Unselected Strain Research Articles

Decreased ivermectin and moxidectin sensitivity in Haemonchus contortus selected with moxidectin over 14 generations

Ivermectin resistance in the nematode Haemonchus contortus has been reported in many parts of the world and many ivermectin resistant isolates have been found to have reduced sensitivity to moxidectin. However, it is unclear whether parasites that are selected with moxidectin would demonstrate reduced sensitivity to ivermectin. In this study, the effects of moxidectin and ivermectin on an unselected strain and a strain of H. contortus derived from the unselected strain but selected over 14 generations with moxidectin, were compared in jirds. The recovery of adult worms and fourth stage (L 4) larvae following treatment were compared between strains and anthelmintics. Moxidectin-selected H. contortus showed reduced sensitivity to ivermectin as well as to moxidectin. Doses of 0.1 mg/kg of moxidectin and 0.4 mg/kg of ivermectin were necessary to obtain an efficacy of 95% or above against the moxidectin-selected strain of H. contortus compared with 0.025 mg/kg for moxidectin and 0.1 mg/kg for ivermectin required for a similar efficacy in the unselected strain.

Histopathological Effects and Growth Reduction in a Susceptible and a Resistant Strain of Heliothis virescens (Lepidoptera: Noctuidae) Caused by Sublethal Doses of Pure Cry1A Crystal Proteins from Bacillus thuringiensis

Two strains of the tobacco budworm Heliothis virescens, one selected in the laboratory for resistance to Cry1Ac crystal protein from Bacillus thuringiensis (for which the mechanism of resistance was not associated with reduced binding) and an unselected control strain, were exposed to sublethal doses of pure Cry1A crystal proteins. A histopathological study was conducted to determine the epithelial damage caused by ingestion of Cry1Ac. Tissue sections of the midgut were obtained after 20, 40 and 60 min of toxin ingestion. Histopathological changes were observed primarily in columnar cells and were time-dependent. However, essentially the same level of damage was observed in the two strains. Toxin feeding tests with Cry1Ac and Cry1Ab, indicated that the toxins retarded growth and inhibited food intake of susceptible larvae, but did not significantly affect larvae of the resistant strain. Since the histopathological damage was similar in both strains, it appears that resistant larvae could repair (or substitute) more readily the damaged cells.

Incidence of skeletal muscle damage in selected and unselected strains of turkey

Incidence of skeletal muscle damage in selected and unselected strains of turkey

Selection at a P-glycoprotein gene in ivermectin- and moxidectin-selected strains of Haemonchus contortus

Resistance to anthelmintics that are used to control parasite populations in domestic animals has become a serious problem worldwide. The development of resistance is an evolutionary process that leads to genetic changes in parasite populations in response to drug exposure. The anthelmintic ivermectin is known to bind to the human membrane transport protein, P-glycoprotein, and P-glycoprotein-deficient mice treated with ivermectin have shown signs of neurotoxicity. P-glycoprotein is believed to be involved in the multidrug resistance phenotype seen in some human cancers and for drug resistance in some protists. We have examined the genetic variation of a P-glycoprotein homologue from the nematode Haemonchus contortus to see if an association exists between specific alleles of this gene and survival to exposure to ivermectin or moxidectin. Two parasite strains passaged without drug treatment and three strains, subjected to anthelmintic selection and derived from the unselected strains, were examined. Allelic variation in the unselected strains showed this locus to be highly polymorphic. χ 2 analyses of allele frequencies showed significant differences between the unselected and the drug-selected derived strains. In all three drug-selected strains, an apparent selection for the same allele was observed. These findings suggest that P-glycoprotein may be involved in resistance to both ivermectin and moxidectin in H. contortus.

