Genetic stability of sexing strains based on the locus sw of Ceratitis capitata

This report deals with the process of improving the stability of medfly, Ceratitis capitata, genetic sexing strains (GSS) based on the swmutation on chromosome 2. This gene affects the rate of development as well as the eye colour and iridescence. The improved sexing strains were produced by mapping swwith deletions and then inducing and screening for new translocations with breakpoints close to the marker. The stability was assessed in large populations over many generations. Twenty-two new Y-2 translocations were identified and polytene chromosome analysis was performed to locate breakpoints. The translocation strains were ranked according to the distance of their breakpoints from sw. The map position of swis region 20D on 2R. As data on the stability of the 22 strains accumulated, Cast191 was shown to be the most promising as no recombination between swand the male sex was found. After rearing the strain for 22 generations under semi-mass rearing conditions, with a population size of 15,000 adults and scoring 1000 flies per generation, only one such event was detected (estimated frequency = 3.1 × 10^–6). Further tests are being carried out with this strain to assess its suitability as a genetic sexing strain for medfly Sterile insect technique (SIT).     

Recombination between homologous autosomes in medfly (Ceratitis capitata) males: type-1 recombination and the implications for the stability of genetic sexing strains

The sterile insect technique (SIT) is an environmentally safe technology to control insect pests. To improve this technology, genetic sexing strains (GSS) have been developed for the Mediterranean fruit fly, Ceratitis capitata. Such strains are based on Y-autosome translocations linking a selectable marker to the male sex and their long-term stability, especially under large-scale mass rearing conditions, is threatened by genetic recombination in the heterozygous males. We have measured male recombination in order to be able to construct GSS that are more stable. Our results show that male recombination occurs at very low frequencies, that is, below 1% per generation. Furthermore, recombination in medfly males occurs premeiotically. By selecting strains where the Y-autosome translocation breakpoint and the selectable marker are closely linked, the deleterious effects of recombination on the stability of GSS can be minimized. In such strains recombination is reduced by ca. 80% as compared to previously studied GSS. Although recombinants still occur at very low frequencies they still pose a threat to the integrity of the sexing system if they possess a selective advantage. Under mass rearing condition such recombinants will accumulate according to their relative fitness and additional measures, such as improved mass rearing strategies, are required to preserve the accuracy of the sexing system. As a conclusion it is shown that current GSS are stable enough to allow mass rearing at levels exceeding 1000 million male medflies per week.     

Isolation and cytogenetic analyses of genetic sexing strains for the medfly,Ceratitis capitata

Over the last 10 years, several genetic sexing strains have been isolated for the Mediterranean fruit fly, Ceratitis capitata, with the aim of improving the Sterile Insect Technique. However, a major problem with the currently used genetic sexing system, which is based on translocations, is their potential genetic instability. Therefore, careful monitoring and chromosome analyses are necessary when new genetic sexing strains are developed. Instability of a genetic sexing strain can be the consequence of recombination or the survival of aneuploid individuals occurring as a consequence of adjacent-1 segregation in the meiosis of males with Y-autosome translocations. Recently, genetic sexing strains have been isolated that show only low levels of recombination. However, many aneuploid flies are produced by these strains. Therefore, we have made an attempt to isolate new genetic sexing strains that show a low percentage of recombination and no survival of aneuploid individuals. We report their genetic behaviour and the polytene chromosome structure of these new strains.     

Chromosomal inversions and genetic control revisited: the use of inversions in sexing systems for higher Diptera

Summary Genetic sexing systems based on sex-linked translocations and deleterious mutations are subject to breakdown from genetic recombination in males. Including inversions in these strains may provide a solution to this problem, by ensuring selective elimination of recombinant products. Inversions could be used either in coupling to or in repulsion to the translocation. The latter system, requiring homozygous-viable inversions, would be more difficult to construct, but would offer several advantages not available with coupled translocation/inversion systems. A system proposed for the blowfly Lucilia cuprina is outlined, which combines homozygous-viable pericentric inversions in repulsion to existing sex-linked translocations. This system should both stabilize the genetic sexing system and increase the suppressive potential of such strains.     

