ) = T ( I I , I R ) C THE SIMULATE DO DO 2 1 1 1 = 1 , 2 0 0 TEMP=T1 (3,4) CALL K0D(T1) IF ( I I I . LT.40) GO TO 9 IF (T1 (3 ,4) .GE. 10. AND.T1 (3 ,4) .GT.TEMP) GO TO 4 IF (T1 (3,4). LT. LAND. T1 (3,4) .LE. TEMP) GO TO 3 9 CONTINUE 2 CONTINUE WRITE(6,102) ( (T1 (II,J) ,11=1 ,3) ,J=1 ,4) ,TEMP,TT ,TB, TX 102 FORMAT(« FELL OUT OF THE BOTTOM OF THE SIM LOOP T IS=« 2,/12F6.0 1 ,'TEMP,TT,TB,TX»,6F10.5) TX=2. WRITE (6, 103) (T1 (IIII,4) , 1 1 1 1 = 1 , 3) , TEM P , B (1) , TK (1) ,TT,TB,TX 103 FORMAT(11F12.3) XY (2, 1) =B (1 ) XY (1 , 1) =TK (1) CALL KLINE (XY,IR,1,1) IF(TT-TB.LT.10) GO TO 6 C ********* i p THIS HAS BEEN DONE HERE BEFORE —BAILOUT IF (FF + TX.EQ.3) GO TO 6 C ********SO WE GO TO THE UP FF=1 OR DOWN FF=0. TK (1) =(TK (1) - (FF*TT- (FF-1 ) *TB) )/1 . 2 + FF*TT- (FF-1) *T B WRITE (6,100)FF,TK (1) ,TB, TT C ********* FLIPPER IS SET-RESET FF= (FF-1) * (-1) GO TO 5 3 TX=0. C TRANSLOCATION DID MAKE IT C WRITE (6, 103) (Tl (I I I I , 4) , 1 1 1 1 = 1 , 3 ) , TEMP , B (1) ,TK (1) ,TT,TB,TX 60 X Y T ( 2 , K ) = B ( 1 ) X Y T (1 , K ) = T K ( 1 ) T T = T K ( 1 ) I F ( T T - T B . i r . 1 0 ) GO TO 6 T K ( 1 ) = ( T K ( 1 ) - T B ) / 2 . + T B GO T O 5 4 T X = 1 C T H E T R A N S L O C A T I O N D I D N T M A K E I T C W R I T E ( 6 , 1 0 3 ) ( T 1 ( I I I I , 4 ) ,1111=1 , 3 ) , T E M P, B (1 ) , T K ( 1 ) , T T , T B , T X X Y B (1 , K ) = T K ( 1 ) X Y B ( 2 , K ) = B ( 1 ) T B = T K ( 1 ) I F ( T T - T B . L T . 1 0 ) GO TO 6 T K ( 1 ) = ( T K (1 ) - T T ) / 2 . + T T 5 C O N T I N U E 6 B ( 1 ) = B ( 1 ) / 1 . 3 I F ( B ( 1 ) . G T . 9.) GO TO 7 K K = K DO 1 0 K = 2 , K K I F ( X Y B ( 1 , K ) + X Y B ( 2 , K ) . N E . Q ) GO TO 1 3 X Y B ( 1 , K ) = X Y B ( 1 , K - 1 ) X Y B ( 2 , K ) = X Y B ( 2 , K - 1 ) 1 3 C O N T I N U E I F ( X Y T (1 , K) + X Y T ( 2 , K) . N E . 0 ) GO T O 10 X Y T ( 1 ,K) = X Y T (1 , K - 1 ) X Y T ( 2 , K ) = X Y T ( 2 , K - 1 ) 1 0 C O N T I N U E C A L L K L I N E ( X Y B , K K , - 1 , 0 ) C A L L K L I N E ( X Y T , K K , - 1 , 0 ) W R I T E ( 7 , 1 0 6 ) T ( 1 , I R ) ,T ( 3 , I R ) C A L L P F L A G 1 0 6 F O R M A T (• ( T T / P P = « , F 4 . 0 , V V F 4 . 0 , •) •) 8 C O N T I N U E X Y ( 1 , 1 ) = X M * 1. 2 X Y ( 2 , 1 ) = 0 . C A L L K L I N E ( X Y , 1 , - 1 , 1 ) GO TO 1 2 9 9 9 C O N T I N U E W R I T E ( 6 , 1 0 5 ) 1 0 5 F O R M A T ( * J O B D O N E * * * * * * * * * * * * * * * * * * * • / / / / ) C A L L E X I T R E T U R N E N D S U B R O U T I N E K O D ( T ) D I M E N S I O N T ( 3 , 4 ) C O MMON D ( 3 ) , B ( 3 ) C T H E A B O V E A R E D E A T H A N D B I R T H R A T E S F O R T T T P AND P P C O M M O N DM ( 3 ) COMMON P O P C C T H E A B O V E A R E T H E S T A T E M A T R I X ( T E N E T I C S T A T E , A G E ) , AND P O P 61 COMMON TK(3),TMAX C ABOVE ARE GENETIC SPECIFIC CARRYING CAPACITIES,AND MAX GROWTH COMMON FINT C ABOVE IS NUMBER OF FLIES INTRODUCED PER STEM C THE ABOVE IS THE NUMBER OF T HOMOS INTRODUCED PER TIME PERIOD COMMON Y,Y1,YSY1 C DEMINISH ADULTS BY DRT. POP=T (2,4)+T (3,4) +T(1,4) DO 4 1=1,3 4 T (1,4) =T (I, 4) - (D (I)-DM (I) * (1.