Haemonchus contortus: Selection at a Glutamate-Gated Chloride Channel Gene in Ivermectin- and Moxidectin-Selected Strains

Blackhall, W. J., Pouliot, J.-F., Prichard, R. K., and Beech, R. N. 1998.Haemonchus contortus: Selection at a glutamate-gated chloride channel gene in ivermectin- and moxidectin-selected strains.Experimental Parasitology90, 42–48. Anthelmintic resistance in nematode parasites of livestock is a serious problem worldwide. Ivermectin, an avermectin, and moxidectin, a milbemycin, are potent endectocides commonly used to control these parasites. The proposed mode of action of avermectins and possibly the milbemycins involves the binding of the drug to the α-subunit of a glutamate-gated chloride channel, which opens or potentiates gating of the channel and leads to the hyperpolarization of the target neuromuscular cell. Glutamate gates the channel by binding to the β-subunit. We have cloned a fragment of a putative α-subunit gene fromHaemonchus contortus.The sequence of the beta subunit is available from GenBank. Genetic variability of this fragment was analysed by single-strand conformation polymorphism in five strains ofH. contortus: two strains passaged without drug selection, two strains selected with ivermectin, and one strain selected with moxidectin. One allele of the putative α-subunit gene appeared to be associated with resistance to the drugs, increasing in frequency in the three drug-selected strains relative to the unselected strains. Another allele appeared to be associated with susceptibility, decreasing in frequency in the three drug-selected strains relative to the unselected strains. A similar analysis of the β-subunit gene showed no significant differences in allele frequencies between the unselected and drug-selected strains. Our findings suggest a correlation between changes in allele frequencies of the putative α-subunit gene and resistance to ivermectin and moxidectin.

Evidence for a genetic variation in the mitochondrial genome affecting traits in White Leghorn chickens.

A mitochondrial Mspl RFLP which was coselected with Marek's disease (MD) resistance in White Leghorn chickens was mapped to the NADH subunit IV. The RFLP was due to a transition, resulting in the change of the low-usage threonine triplet ACT (Mspl- allele) to the high usage triplet ACC (Mspl+ allele). Trait association studies within an unselected strain revealed that the Mspl- allele whose frequency was reduced in MD resistant strains was associated with high body weight and high egg specific gravity (a measure of eggshell thickness). Analysis at three different time points indicated a significant interaction between the mitochondrial genotype and the growth hormone genotype in early but not in late adulthood. The analysis indicates that mitochondrial variants may contribute to phenotypic variation in chickens and that such contributions may be dependent on the genetic background.

Ivermectin resistance in nematodes may be caused by alteration of P-glycoprotein homolog

Resistance to ivermectin and related drugs is an increasing problem for parasite control. The mechanism of ivermectin resistance in nematode parasites is currently unknown. Some P-glycoproteins and multidrug resistance proteins have been found to act as membrane transporters which pump drugs from the cell. A disruption of the mdr1a gene, which encodes a P-glycoprotein in mice, results in hypersensitivity to ivermectin. Genes encoding members of the P-glycoprotein family are known to exist in nematodes but the involvement of P-glycoprotein in nematode ivermectin-resistance has not been described. Our data suggest that a P-glycoprotein may play a role in ivermectin resistance in the sheep nematode parasite Haemonchus contortus. A full length P-glycoprotein cDNA from H. contortus has been cloned and sequenced. Analysis of the sequence showed 61–65% homology to other P-glycoprotein/multidrug resistant protein sequences, such as mice, human and Caenorhabditis elegans. Expression of P-glycoprotein mRNA was higher in ivermectin-selected than unselected strains of H. contortus. An alteration in the restriction pattern was also found for the genomic locus of P-glycoprotein derived from ivermectin-selected strains of H. contortus compared with unselected strains. P-glycoprotein gene structure and/or its transcription are altered in ivermectin-selected H. contortus. The multidrug resistance reversing agent, verapamil, increased the efficacy of ivermectin and moxidectin against a moxidectin-selected strain of this nematode in jirds ( Meriones unguiculatus). These data indicate that a P-glycoprotein may be involved in resistance to ivermectin and other macrocyclic lactones in H. contortus.

