The enzymatic degradation of malathion in organophosphate resistant and susceptible strains of Musca domestica

The metabolic fate of malathion in susceptible and organophosphate resistant strains of houseflies was studied by means of radioisotope-techniques, in vivo and in vitro. The main interstrain difference was the superior ability of the two malathion resistant strains to degrade malathion to its monocarboxylic acid derivative. Attempts were made to characterize the carboxyesterases responsible for this difference with respect to heat stability, pH dependency and organophosphate inhibition, and to estimate their importance in causing resistance by means of cross-resistance and synergism experiments. The relation to, and possible identity of the carboxyesterases with, the malaoxon degrading enzymes previously found in the malathion resistant strains is discussed.

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Zusammenfassung Der Malathionmetabolismus wurde mit Hilfe der Radioisotopentechnik in normal empfindlichen und organophosphatresistenten Stubenfliegenstämmen in vivo und in vitro untersucht Die beiden malathionresistenten Stämme konnten Malathion besser in seinen Monocarboxylsäure-Abkömmling abbauen. Es wurde versucht, die hierfür verantwortliche Carboxyesterase hinsichtlich ihrer Hitzestabilität, pH-Abhängigkeit und Hemmbarkeit durch Organophosphate zu charakterisieren und ihre Bedeutung in der Resistenzbildung durch Kreuzungsresistenz- und Synergismusversuche zu bestimmen. Die Beziehung der besonders in den malathionresistenten Stämmen gefundenen malaoxonabbauenden Enzyme zu den Carboxyesterasen und ihre mögliche Identität mit denselben wird diskutiert.

     

Somatic hybridization between potato and Nicotiana plumbaginifolia

Summary Electrofusion was carried out between mesophyll protoplasts from the transformed diploid S. tuberosum clone 413 (2n=2x=24) which contains various genetic markers (hormone autotrophy, opine synthesis, kanamycin resistance, -glucuronidase activity) and mesophyll protoplasts of a diploid wild-type clone of N. plumbaginifolia (2n=2x=20). Hybrid calli were obtained after continuous culture on selection medium containing kanamycin. Parental chromosome numbers, determined at 2 months after fusion, revealed hybrid-specific differences between the individual calli. On the basis of these differences three categories of hybrids were distinguished. Category I hybrids contained between 8 and 24 potato chromosomes and more than 20 N. plumbaginifolia chromosomes; category II hybrids had between 1 and 20 N. plumbaginifolia chromosomes and more than 24 potato chromosomes; category III hybrids contained diploid or subdiploid numbers of chromosomes from both parents. The hybrids were evenly distributed over the three categories. After a 1-year culture of 24 representative hybrid callus lines on selection medium the karyotype of 10 hybrids remained stable, whereas 8 hybrids showed polyploidization of the genome of one parent, together with no or minor changes of the chromosome numbers of the other parent. Six hybrids showed slight changes in the hybrid karyotype. The elimination of chromosomes of a particular parent was not correlated to their metaphase location. The processes of spontaneous biparental chromosome elimination leading to the production of asymmetric hybrids of different categories are discussed.     

Potential cause of lethality of an allele implicated in social evolution in fire ants

The gene Gp-9 is believed to have a major effect on colony social organization in fire ants, with the presence of b-like alleles in a colony associated with multiple-queen (polygyne) organization. Queens and workers of polygyne Solenopsis invicta homozygous for the b-like allele designated b suffer reduced viability compared to other genotypes, and bb queens do not survive to become egg-layers. Thus, the b allele effectively acts as a recessive lethal. This allele differs from the remaining b-like alleles (designated b′), as well as all other Gp-9 alleles, by encoding a lysine at position 151 in the protein product, suggesting that this substitution is responsible for its deleterious effects. We tested this hypothesis by comparing frequencies of b′b′ and bb homozygotes, first in queens of Solenopsis richteri and S. invicta, then in S. invicta workers from populations polymorphic for the two b-like alleles. We found that almost 20% of S. richteri queens were b′b′ homozygotes, compared to the virtual absence of bb homozygotes among S. invicta queens, and that 5–18% of S. invicta workers bore genotype b′b′, compared to the apparent lack of bb workers in the same populations. While we cannot entirely rule out involvement of other genes in complete gametic disequilibrium with Gp-9, our data are consistent with the hypothesis that the Lys¹⁵¹ residue in GP-9 protein confers the deleterious effects of the b allele in homozygous condition, possibly by impairing the protein’s function through interference with ligand binding/release or hindrance of dimer formation.     