Genetic effects induced in Sacharomyces cerevisiae by cyclophosphamide in vitro without liver enzyme preparations

Cyclophosphamide induced forward mutation in Saccharomyces cerevisiae strain S288C and mitotic recombination in strains D3 and D5 but not in strain D4. The yeast cells were treated with the compound in phsphate buffer without recourse to metabolic activation protocols. Elevation of the treatment temperature increased the genetic activity of cyclophosphamide. Respiration-deficient isolates of strains S288C and D3 were more sensitive than the respiratory competent parent strains were for inducing forward mutation and mitotic recombination, respectively. Cyclophosphamide was incubated in phosphate buffer alone for increasing time intervals; strain D3 cells were added to aliquots for each time interval and incubated for an additional 30 min. The frequency of induced recombination increased as the time of compound incubation increased, showing that spontaneous degradation of cyclophosphamide to genetically active breakdown products was responsible for the genetic damage induced in the yeast cells.     

Mass rearing of temperature sensitive genetic sexing strains in the Mediterranean fruit fly (Ceratitis capitata)

Genetic sexing strains (GSS) based on the temperature sensitive lethal(tsl) mutation are being used to produce sterile male medflies for large scale sterile insect technique (SIT) programmes for this pest. The use of male-only strains increases the overall efficiency of the technique. Currently more than 1.4 billion sterile male-only pupae are produced per week in different facilities around the world. Due to the mutations used to construct these strains, that is, translocations and selectable markers, they require different and more careful mass rearing procedures than do bisexual strains (BSS). The basic rearing technology has been developed and can be used to produce only males on a predictable basis to a level of 99.9% accuracy. If specific rearing procedures are followed, then tsl-based GSS has a rearing efficiency that is equal to that of a BSS and it is already know that males produced by the tsl-based GSS are of equal quality to males produced by BSS. Based on current rearing technology the cost of production of male pupae is about the same for both types of strain. This is due to the large colony that is required for the tsl-based GSS. This paper discusses the considerations that need to be taken into account during mass rearing of GSS and identifies the most efficient production processes that are currently available.     

Comparison of two sterile Mediterranean fruit fly (Diptera: Tephritidae) strains released in California's Preventative Release Program

At Mediterranean fruit fly rearing centers, genetic sexing strains such as the male-only temperature sensitive lethal (tsl) strains, are replacing many of the older bisexual strains, such as Maui-93. Three tests were performed to compare the performance of the Vienna-4 (which carries tsl) and Maui-93 strains. Using standard laboratory quality control tests, the Maui-93 strain had higher percent fliers and a lower percentage deformed and partially eclosed flies. Analyses of data from field mark-recapture tests with Jackson traps failed to find significant differences in recapture numbers between the two strains. In field cage survivorship tests, the Vienna-4 strain showed significantly lower mortality in comparison to the Maui-93 strain. These results suggest that although the quality of Vienna-4 flies is lower than Maui-93 at the time of adult emergence, it is comparable with or better than Maui-93 several days later, because weaker flies have been eliminated from the cohort. The benefits of releasing only sterile males, instead of both males and females, was not factored into the quality assessment in this study, but would be an additional benefit in using a strain that carries a tsl mutation.     

Cytogenetic analysis of three genetic sexing strains of Ceratitits capitata

Summary Polytene chromosomes of three genetic sexing strains of Ceratitis capitata were analyzed. The genetic sexing mechanism is based on a pupal color dimorphism (white-brown) and is the result of a reciprocal translocation between the Y chromosome and the autosome bearing the w locus (white pupal case). The analyzed polytene chromosomes were derived from two different pupal tissues, the orbital bristle and fat body cells. The Y chromosome is visible in both tissues, while the autosomes present a different banding pattern. Based on these features, the autosome breakpoints in the three Y; autosome translocations were mapped, and the homology of the translocated autosome in both tissues was established. In addition, the location of the break-points was compared to the stability of these three strains.     

不同饲养温度对橘小实蝇蛹色伴性遗传品系的影响

分别在19℃,22℃,25℃,28＇t2,31℃下饲养橘小实蝇蛹色伴性遗传品系,统计各温度下卵到蛹的的获得率、蛹重、羽化率、存活率及飞出率。结果表明卵到蛹的获得率28℃最大,为62．12％,19℃最小,为43．12％;蛹重25℃最重为1．3854g／100粒,19℃最轻,为1．2610g／100粒;羽化率25℃最高,为93％,31℃最低,仅82．67％;存活率31℃最高,为66％,19℃最低,仅为4．67％;飞出率25℃最高,为65％,31℃最低,为16％。综合不同温度下的卵到蛹的的获得率、蛹重、羽化率、存活率及飞出率得出25℃-28℃是大量饲养橘小实蝇蛹色伴性遗传品系的最佳温度。     

Dramatic developmental changes in larval knockdown response enhance genetic sexing based on DDT resistance in Anopheles stephensi (Diptera: Culicidae)