Genetic Variation in Larval Survival and Growth and Response to Selection by Colorado Potato Beetle (Coleoptera: Chrysomelidae) on Tomato

Leptinotarsa decemlineata (Say) originating from potato in Massachusetts (UM strain) differed from those originating from potato in New York (NY strain) in their ability to use tomato, Lycopersicon esculentum Miller, as a host. Larvae from the UM strain had higher survival and weighed more on tomato than larvae from the NY strain. Both strains responded to selection for adaptation to tomato. Larval survival to 4-d and the weight of 4-d-old larvae significantly increased in the NY strain after adults and larvae were maintained on tomato for 3 generations. The UM strain did not respond to selection during 4 generations; but 4-d-old larvae reared on tomato were significantly larger after 12 generations of selection than in the unselected control. In both strains, weights of larvae reared on tomato increased when selection on tomato occurred only during the larval stage. Reciprocal crosses between the selected and unselected strains provided no evidence for sex linkage or cytoplasmic inheritance of the differences in larval survival and larval weight on tomato. Similarly, there was no evidence that the food plant of the mother affected the survival of her offspring on either tomato or potato. However, there was evidence that the choice of food plant made by the mother during larval and adult stages affected the 4-d weight of her offspring on tomato; this effect was observed in the unselected NY strain, but not in a UM strain that had been selected in the larval stage on tomato for 8 generations. Increased adaptation to tomato was not associated with a decrease in adaptation to potato, as measured by weight and survival of 4-d-old larvae.

Individual Differences in Plasma Catecholamine and Corticosterone Stress Responses of Wild-Type Rats: Relationship With Aggression

Plasma noradrenaline (NA), adrenaline (A), and corticosterone (CS) responses to social and nonsocial stressors were studied in male members of a strain of wild-type rats, widely differing in their level of aggression. The aggressiveness was preliminarily established by measuring the latency time to attack (ALT) a male intruder in a standard resident-intruder test. Animals were then provided with a jugular vein cannula for blood sampling during stress exposure. Implanted rats were randomly assigned to 3 experimental treatments: social stress (defeat experience, SD), nonsocial stress (presentation of a shock-prod, SP) and control (animals undisturbed in their home cages, CTR). A significant correlation was found between ALT and the amount of time spent in burying the probe in SP rats: the more aggressive the animal, the higher the rate of burying behavior. SD induced a much stronger effect on plasma NA, A, and CS concentrations than SP. A significant negative correlation was found between ALT scores and values of the area under the response time curve for NA and A, in both SD and SP situations: the more aggressive the animal, the higher the catecholaminergic reactivity to the stressors. On the contrary, no evidence of a correlation between aggressiveness and plasma corticosterone responses was found, neither in SD nor in SP rats. These findings in an unselected strain of wild-type rats confirmed that an aggressive/active coping strategy is associated with a high sympathetic-adrenomedullary activation and support the concept of individual differentiation in coping styles as a coherent set of behavioral and neuroendocrine characteristics.

Insecticide Susceptibility and Detoxication Enzyme Activities in Permethrin-Selected Diamondback Moths

Selection of larvae of the diamondback moth,Plutella xylostella(L.), with permethrin increased resistance. After 21 generations of continuous selection pressure, resistance to permethrin was over 600-fold in this strain compared with the unselected parental strain. The permethrin-selected strain was also cross-resistant to all pyrethroids tested, but remained susceptible to organophosphorus, carbamate, cyclodiene, chloronicotinyl, avermectin, and microbial insecticides tested. Detoxication enzyme assays revealed that activities of microsomal oxidases (epoxidases, hydroxylases, sulfoxidase,N-demethylase, andO-dealkylases), glutathioneS-transferases (DCNB, CDNB, and PNPA conjugation), hydrolases (general esterase, carboxylesterases, β-glucosidase, acetylcholinesterase, and carboxylamidase), and reductases (juglone reductase and cytochrome c reductase) and levels of cytochrome P450 and cytochrome b5were similar in the permethrin-selected and parental strains. There was no difference in the rate of cuticular penetration of permethrin between the two strains. Synergist studies showed that neither piperonyl butoxide (microsomal oxidase inhibitor) norS,S,S-tributyl phosphorotrithioate (DEF, esterase inhibitor) reduced the level of resistance to permethrin. The results indicated that pyrethroid resistance observed in this strain was most likely attributed to decreased target site sensitivity.