Intergeneric hybridization between Pleurotus ostreatus and Schizophyllum commune by PEG-induced protoplast fusion

Intergeneric hybridization between Pleurotus ostreatus and Schizophyllum commune was studied using PEG-induced fusion. The fusion of protoplasts from auxotrophic mutant strains resulted in the formation of fusion hybrids in the frequencies of 3.6 to 7.3×10^–5. Most of these fusion hybrids were monokaryotic and sterile and no heterokaryosis occurred. Most fusants showed a significantly higher nuclear DNA content when compared to parental strains and no diploids (parent 1 genome plus parent 2 genome) were found. Some fusion hybrids revealed both parental fragments in nuclear and mitochondrial rDNA PCR profiles. AP-PCR (Arbitrarily-primed Polymerase Chain Reaction) fingerprints also indicated that most of the fusion products were recombinant hybrids.     

A cluster of esterase genes on chromosome 3R ofDrosophila melanogaster includes homologues of esterase genes conferring insecticide resistance inLucilia cuprina

We identify an esterase isozyme inDrosophila melanogaster, EST 23, which shares biochemical, physiological, and genetic properties with esterase E3, which is involved in resistance to organophosphate insecticides inLucilia cuprina. Like E3, theD. melanogaster EST 23 is a membrane-bound -esterase which migrates slowly toward the anode at pH 6.8. Both enzymes have similar preferences for substrates with shorter acid side chain lengths. Furthermore, on the basis of their high sensitivity to inhibition by paraoxon and their insensitivity to inhibition by eserine sulfate, both enzymes were classified as subclass I carboxylesterases. The activity of each enzyme peaks early in development and, again, in the adult stage. Both enzymes are found in the male reproductive system and larval and adult digestive tissues, the latter being consistent with a role for these enzymes in organophosphate resistance. Fine structure deficiency mapping localizedEst 23 to cytological region 84D3 to E1-2 on the right arm of chromosome 3. Moreover, we show that the genes encoding three other esterase phenotypes also map to the same region; these phenotypes involve allozymic differences in EST 9 (formerly EST C), ali-esterase activity, defined by the hydrolysis of methyl butyrate, and malathion carboxylesterase activity, defined by hydrolysis of the organophosphate malathion. This cluster corresponds closely to that encompassing E3 and malathion carboxylesterase on chromosome 4 inL. cuprina, the homologue of chromosome 3R inD. melanogaster.     

Genetic variation of an acid phosphatase (Acp-2) in the laboratory rat: Possible homology with mouse AP-1 and human ACP2

A genetic locus controlling the electrophoretic mobility of an acid phosphatase in the rat (Rattus norvegicus) is described. The locus, designed Acp-2, is not expressed in erythrocytes but is expressed in all other tissues studied. The product of Acp-2 hydrolyzes a wide variety of phosphate monoesters and is inhibited by l(+)-tartaric acid. Inbred rat strains have fixed either allele Acp-2^a or allele Acp-2^b. Codominant expression is observed in the respective F₁ hybrids. Backcross progenies revealed the expected 1:1 segregation ratio. Possible loose linkage was found between the Acp-2 and the Pep-3 gene loci at a recombination frequency of 0.36±0.06.Supported by the Deutsche Forschungsgemeinschaft (Grant Be 352/15) and by a grant from the Alexander-von-Humboldt-Stiftung (VB2-FLF).     