-POP/TK (I) ) )*T(I,4) C MOVE UP LARVAE DO 1 1=1,3 J = 5-I II=J-1 T (2, J) =T (2, J) +T(2,II) T (3,3) =T (3, J) +T (3, II) T(1,J)=T(1,J)+T(1,II) T (2,11) =0. T (3,11) =0. 1 T(1,II)=0. C HERE WE INTRODUCE THE INTRODUCTION FUDGE FACTOR 1(1,4) =T (1,4) +FINT C ADD ON NEW BABIES T (2,1) = B (2) *T (2,4) * (. 25* (T (2, 4) /POP) +. 125* (T (3, 4) /POP) + 2 .125*(T(1,4)/POP))+ 1 B (1) *T (1,4)* (.5* (T (3, 4) /POP) ) +B (3) *T (3, 4) * (. 5*T (1 ,4) /POP) T (1, 1) =B (1) *T (1 ,4) * ( (T (1 ,4) /POP) + . 123* (T (2,4) /POP) ) 1 +B (2) *T (2, 4) * (. 0625*T (2, 4)/POP+. 125*T (1,4) /POP) T(3,1)=B (3) *T(3,4) * ( (T (3,4)/POP) +. 125* (T (2,4)/POP) ) 1 +B (2) *T (2, 4) * ( (T (2,4) /POP) *. 0625+ (T (3 ,4 )/POP) *. 125) C WE CALCULATE THE EFFECTOF POPULATION LIMITATION ON BIRTHS Y1=T(2,1) +T (1,1) +T(3,1) DO 3 1=1,3 Y=TMAX*(1-POP/TK (I)) IF (Y. GT. Y 1) GO TO 3 IF(Y.LE.O.) Y=.0001 CANT LET THE NUMBER OF LARVAE GO NEGATIVE YSY1=Y/Y1 T (I,1)=T (I, 1)*YSY1 3 CONTINUE RETURN END SENDFILE 80. 80. 80. BIRTH RTS .4 .4 .4 DEATH RTS .3 .3 .3 ABOV - THIS=MIN DEATH RT 98. 0. 2. ADULT STARTING POPS OF TT TP PP 94. 0. 6 ADULT STARTING POPS OF T l TP PP 86. 0. 1 ADULT STARTING POPS OF TT TP PP 70. 0. ADULT STARTING POPS OF TT TP PP 62 2000. 2000. 20 CARRYING CAPACITIES OF TT TP PP 300 . 0. TM AX FINTRO SUBROUTINE KGA(MAXX,MAXY) C* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * C** THE FOLLOWING I S A ROUTINE TO DRAW THE GRAPH AXIES TO A** C** POINTS BETWEEN 0.0 AND THE STATED MAXIMA , MAXX AND M** C** AXIES ARE LABELED WITH ROUNDED OFF VALUES IN F5.2 WHERE ** C** ELSE WHERE 1 E ' FORMAT IS USED ** C** * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * REAL MAXX,MAXY XIN=10. YIN=7. XIN=8. YIN=6. 101 FORMAT(2F10.3) CALL P1130 X=XIN/MAXX Y=YIN/MAXY YP=-MAXY/7.0 XP=-MAXX/5. CALL SCALF(X,Y,XP,YP) C** DRAW X AXIS U=MAXX/5.0 CALL FGRID(0,0.0,0.0,U,5) C** PUT NUMBERS ON X AXIS YN=-MAXY/23. XN=MAXX DO 2 1=1,6 I F (1-6) 3,4,4 4 XN=0.0 3 CALL FCHAR(XN,YN,0.10,0.15,0.0) , I F (MAXX-99. 99) 10, 10, 1 1 10 WRITE(7,102) XN CALL PFLAG 102 FORMAT(F5.2) GO TO 12 11 WRITE(7,100)XN CALL PFLAG 12 CONTINUE C** PUT NUMBERS ON Y AXIS 2 XN=XN-MAXX/5. XN=-MAXX/9. YN=0.000 DO 1 1=1,6 I F ( I - 1 ) 5,6,5 6 YN=0.0 5 CALL FCHAR(XN,YN,.10,.15,0.0) YN=YN*.00005+YN I F (MAXY-99. 99) 7,7,8 7 WRITE (7,102) YN CALL PFLAG 63a GO TO 9 8 WRITE(7,100) YN CALL PFLAG 100 FORMA T(E8.2) 9 CONTINUE YN=YN + MAXY/5. 1 CONTINUE C** DRAW Y AXIS U=MAXY/5 CALL FGRID(3,0.0,MAXY,U,5) RETURN END SUBROUTINE KLINE (XY,NP,J,K) C** * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * C** K CONT ROLES WHETHER A LINE WILL BE PLOTTED BE TWEE ** C** ON ENTRANCE 0=LINE 1=NOLIME ** C** XY 2 BY NP ARRAY OF DATA TO,BE PLOTTED * C** J INDICATES WHAT TYPE OF MARKER TO BE ON THE LIN * C** * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * DIMENSION XY(2,1) X=XY(1,1) Y=XY(2,1) CALL FPLOT (K,X,Y) DO 1 1=1,NP X=XY (1 , 1 ) Y=XY ( 2 , 1 ) CALL FPLOT (0,X,Y) CALL FPLOT(2,X,Y) CALL POINTS (J) 1 CALL FPLOT(K,X,Y) CALL FPLOT(1,X,Y) RETURN END SUBROUTINE KLABL {XM , YM , J) YIN=6. XIN=8. GO TO (1 , 2 ) , J 1 XN=0.0 YN=(-YM/ (1.2*YIM))*.9 D=0. 