Progress 10/01/99 to 10/01/03
Outputs
1. A cytochrome c oxidase subunit I gene (COXI), was identified and isolated as a differentially expressed gene between insecticide susceptible ACY and resistant Apyr-R German cockroach strains using PCR-selected subtractive hybridization and cDNA array techniques. The cDNA sequence of COXI has an open ready frame of 1533 nucleotides encoding a putative protein of 511 amino acid residues. Northern blot analysis indicated that levels of COXI expression were similar in three life stages (eggs, nymphs, and adults) of the susceptible ACY strain. The expression of COXI in the resistant Apyr-R strain was developmentally regulated, with low expression in eggs, an increase (~1.4-fold) in nymphs, and rose to a maximum (~3-fold) in both adult females and males. Comparison of COXI expression between ACY and Apyr-R strains indicated that there was no difference in the eggs of the two strains, but expression was higher (~1.5-fold) in nymphs and much higher (~3- to 4-fold) in adult
males and females of the Apyr-R strain. Levels of COXI mRNA were about 1.4- and 1.7-fold increases in the abdomen tissues compared with the head+thorax tissues of ACY and Apyr-R strains, respectively (Fig. 4A, B). Although expression patterns of COXI in head+thorax and abdomen tissues were similar (i.e. lower in the head+thorax tissues and higher in the abdomen tissues) in both the ACY and Apyr-R strains, the expression of COXI was about 2.5-fold higher in the head+thorax and ~3-fold higher in abdomen tissues of the Apyr-R strain compared with the corresponding ACY samples. The overexpression of COXI in resistant German cockroaches merits the investigation of the importance of the gene in insecticide resistance of German cockroaches. 2. Two cytochrome P450 genes, CYP4AB1 and CYP4AB2, and the Gp-9 gene were identified as being specifically overexpressed in workers of the red imported fire ant using PCR-selected subtractive hybridization and cDNA array techniques. Full-length CYP4AB1
and CYP4AB2 were cloned and sequenced. The cDNA sequences of CYP4AB1 and CYP4AB2 have open reading frames of 1389 and 1533 nucleotides encoding proteins of 463 and 511 amino acid residues, respectively. Northern blot analysis was performed to compare expression levels of CYP4AB1, CYP4AB2, and Gp-9 for different developmental stages and castes of fire ants. We demonstrate that the expression of these three genes is developmentally and caste specifically regulated in red imported fire ants. Levels of CYP4AB1 mRNA were undetectable in 3rd+4th instars, worker pupae, and alate (mixed sex) pupae; readily detectable in male and female alates; increased (~3-fold) in the queens; and rose to a maximum (13-fold) in workers. Similarly, the expression of CYP4AB2mRNA was undetectable in 3rd+4th instars, worker pupae, and alate pupae; low in male and female alates and queens; and increased (~7-fold) in workers. Levels of Gp-9 mRNA were readily detectable in male alates; increased (~3-fold) in female
alates; and reached a maximum (~12-fold) in workers. Their caste-specific overexpression suggests the functional importance of CYP4AB1, CYP4AB2, and Gp-9 in workers of the red imported fire ant.
Impacts
Our studies will offer tremendous potential in broadening our understanding of the molecular basis of insecticide resistance and, in turn, will provide important information for designing novel strategies to manage insect pests. Our studies will also lead to a understanding of the molecular basis of development, reproduction and defense mechanisms for the red imported fire ant, which is of fundamental biological interest and may result in the discovery of a better way to manage the red imported fire ant.
Publications
- Pridgeon, J. W., L. Zhang, and N. Liu. 2003. Overexpression of CYP4G19 associated with pyrethroid resistant German cockroaches. Gene 314, 157-163_____________________________________________________ Pridgeon, J. W. and N. Liu. 2003. Overexpression of the cytochrome c oxidase subunit I gene associated with a pyrethroid resistant strain of German cockroaches, Blattella germanica. Insect Biochem. Mol. Biol. 33, 1043-1048. ____________________________________________________ Liu, N and L. Zhang. CYP4AB1, CYP4AS1 and Gp-9 overexpression in workers of the red imported fire ant, Solenopsis invicta. Gene 327, 81-81.