Insecticide resistance and malathion carboxylesterase in the sheep blowfly,Lucilia cuprina

Resistance to the organophosphorus insecticide malathion in genetically related strains of the Australian sheep blowflyLucilia curprina was examined. Separate lines of blowflies were established by homozygosis of the fourth chromosome of the parental RM strain. Both the RM and the derived resistant (der-R) strains are approximately 100 times more resistant to malathion than the related susceptible der-S strain, resistance being correlated with a 45- to 50-fold increase in a malathion carboxylesterase (MCE) activity. MCE has a pH optimum ranging between 6.6 and 8.0 and is strongly inhibited by the carboxylesterase inhibitors triphenyl phosphate, paraoxon, and diiospropylfluorophosphate. Subcellular fractionation revealed that MCE was localized predominantly to the cytosol and mitochondria in both resistant and susceptible blowflies. A single MCE was purified to homogeneity from RM blowflies. It has a pI of 5.5, is a monomer of 60.5 kDa, and hydrolyzes malathion with aV _max of 755 nmol/min/mg protein and aK _m of 11.0 µM. L. cuprina have thus evolved a remarkable MCE which is faster and more efficient at hydrolyzing a specific insecticide than any other insect esterase yet described.     

Analysis of somatic hybrids between two sterile dihaploid Solanum tuberosum L. breeding lines. Restoration of fertility and complementation of G. pallida Pa2 and Pa3 resistance

Fourteen somatic hybrids generated by electrofusion of mesophyll protoplasts from a non-flowering dihaploid S. tuberosum clone, DHAK-11, and a male-sterile dihaploid clone S. tuberosum, DHAK-33, were grown in the greenhouse and subjected to morphological assessments and tests for fertility and resistance to the white potato cyst nematode Globodera pallida pathotypes Pa2 and Pa3. The ploidy level of the hybrids ranged from 38 to 63 chromosomes. All hybrids developed flowers with violet petals except for one, hy-56, that possessed red petals. The colour of the tuber skin was purple in all hybrids except in hy-56 where the tuber skin was red. All of the hybrids were female fertile and generated viable seeds. Near-tetraploid hybrids produced the highest number of seeds per fruit and these seeds had a normal size. Hybrids with 58 or more chromosomes produced smaller seeds and less seeds per fruit. The germination frequency of the seeds was not influenced by the chromosome number of the hybrids. Pollen viability was determined and the male fertility of three hybrids was tested. Pollination with these three hybrids gave rise to fruit development, but only one produced viable seeds. The hybrids were tested for resistance to G. pallida pathotypes Pa2 and Pa3. A high level of resistance to Pa3, inherited from one parental clone, DHAK-11, and a high level of resistance to Pa2, inherited from the other parental clone, DHAK-33, was combined in four hybrids. These results demonstrate, that protoplast fusion is an efficient method for restoring the fertility of somatic hybrids generated from sterile parent clones, and is a powerful procedure for the complementation of multigenetic disease resistance traits in potato breeding lines.     

Inheritance of morphological and chemical characters in interspecific hybrids between Papaver bracteatum and Papaver pseudo-orientale

Summary The alkaloid profiles and morphological traits of the capsules of Papaver bracteatum, P. pseudo-orientale, and their hybrids were studied. Dominance of the hexaploid parent P. pseudo-orientale was observed for various characters. A genetic model assuming allelic additive effects and polysomic inheritance was elaborated for the control of isothebaine content in the capsules. The distribution of thebaine content in the segregating generations, F₂ and BCF₁ was evidence of the transfer of genes from the diploid parent P. bracteatum in the gametes of the interspecific hybrid and their expression in its progenies. These findings indicate the potential use of inter-specific hybrids between the Oxytona species in the breeding of cultivars for industrial or ornamental purposes.Contribution No. 3066-E, 1990 series from The Agricultural Research Organization, The Volcani Center, Bet Dagan 50 250, Israel     

Isolation and characterization ofAcinetobacter sp. mutants defective in exopolysaccharide biosynthesis

Nitrosoguanidine-induced mutants ofAcinetobacter sp. defective in exopolysaccharide biosynthesis did not differ from the parent strain in distinguishing physiological and biochemical properties, such as requirements for growth factors, utilization of mono- and disaccharides, and resistance to antibiotics. The genetic relation of parent and mutant strains was shown by 16S rRNA PCR analysis. The comparative study of parent and mutant strains with respect to resistance to unfavorable environmental factors confirmed our hypothesis thatAcinetobacter sp. exopolysaccharides perform protective functions. Hybridization experiments revealed the conjugal transfer of plasmid R68.45 fromPseudomonas putida BS228 (R68.45) to mutant but not to the parentAcinetobacter sp. strains. The role of theAcinetobacter sp. exopolysaccharides in providing the genetic stability of this bacterium is discussed.     