0 GO TO 3 2 YN=0.0 D=1.5708 XN=(-XM/ (.80*XIN) ) *1 .4 3 CALL FCHAR(XN,YN , .2, .2,D) RETURN END $SIG 63© APPENDIX HJ_ ESTIMATION OF KARYOTYPE WITHOUT MARKERS^ The purpose of t h i s appendix i s to d e s c r i b e a method to estimate the karyotype, i . e . T/T, T/+, +/+, of i n d i v i d u a l male i n s e c t s . Male f l i e s of unknown genotype were s i n g l y mated to females of known wi l d karyotype, and the progeny production recorded. T h i s data was arranged i n the t a b u l a r graphic form of Figure 11, (top f o u r t a b l e s ) . For purposes of normality these top four t a b l e s were summed to produce the bottom t a b l e , ( e n t i t l e d TOTAL) f i g u r e 11, bottom shows three normal d i s t r i b u t i o n s , o v e r l a p p i n g s l i g h t l y i n t h e i r t a i l s . The procedure f o r e s t i m a t i n g genotype i s then to c o n s i d e r as I have t h a t the l e f t - m o s t normal curve i s the r e s u l t of of T/+ X •+/+ matings, the center curve i s the r e s u l t of T/T X +/+ matings, and that the r i g h t - m o s t curve i s the r e s u l t of +/+ X +/+ matings. The assumptions were confirmed by c y t o l o g i c a l methods as f o l l o w s . I examined a high producing TVR10, three medium producers, TVR34, 39, and 19, and examples from a l l f o u r t r a n s l o c a t i o n i n low producers. In a l l i n s t a n c e s the c y t o l o g i c a l methods agree with my assumptions. Using these t e s t e d assumptions and making the f u r t h e r assumption t h a t there w i l l be no " s l o p " about the d i v i d i n g l i n e s , we can c o n s t r u c t the f i n a l t a b l e , t a b l e 4. F i g u r e . 1 1 . Procedure f o r e s t i m a t i o n of karyotype without the use of v i s i b l e marker genes. The 3 mormal d i s t r i b u t i o n s on the bottom a x i s are assumed to be the r e s u l t of T/+, T/T, and +/+ male parents, r e s p e c t i v e l y . R e s ults must be confirmed c y t o l o g i c a l l y . FREQUENCY/DIVSION 6&~ 1 tj D 0 D 5 Dl 5 . D % Dl v D b. D • 0 0 « a o —I > Iv Cu XjJ lo Cw oo a co "3 «0 - f Oi w — < 70 " 1 CD >o cn K> <o fr) ^ t4 © I S JN 1^ t6 to 0 Nl NJ VI Nj *<i «« *> «, CC) oo Ui 0 O CO •fc CU o o -° -e -P -r -f u, >J — oo r-V J . N I 05 <« 0) CO -0 CO 9 o o ° O g o 73 co co c* c - O - Q -e -p- ~f-C _5 w r- r-C A A O C-o °» r. W O N / -•sj <ac* 5 co CO o JN o CO o o o u These r e s u l t s i n d i c a t e t h a t TVR19 i s a f a r s u p e r i o r competitor with the Oregon-r stock than are the other t r a n s l o c a t i o n s . 

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