Genetic sexing systems based on a conditional lethal require good discrimination between the different phenotypes. DDT resistance in the early instars of Anopheles stephensi Liston is not a good candidate when based on mortality, but this study shows that the knockdown response gives exceptional discrimination between heterozygous resistant and homozygous susceptible individuals. One- and two-day-old larvae of the DlDDT strain showed high (417-fold) resistance to knockdown by DDT, but very low resistance to mortality (3.3-fold). This changes with the onset of the third instar, so that by the fourth instar, mortality resistance is high (108-fold) and knockdown resistance is low (6.5-fold). Susceptibility to DDT decreases from first to fourth instar in the susceptible strain by 443-fold for knockdown and 15-fold for mortality and in the resistant strain by 8.5-fold for knockdown and 491-fold for mortality. The DDT knockdown response in young larvae was successfully used to identify two Y-autosome translocations linked to the resistance gene, DDT. T(Y-3)69 and T(Y-3)72 gave recombination values between the translocation breakpoint and the DDT locus of 4.1 and 10.1 crossover units, respectively. T(Y-3)69 proved to be an adequate genetic sexing system for laboratory studies.     

Genetic sexing strains in medfly,Ceratitis capitata,sterile insect technique programmes

The introduction of genetic sexing strains (GSS) into medfly, Ceratitis capitata(Wiedemann), sterile insect technique (SIT) programmes started in 1994 and it was accompanied by extensive evaluation of the strains both in field cages and in open field situations. Two male-linked translocation systems, one based on pupal colour, wp, and the other based on temperature sensitivity, tsl, have been used in medfly SIT programmes and they have quite different impacts on mass rearing strategy. In strains based on tsl, female zygotes are killed using high temperature and for wpstrains, female and male pupae are separated based on their colour. In all these systems the colony females are homozygous for the mutation requiring that the mutation is not too deleterious and the males are also semi-sterile due to the presence of a male-linked translocation. Managing strain stability during large-scale mass rearing has presented some problems that have been essentially solved by selecting particular translocations for GSS and by the introduction of a filter rearing system (FRS). The FRS operates by removing from the colony any recombinant individuals that threaten the integrity of the strain. The use of GSS opens up the possibility of using the SIT for suppression as opposed to eradication and different radiation strategies can be considered. Some of the many field trials of the strains that were carried out before the strains were introduced into operational programmes are reviewed and an overview is given of their current use.     

Translocations in DICTYOSTELIUM DISCOIDEUM

Fourteen translocations of independent origin were identified in Dictyostelium discoideum on the basis of segregation anomalies of diploids heterozygous for these chromosome rearrangements, all of which led to the cosegregation of unlinked markers. Many of these translocations were discovered in strains mutagenized with MNNG or in strains carrying mutations affecting DNA repair; however, spontaneous translocations were also obtained. Haploid mitotic recombinants of the rearranged linkage groups were produced from diploids heterozygous for the translocations at frequencies of up to 5% of viable haploid segregants; this is at least a ten-fold higher frequency than that seen with diploids not heterozygous for translocations (approximately 0.1%). These haploid recombinants included both translocated and nontranslocated strains. The T354(II, VII) translocation and possibly the T357(IV, VII) translocation reduce the chromosome number to n = 6; haploids carrying 11 other translocations all have karyotypes with n = 7. Genetic characterization of the T357(IV, VII) translocation showed that the bwnA and whiC loci normally found on linkage group IV were physically linked to the linkage group VII loci couA, phgA, bsgB and cobA.     

Genetic Recombination in Mycobacteria

Evidence for genetic recombination between Mycobacterium smegmatis strain Rabinowitchi (Rab) and strain Jucho or PM5 is presented. Backcrosses of recombinants by either parental strain indicated four different types of mating behavior, suggesting that the mycobacterial compatibilities are controlled by at least two different factors. No sex factor that transfers at a high frequency or that is sensitive to acridine dyes was detected. Analysis of segregation of unselected markers revealed that strain Jucho, or PM5, contributes the majority of alleles in almost all recombinants obtained from different selective media. Efforts to construct linkage maps for the markers employed failed because of ordering ambiguities. Mating medium containing streptomycin prevented genetic recombination when strain Rab was resistant to the antibiotic and Jucho, or PM5, was sensitive, but it did not prevent recombination when Rab was sensitive to streptomycin and Jucho, or PM5, was resistant. Very low frequency of recombinant formation was observed when Jucho, or PM5, had been treated with streptomycin, whereas recombinants were formed at fairly high frequencies when Rab had been treated with the antibiotic, suggesting that the roles of parental strains in zygote formation were not identical. The results suggest a polar transfer of genetic material from Rab to Jucho, or PM5, although an alternative possibility of cell fusion followed by exclusion could not be excluded.     