Quantitatively-inherited resistance toGlobodera pallida is dominated by one major locus inSolanum spegazzinii

A high level of resistance toGlobodera pallida pathotypes Pa2 and Pa3 exists inSolanum spegazzinii, a wild relative of potato (S. tuberosum ssp.tuberosum). Here we report the mapping of loci involved in quantitatively-inherited nematode resistance with the use of RFLPs. One major locus,Gpa, was mapped on chromosome 5 and two minor loci on chromosomes 4 and 7 ofS. spegazzinii. Additionally, the contribution of the susceptible parent to nematode resistance was determined. TheGpa locus was solely responsible for the high resistance level found in the segregating population. However, the RFLP marker closely linked to this resistance locus showed a distorted segregation, with a shortage of plants having the resistance linked allele. Our results indicate that a prediction of the genetic constitution of a quantitative trait based solely on phenotypic observations can lead to erroneous conclusions.     

A locus controlling the activity of UDP-galactose:GM2(NeuGc) galactosyltransferase in mouse liver is linked to the H-2 complex

Genetic polymorphism in the expression of the G_M1(NeuGc) ganglioside has been shown in the liver of inbred strains of mice. Through analysis of the gangliosides of H-2 congenic and recombinant strains, this polymorphism was demonstrated to be controlled by a locus mapped left outside of the H-2 complex on chromosome 17, and the locus was assumed to control the level of the activity of G_M1(NeuGc) synthetase, UDP-galactose:G_M2(NeuGc) galactosyltransferase (E.C.2.4.1.62) [Hashimotoet al., J Biochem (1983) 94:2049-54].In the present study we analyzed the genetic linkage between the activity of the galactosyltransferase and the H-2 haplotype. For this purpose, we selected two inbred strains of mice, WHT/Ht and BALB/c, because they have different levels of the transferase activity and show different H-2 haplotypes; the specific activity of the transferase obtained with BALB/c was one-eighth of that with WHT/Ht, and BALB/c expressed the la.7 antigen as one of the products encoded in their H-2^d complex, whereas WHT/Ht did not. To analyze the linkage between these two phenotypes, WHT/Ht were mated with BALB/c to obtain the F₁ mice, and the female F₁ mice were then backcrossed to WHT/Ht. It was found that one half of the backcross generation expressed the la.7 antigen derived from BALB/c and had a significantly lower specific activity of the transferase than that of WHT/Ht, while the other half did not express the la.7 antigen but had the same specific activity of the transferase as that obtained with WHT/Ht.These results suggest that the locus controlling the level of the transferase activity in mouse liver is linked to the H-2 complex on chromosome 17.Abbreviations NeuGc N-glycolylneuraminic acid The ganglioside nomenclature is based on the system of Svennerholm, J Neurochem (1963) 10:613-23. The sialic acid species present is shown in parentheses after the ganglioside abbreviation.     

Interspecific hybridization between Vigna radiata (L.) Wilczek and V. glabrescens

Summary Interspecific hybrids of the mungbean, Vigna radiata (L.) Wilczek (2n=22) and V. glabrescens (2n=44) were generated with the aid of embryo culture. V. glabrescens x V. radiata hybrids were recovered via germination of the immature embryos. Reciprocal hybrids were obtained via shoot formation from embryonic callus. The authenticity of the hybrids was determined by morphological characteristics, chromosome number, and isozyme patterns. The hybrids were highly sterile upon selfing, but backcrossing to the diploid parent yielded viable seeds. Some of the plants resembled the diploid parent morphologically while others resembled neither parent. The backcross plants were sufficiently fertile to give a large number of mature, selfed seeds. Plants obtained differed morphologically and in their isozyme patterns from either parent, indicating introgression. These progeny populations will be used as bridging materials to transfer pest resistance from the wild tetraploid to the cultivated mungbean.     