Lack of effect of dopamine d 2 blockade on ethanol intake in selected and unselected strains of rats

Previous research has suggested that brain catecholamines may be involved in regulating ethanol intake. This study was designed to look more specifically at dopamine (DA) and whether DA D 2 receptor blockade with the antagonist pimozide would alter ethanol consumption in rats. Subjects were male Maudsley Reactive and Wistar rats, the former previously shown to consume larger amounts of ethanol than the latter. Both strains were screened for ethanol intake by presentation of ethanol solutions (free choice with water) in increasing steps from 2% to 10% (v/v) on an alternate-day schedule. Following the screening period, animals were switched to a schedule of everyday presentation of the 10% (v/v) ethanol solution (free choice with water) for 10 baseline days. Animals were then divided into high and low drinking levels according to whether their mean baseline ethanol intake (g/kg) fell within ±0.5 SD of the mean intake of their group (Maudsley Reactives: mean = 2.55 g/kg, low drinkers < 1.63, high drinkers > 3.47; Wistars: mean = 2.17 g/kg, low drinkers < 1.53, high drinkers > 2.82). The animals were assigned to one of five treatment groups for 5 subsequent days where they received IP injections of pimozide (0.08, 0.24, or 0.48 mg/kg), tartaric acid, or saline. Following the treatment period, ethanol consumption was recorded for 5 posttreatment days. No significant differences due to treatment were observed for either intake or preference of ethanol across treatments, drinking groups, or strains. The results obtained in the present study suggested that interference in DA neurotransmission through administration of the D 2 antagonist pimozide does not significantly alter ethanol consumption in either MR or Wistar animals.

Origin and implications of bovine spongiform encephalopathy.

All spongiform encephalopathies in animals, including humans, are slow developing infectious diseases. The current working theory links the origin of bovine spongiform encephalopathy (BSE) to the feeding of cattle with meat and bone meal prepared from scrapie-infected sheep remains. Recycling of cattle meat and bones (MBM) essentially resulted in the selection of a single strain from the "wild type", a mixture of 20 strains. The BSE agent is easily transmitted through ingestion, with some evidence of vertical transmission. Paradoxically, cattle have selected a major new strain which appears to be more virulent than an unselected strain found in scrapie sheep. The same strain of BSE agent is implicated in the occurrence of spongiform encephalopathy in domestic cats, tiger, and some exotic species of ruminants in zoos. The properties of BSE and its spread into cattle are still disputed. Since our understanding of the disease and its transmissibility in humans must await observations that will be made over some years to come, it is important to keep a reasonable perspective and ensure that any speculative comment is consistent with fact. In risk assessment in such circumstances, it is tempting give too much credence to persuasive parallels when direct relevant information is not available. On the other hand, it would also not be wise to assume that the disease will die by itself and will have no effect on humans.

Polygenic mutation in Drosophila melanogaster: non-linear divergence among unselected strains.

A highly inbred strain of Drosophila melanogaster was subdivided into 20 replicate sublines that were maintained independently with 10 pairs of randomly sampled parents per generation for 180 generations. The variance between lines in abdominal and sternopleural bristle number increased little after 100 generations, in contrast to the neutral expectation of a linear increase; and the covariances of line means between different generations declined with increasing number of generations apart, in contrast to the neutral expectation of constant covariance. Thus, under a neutral model, the estimates of mutational variance were lower than for previous estimates from the first 100 generations of subline divergence. An autoregressive model was fitted to the variance of line means that indicated strong natural selection. There is no single unequivocal explanation for the results. Possible and nonexclusive alternatives include stabilizing selection on bristle number and deleterious effects on fitness of bristle mutations. The inferred strengths of selection on both traits are too high for stabilizing selection alone, and the between-line variance did not continue to increase sufficiently for pleiotropy alone to account for the observations. A third potential explanation that does not invoke selection is duplicate epistasis between mutations affecting bristle number.