Towards a male-only release system for SIT with the Queensland fruit fly,Bactrocera tryoni,using a genetic sexing strain with a temperature-sensitive lethal mutation

Flies that are homozygous for the recessive autosomal mutation bent wingshave a limited ability to fly and are less tolerant of high temperatures than normal flies in both the egg and puparial stages. The differences between the mutant and normal flies were found sufficient to be the basis of a genetic sexing strain. Genetic sexing strains were created using translocations of the autosome bearing the wild-type allele of bent wings(chromosome 2) to the Y chromosome, and crossing male flies carrying the translocation to mutant bent wingsfemales. In the resulting strain, the females were homozygous for the bent wingsmutation and the males were phenotypically normal for wing characters. Several translocations were recovered after irradiation, but only one translocation involving chromosome 2 was both stable and expressed in a stock that was vigorous enough for long-term viability. Unfortunately, all stocks containing the translocation showed high levels of temperature-dependent lethality, including, inexplicably, both males and females. Translocation stocks showing this effect included bent wings, another second chromosome mutation, white marks, and an otherwise normal stock. This phenomenon is probably rare, as it has not been reported before. It is likely that bent wingscould be suitably used with another translocation.     

Unequal genetic exchange in Escherichia coli tandem duplications may represent a special pathway of homologous recombination

Heterozygous tandem duplications that appear in Escherichia coli conjugation matings segregate different types of haploid and diploid recombinants because of unequal crossing over between sister chromosomes. As shown previously, the frequency of segregants in the extended duplication D104 (approximately 150 kb or more than 3 min of the genetic map) heterozygous for E. coli deo-operon genes (deoA deoB::Tn5/deoC deoD) is not decreased in strains with defective RecBCD and RecF recombination pathways. Analysis of a shorter duplication of this type (approximately 46 kb) showed that the frequency of segregants in the strain recBC sbcBC recF was similar to that in a strain with undamaged system of recombination. Thus, genetic exchange between direct DNA repeats in tandem duplications may follow a special pathway of homologous recombination, which is independent of the recBC and recF genes.     

Host defenses in experimental scrub typhus: mapping the gene that controls natural resistance in mice

Natural resistance of mice to lethal ifections of Rickettsia tsutsugamushi, strain Gilliam, is controlled by a single, autosomal, dominant gene, which we have designated Ric, with r and s representing the resistant nd susceptible alleles, respectively. Using three sets of recombinant inbred mouse strains (BXD, BXH, and BXJ), the Ric locus was mapped to Chromosome 5 closely linked to the retinal degeneration (rd) locus. This linkage was confirmed by a backcross analysis. Based on the RI strains and the C57BL/6Ty-le congenic strain (the only proven Ric-rd cross-over), we estimate the recombination frequency between Ric and rd to be 0.015. Three presumptive Ric-rd recombinants detected among 93 backcross mice may represent caes of incomplete penetrance of the resistance allele rather than recombination. Analyis of th C57BL/6JTy-le congenic strain indicates that Ric is proximal to rd on Chromosome 5. If so, the correct gene order is Pgm-1-W-Ric-rd-Gus.     

Viability and behaviour of translocations that suppress female recombination in the Mediterranean fruit fly,Ceratitis capitata (Wied.)

Summary The viability of a series of recombination suppressor (RS) strains in Ceratitis capitata, all previously found to contain a reciprocal autosomal translocation, was assessed for egg hatchability and adult emergence in both the homozygous and heterozygous state. Except in T 30C, which contains a Y-autosome translocation in addition to the A-A translocation, egg hatch was significantly reduced in all heterozygous translocation strains, and ranged from 42.4% to 58.5% in seeded eggs compared to a control value of 82.8%. Adult emergence from hatched eggs was affected to a lesser extent, but with a range of 59.5% to 84.2%, compared to the control value of 83.1%, remained significantly reduced in 4 of the 6 translocation strains, as well as in the male line of T 30C. In the homozygous configuration all strains, except T 19 and T 109, showed a significant reduction in egg hatchability, whereas adult emergence was not adversely affected. A significant reduction in the egg hatchability of the translocation heterozygotes compared to that of the homozygotes was observed in 5 of the 7 strains, the observed reduction in T 55/109 being non-significant while that of T 30C was significantly increased. The behaviour of translocations as recombination suppressors and their suitability for inclusion in breeding schemes for the isolation of induced recessive mutations is discussed.This work forms part of a Joint FAO/IAEA research programme on the development of genetic sexing mechanisms for the Mediterranean fruit fly, Ceratitis capitata (Wied.)     