Biochemical markers in a species endangered by introgression: The red wolf

The red wolf (Canis rufus), native to much of the southeastern United States, is endangered by man's activities and by hybridization with other species of the genus Canis. The absence of diagnostic morphological markers to distinguish the red wolf from its hybrids has led to the application of the methods of biochemical genetics to this problem. The finding of a unique electrophoretically determined allele with a distribution congruent with the geographical distribution of the remaining red wolf population is reported.This research was supported by NIH Public Health Service Grants GM 19513 and CA 19311 and by the State of Texas.     

Variable responses of insects to hybrid versus parental sagebrush in common gardens

Both ecological and genetic mechanisms have been proposed to explain patterns of herbivore attack on interspecific plant hybrids, but distinguishing among them can be difficult in natural hybrid zones. We performed a common-garden experiment to evaluate four genetic hypotheses: dominance, additivity, elevated hybrid susceptibility, and elevated hybrid resistance. Censuses and cage experiments were used to compare insect responses to basin big sagebrush (Artemisia tridentata spp. tridentata), mountain big sagebrush (A. t. vaseyana), and their F₂ progeny. After two growing seasons, hybrid shrubs resembled mountain big sagenbrush in size, but were more similar to basin big sagebrush in flower production. Censuses of naturally colonizing insects (the gall midge Rhopalomyia obovata, the bagworm moth Apterona helix, and the aphid Obtusicauda coweni) tended to support the dominance hypothesis: if the insect clearly discriminated between the two parents, its frequency on hybrids closely resembled that on one of the parents. Moreover, colonization of hybrids in all three cases suggested a dominance deviation toward the susceptible parent rather than toward the resistant parent. In contrast to the censuses, cage experiments involving two insects supported the hybrid-susceptibility hypothesis; both survival and growth of the grasshopper Melanoplus sanguinipes and growth of the leaf beetle Trirhabda pilosa were higher on hybrid shrubs than on either parent. Because many secondary compounds have been determined to occur at intermediate concentrations in F₂ shrubs, dominance for susceptibility may indicate that insects respond to plant traits (e.g., oviposition stimulants and deterrents) in a threshold manner. Mechanisms underlying increased hybrid susceptibility are less clear, but our experimental design makes environmental explanations (e.g., the plant-stress hypothesis) unlikely. Although we eliminated several confounding factors, our results agree with the conclusion from natural hybrid zones that insect responses to hybrid plants are likely to be idiosyncratic; even congeneric species did not respond similarly to hybrid and parental plants.     

Intergeneric hybridization between Diplotaxis siifolia,a wild species and crop brassicas

Summary Attempts were made to obtain intergeneric hybrids between Diplotaxis siifolia, a wild species, and cultivars of Brassica (B. campestris, B. juncea, and B. napus). The crosses showed unilateral incompatibility. When the wild species was used as female parent, pollen germination and pollen tube growth were normal, but hybrid seeds aborted due to post-fertilization barriers. Reciprocal crosses (cultivars as female parent) showed strong pre-fertilization barriers; although pollen grains showed germination, pollen tubes failed to enter the stigma. Hybrids were realized in two of the crosses, D. siifolia x B. juncea and D. siifolia x B. napus, through ovary culture. The hybrids were multiplied in vitro by multiplication of axillary shoots, or somatic embryogenesis. Detailed studies were carried out on the hybrid D. siifolia x B. juncea. F₁ hybrids had shrivelled anthers and were pollen sterile. Amphiploids of this hybrid showed 60% pollen fertility and produced seeds upon self-pollination as well as backcross pollination with the pollen of B. juncea.     