Laboratory Selections of Populations in the Citrus Red Mite, &lt;i&gt;Panonychus citri&lt;/i&gt; (MCGREGOR), with Hexythiazox and Their Cross Resistance Spectrum

Laboratory selection experiments for resistance and for susceptibility with hexythiazox were conducted using Haibara strain of citrus red mite, Panonychus citri MCGREGOR collected from a citrus orchard at Haibara Agricultural Research Station (Nippon Soda Co., Ltd.) in Shizuoka Prefecture, Japan. The mites had been selected with hexythiazox 17 times in the orchard. A high level of hexythiazox resistance developed with 6 successive laboratory selections for resistance, and the resistance ratios at LC 50 were 23,000 and 4450 compared with the unselected strain and parental generation for laboratory selection, respectively. After 5 selections for susceptibility, the strain became more susceptible to hexythiazox, and the susceptibility ratio at LC 50 was 0.59. In the resistant strain, the levels of the resistance were high at all developmental stages including eggs laid by treated adult females. No synergistic effects of piperonyl butoxide with hexythiazox were observed on the activity against eggs and eggs laid by treated females in the resistant strain. Among 20 miticides investigated about their efficacies against the hexythiazox resistant strain, clofentezine, flufenoxuron and flucycroxuron showed low activities to the resistant strain.

Voluntary ethanol consumption in rats

The present investigation examined two methods of ethanol presentation to laboratory rats that have been used to examine the mechanisms mediating voluntary ethanol intake in animals. Experiment I examined whether a restricted access procedure had any significant and meaningful relationship in individual animals to drinking behavior in an unrestricted 24h paradigm. An unselected strain of rats was given free access (unrestricted 24h free choice) to ethanol and water, and later exposed to a restricted 10min access to ethanol. A significant positive relationship between the absolute amount of ethanol consumed in the 24h access paradigm and the amount ingested by the same animals in the restricted access procedure was demonstrated. Experiment 2 examined the extent to which a forced choice preference testing procedure, commonly used in screening ethanol-preferring P rats, was in and of itself sufficient to produce increased levels of ethanol consumption in unselected Long-Evans rats. Results indicated that subjects receiving only 4 days of forced exposure to 10% ethanol consumed, over the next eight ethanol presentations, levels of ethanol exceeding 5g/kg with a 0.60 preference ratio. A microstructural analysis of the pattern of free choice ethanol intake following forced ethanol exposure (Experiment 3) revealed that rats consumed ethanol within short discrete bouts with the largest of these daily bouts consisting of approximately 4ml (0.75g/kg) of 10% ethanol. The amount consumed during the restricted access bout of Experiment 1 was seen to be within the range of the bouts recorded in Experiment 3. These results suggest that consumption of ethanol during the restricted access may simulate an individual bout of ethanol intake during non-restricted access. The results support the notion that many of the different ethanol drinking models used may have a common basis and that the assessment of the amount and pattern of intake across methods and strains may represent different but equally valid approaches to the study of the same underlying mechanisms.

Reversal of resistance to Bacillus thuringiensis in Plutella xylostella.

Continued success of the most widely used biopesticide, Bacillus thuringiensis, is threatened by development of resistance in pests. Experiments with Plutella xylostella (diamondback moth), the first insect with field populations resistant to B. thuringiensis, revealed factors that promote reversal of resistance. In strains of P. xylostella with 25- to 2800-fold resistance to B. thuringiensis compared with unselected strains, reversal of resistance occurred when exposure to B. thuringiensis was stopped for many generations. Reversal of resistance was associated with restoration of binding of B. thuringiensis toxin CryIA(c) to brush-border membrane vesicles and with increased biotic fitness. Compared with susceptible colonies, revertant colonies had a higher proportion of extremely resistant individuals. Revertant colonies responded rapidly to reselection for resistance. Understanding reversal of resistance will help to design strategies for extending the usefulness of this environmentally benign insecticide.