High-frequency RNA recombination of murine coronaviruses.

The RNA genome of coronaviruses consists of a single species of nonsegmented RNA. In this communication, we demonstrate that the RNA genomes of different strains of murine coronaviruses recombine during mixed infection at a very high frequency. Susceptible cells were coinfected with a temperature-sensitive mutant of one strain of mouse hepatitis virus (MHV) and a wild-type virus of a different strain. Of 21 randomly isolated viruses released from the coinfected cells at the nonpermissive temperature, 2 were recombinants which differed in the site of recombination. After three serial passages of the original virus pool derived from the mixed infection, the majority of the progeny viruses were recombinants. These recombinant viruses represented at least five different recombination sites between the two parental MHV strains. Such a high-frequency recombination between nonsegmented RNA genomes of MHV suggests that segmented RNA intermediates might be generated during MHV replication. We propose that the RNA replication of MHV proceeds in a discontinuous and nonprocessive manner, thus generating free segmented RNA intermediates, which could be used in RNA recombination via a copy-choice mechanism.     

Trigenomic chromosomes by recombination of <Emphasis Type="Italic">Thinopyrum intermedium </Emphasis>and <Emphasis Type="Italic">Th. ponticum </Emphasis>translocations in wheat

Rusts and barley yellow dwarf virus (BYDV) are among the main diseases affecting wheat production world wide for which wild relatives have been the source of a number of translocations carrying resistance genes. Nevertheless, along with desirable traits, alien translocations often carry deleterious genes. We have generated recombinants in a bread wheat background between two alien translocations: TC5, ex-Thinopyrum (Th) intermedium, carrying BYDV resistance gene Bdv2; and T4m, ex-Th. ponticum, carrying rust resistance genes Lr19 and Sr25. Because both these translocations are on the wheat chromosome arm 7DL, homoeologous recombination was attempted in the double hemizygote (TC5/T4m) in a background homozygous for the ph1b mutation. The identification of recombinants was facilitated by the use of newly developed molecular markers for each of the alien genomes represented in the two translocations and by studying derived F₂, F₃ and doubled haploid populations. The occurrence of recombination was confirmed with molecular markers and bioassays on families of testcrosses between putative recombinants and bread wheat, and in F₂ populations derived from the testcrosses. As a consequence it has been possible to derive a genetic map of markers and resistance genes on these previously fixed alien linkage blocks. We have obtained fertile progeny carrying new tri-genomic recombinant chromosomes. Furthermore we have demonstrated that some of the recombinants carried resistance genes Lr19 and Bdv2 yet lacked the self-elimination trait associated with shortened T4 segments. We have also shown that the recombinant translocations are fixed and stable once removed from the influence of the ph1b. The molecular markers developed in this study will facilitate selection of individuals carrying recombinant Th. intermedium–Th. ponticum translocations (Pontin series) in breeding programs. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of nucleotide excision repair in human cells on intrachromosomal homologous recombination induced by UV and 1-nitrosopyrene.

To study the role of nucleotide excision repair in the induction of intrachromosomal homologous recombination in mammalian cells, we introduced a plasmid containing a substrate for recombination into three human cell lines that differ in their repair capacity and compared the frequency of recombination induced by UV radiation and by 1-nitrosopyrene. One strain had a normal capacity for nucleotide excision repair, the second exhibited an intermediate rate of repair, and the third, derived from a patient with xeroderma pigmentosum, had no ability to repair UV- or 1-nitrosopyrene-induced DNA damage. The endogenous thymidine kinase genes in these cell strains had been inactivated, and the cells contained an integrated copy of a plasmid carrying duplicated copies of the herpes simplex virus type 1 thymidine kinase (Htk) gene, each inactivated by an 8-base-pair XhoI site inserted at a unique site. A functional tk gene can only be generated by a productive recombination event between the two Htk genes. In all three stains, UV and 1-nitrosopyrene induced dose-dependent increases in the frequency of recombinants. However, the doses required to cause a specific increase in recombination in the repair-deficient strains were 10 to 30 times lower than the dose required for the cell strain with a normal capacity for repair. These results strongly suggest that unexcised DNA lesions, rather than excision repair per se, stimulate intrachromosomal homologous recombination. Southern blot analysis of DNA from representative recombinants indicated that in all cases one of the two Htk genes had become wild type (XhoI resistant). The majority (90%) retained the Htk duplication, consistent with nonreciprocal transfer of genetic information (gene conversion).