Evolutionary Processes Associated with Biological Invasions in the Brassicaceae

This review highlights evolutionary processes in the Brassicaceae which cause, accompany or are a consequence of biological invasions. Case studies in Capsella and Diplotaxis address the genetics of colonising species. The greatest colonising ability in Capsella bursa-pastoris is associated with polyploidy, predominant selfing, and high genetic diversity. Success of colonial populations seems to be due to the introduction of preadapted genotypes. Colonising species in Diplotaxis reveal contrasting evolutionary patterns. Genetic attributes in D. muralis include annuality, polyploidy, and predominant selfing. Very different from Capsella, D. muralis is nearly devoid of genetic diversity as revealed by molecular markers. In contrast to D. muralis, the colonising D. tenuifolia is perennial, diploid, self-incompatible, and displays high genetic diversity. Hybrid speciation, establishment of the hybrids in man-made habitats, stabilisation of their reproductive system, and reproductive isolation from the parent populations were analysed in Cardamine. The Nasturtium example highlights the importance of hybridisation for the evolution of invasiveness. The last case study concentrates on the evolutionary consequences of hybridisation between native and invading Rorippa species. Introgressive hybridisation between the invasive Rorippa austriaca and native Rorippa species is common and widespread in central Europe, and interspecific gene transfer has led to the formation of a new invasive genotype. Each successful invasion presents new aspects and sound case studies are needed in order to understand the ecology and evolution of the colonisation process and to enable us to assess the evolutionary consequences of biological invasions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Partial resistance to late blight (Phytophthora infestans) in hybrid progenies of four South American Solanum species crossed with diploid S. tuberosum

Resistant genotypes of the diploid tuber-bearing South American species Solarium arnezii x hondelmannii, S. berthaultii, S. leptophyes and S. microdontum were crossed with three diploid genotypes of S. tuberosum that varied in resistance and maturity type. The progenies were field tested for 2 years for resistance to a complex race of Phytophthora infestans. A wealth of genetic variation for resistance was found in most of the progenies. At least two susceptibility groups could be distinguished in some progenies of S. microdontum. This could be explained by the presence of several major resistance genes in the wild parent and, unexpectedly, in the susceptible parent SH 82-44-111. In most of the wild parents and in the susceptible parent SH 77-114-2988 there appeared to be minor resistance genes. General combining ability effects were predominant; small specific combining ability effects were detected in some crosses of S. microdontum. Gene action appeared dominant in some crosses.     

An acid phosphatase locus expressed in mouse kidney (Apk) and its genetic location on chromosome 10

A genetic locus controlling the electrophoretic mobility of an acid phosphatase in mouse kidney is described. This locus, called acid phosphatase-kidney (Apk), is not expressed in erythrocytes, liver, spleen, heart, lung, brain, skeletal muscle, stomach, or testes. The product of Apk hydrolyzes the substrate naphthol AS-MX phosphoric acid but is not active on a-naphthylphosphate or 4-methylumbelliferylphosphate. It is not inactivated by 50 C for 1 hr, nor is its electrophoretic mobility altered by incubation with neuraminidase. The locus is invariant among 31 inbred strains (Apk ^a), with a variant allele (Apk ^m) observed only in Mus musculus molossinus. Codominant expression was observed in F₁ hybrids of M. m. molossinus and inbred strains. Apk was mapped on Chr 10, near the neurological mutant waltzer (v).This work was supported by Contract NO1-ES42159 from the National Institute of Environmental Health Sciences and by Grant 1-476 from the National Foundation—March of Dimes. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Unique chromosome behavior and genetic control in Brassica×Orychophragmus wide hybrids: a review

Researchers recognized early that chromosome behavior, as other morphological characters, is under genetic control and gave some cytogenetical examples such as the homoeologous chromosome pairing in wheat. In the intergeneric sexual hybrids between cultivated Brassica species and another crucifer Orychophragmus violaceus, the phenomenon of parental genome separation was found under genetic control during mitosis and meiosis. The cytogenetics of these hybrids was species-specific for Brassica parents. The different chromosome behavior of hybrids with three Brassica diploids (B. rapa, B. nigra and B. oleracea) might contribute to the different cytology of hybrids with three tetraploids (B. napus, B. juncea and B. carinata). The finding that genome-specific retention or loss of chromosomes in hybrids of O. violaceus with B. carinata and synthetic Brassica hexaploids (2n=54, AABBCC) is likely related to nucleolar dominance gives new insight into the molecular mechanisms regarding the cytology in these hybrids. It is proposed that the preferential expressions of genes for centromeric proteins from one parent (such as the well presented centromeric histone H3) are related with chromosome stability in wide hybrids and nucleolar dominance is beneficial to the production of centromere-specific proteins of the rRNAs-donor parent and to the stability of its chromosomes.