Selective breeding ofSteinernema feltiae(Filipjev) (Nematoda: Steinernematidae) for improved efficacy in control of a mushroom fly,Lycoriella solaniWinnertz (Diptera: Sciaridae)

A method of selecting a Steinernema feltiae strain that is effective against a mushroom fly, Lycoriella solani, is described in detail. The pest control efficacy of the selected nematode strain was evaluated and compared with the efficacy of two unselected strains. The selection procedure was designed to give preference to nematode individuals with the greatest ability (1) to search effectively for the target insect larvae in their natural habitat, (2) to infect them shortly after application and (3) to reproduce in their haemocoel. Thirty‐four rounds of selection achieved a 4‐fold improvement in nematode ability to find and parasitize third‐ and fourth‐instar larvae of the pest in the mushroom substrate. In 24‐h laboratory experiments, mortality of the insect caused by nematode juveniles rose from 22.5%, recorded for the original unselected isolate, to 92.5% for the selected strain. In a 51‐day experiment conducted on a mixed age mushroom house population of L. solani, the enhanced pest control ability o...

Biological differences between strains of Tribolium castaneum selected for resistance to hypoxia and hypercarbia, and the unselected strain

Abstract. A strain of the red flour beetle Tribolium castaneum (Herbst) was selected separately for resistance to hypoxia and hypercarbia. At the 21st generation, adults and pupae of the line selected for resistance to hypoxia were significantly heavier than those selected for resistance to hypercarbia and the unselected line. It also exhibited a more prolonged developmental range than the unselected line. The line selected for resistance to hypercarbia was also significantly heavier than the unselected line. When the selected and unselected lines were subjected to starvation at the 26th generation, metabolism in both selected lines proceeded at a lower rate than in the unselected line and consequently the selected lines survived for much longer periods.

Increase in cold‐shock tolerance by selection of cold resistant lines in Drosophila melanogaster

Abstract. In Drosophila melanogaster, the cold‐shock tolerance of adult flies at ‐7°C increased 22% after a prior 2h exposure to 4°C as measured by LD50, the dose (degree minutes of exposure to subzero temperature) which resulted in 50% mortality. Cold‐shock tolerance was further significantly increased by selecting cold resistant lines by exposure of adults (1) to 4°C for 2 h (short‐term chilling), or (2) to ‐7°C for 80–120 min (cold shock), or (3) to short‐term chilling followed by cold‐shock. After ten generations of selection, the greatest increase in cold‐shock tolerance was found in flies selected using the combined exposure of short‐term chilling and cold shock. LD50s increased 33% in comparison with the unselected control strain when no chilling pre‐treatment was given prior to cold shock at ‐7°C. The rapid cold‐hardening response increased 82% in the line selected by the short‐term chilling and cold‐shock regime. The enhanced cold‐shock tolerance was relatively stable since no decrease was observed after four generations without selection. This report shows the role of short‐term adaptation as well as selection in the capacity to survive low temperatures in non‐diapausing stages of insects.

Associations Between Major Histocompatibility Complex Genes and Production Traits in White Leghorns

The frequency of the MHC haplotype B15 had been found in a previous study to be more than two times higher in a White Leghorn line selected for high egg production compared with the unselected control strain. To further evaluate these findings, matings were performed between chickens with the same heterozygous B genotypes, being combinations of the most frequent haplotypes, i.e., B15, B19, and B21. In total, more than 1,300 observations from two generations were analyzed. In each generation, approximately one half of the chickens were derived from the line selected for total egg mass, the other half from the control strain. The MHC genotypes were determined serologically. Additive and dominance effects of B haplotypes on production traits were analyzed using an individual animal model. The estimation of genotypic values, together with the analysis of gene substitution effects, showed that the B15 haplotype was associated with early sexual maturity and low egg production during the late production period, i.e., between 43 and 63 wk of age, whereas B19 was associated with later onset of sexual maturity. The association of B15 with early sexual maturity would thus explain the high frequency of the B15 haplotype previously observed in a line selected for high early egg production. No dominance effect of the B system was observed for any of the traits, suggesting that the present results were due predominantly to additive gene effects.