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Progress 01/01/02 to 12/31/02
Outputs
1. We have investigated mechanisms of pyrethroid resistance in SeALHF and ALHF house flies. Our study indicated that P450 monooxygenase-mediated metabolism plays a major role in permethrin resistance in SeALHF, while hydrolytic metabolism has a minor contribution to resistance. A 392 bp para-type sodium channel gene fragment, where kdr (L1014F) and super-kdr (M918T) mutations reside, was generated from ALHF and SeALHF. The M918T mutation was not detected in ALHF or SeALHF. The L1014F mutation was present in ALHF, but not in SeALHF, suggesting that the kdr mutation is an important factor in pyrethroid resistance in ALHF. A leucine to histidine (L1014H) substitution at the position corresponding to kdr mutation was detected in SeALHF. 2. A new cytochrome P450 gene, CYP4G19, was identified and isolated as a differentially expressed gene between insecticide susceptible ACY and resistant Apyr-R German cockroach strains. Northern blot analysis indicated that levels of
CYP4G19 expression were low in eggs, nymphs, and adults of the susceptible ACY strain with a similar expression pattern. The expression of CYP4G19 in the resistant Apyr-R strain was developmentally regulated, with very low expression in eggs, increasing in nymphs, and reaching a maximum in both female and male adults. Comparison of CYP4G19 expression between ACY and Apyr-R strains indicated that there was no difference in their eggs, but expression was higher (1.7-fold) in the nymphs, and much higher (5-fold) in the male and female adults of the Apyr-R strain. Although expression patterns of CYP4G19 in head+thorax and abdomen tissues were similar (i.e. lower in head+thorax tissues and higher in abdomen tissues) in both the ACY and Apyr-R strains, the overexpression was more evident in the Apyr-R strain in both head+thorax and abdomen tissues than in the ACY strain. 3. We obtained 17 cDNA fragments that were specifically overexpressed in queens and 2 cDNA fragments overexpressed in
workers. Northern blotting results indicated that 7 of the 17 candidate clones showed consistent overexpression in fire ant queens and 2 clones were overexpressed in workers. These clones showed amino acid sequence homology with the vitellogenin gene (Vg1), RNA helicase, RAD16 protein, SPARC, cytochrome P450 genes (family 4), and pheromone binding protein (PBP). Our results showed that the cytochrome P450 gene was overexpressed in workers; the vitellogenin gene was only expressed in female alates and queens; and the putative pheromone binding protein was expressed in workers, female alates, and queens, with much higher expression in workers.
Impacts
Our studies will offer tremendous potential in broadening our understanding of the molecular basis of insecticide resistance and, in turn, will provide important information for designing novel strategies to manage insect pests. Our studies will also lead to a understanding of the molecular basis of development, reproduction and defense mechanisms for the red imported fire ant, which is of fundamental biological interest and may result in the discovery of a better way to manage the red imported fire ant.
Publications
- Liu, N. and J. W. Pridgeon. 2002. Metabolic detoxication and the kdr mutation in pyrethroid resistant house flies, Musca domestica (L.). Pestic. Biochem. Physiol. 73, 157-163.
- Pridgeon, J. W., A. G. Appel, W. J. Moar, and N. Liu. 2002. Variability of resistance mechanisms in three strains of pyrethroid resistant German cockroach, Blattella germanica (L.). Pestic. Biochem. Physiol. (Pestic. Biochem. Physiol. 73, 149-156.
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Progress 01/01/01 to 12/31/01
Outputs
1. To investigate pyrethroid resistance in H. zea, and to continue the successful use of Bt cotton and pyrethroid insecticides, we tested susceptibility of field population of H. zea to pyrethroid insecticides in Alabama (Tennessee Valley Substation, Limestone County; Wiregrass Experimental Station, Headland; E.V. Smith Research Center, Lee County) since 1998. Our study has indicated that 14 to 26% field population of H. zea in the area of Lee County, 3 to 14% in Limestone County, and 6 to 9 % in Headland developed resistance to pyrethroid insecticides. Although these levels of survival of H. zea might not cause the economic problems, these results have indicated the potential of pyrethroid resistance in H. zea. in the Alabama State. 2. I have focused my research on the genetics and molecular mechanisms of resistance in house flies. We have determined that multi-mechanisms are involved in pyrethroid resistance and cross-resistance in an Alabama house fly strain
(ALHF) and one of the mechanisms is increased cytochrome P450-mediated detoxication. A paper documenting this work has been published (Liu and Yue 2000); we have identified that resistance in ALHF is linked to autosomes 1, 2, 3, and 5 (Liu and Yue, 2001); we have isolated two new cytochrome P450 genes, CYP28B1 and CYP4G13v1, and their 5' flanking regions from ALHF (Liu and Zhang, 2002, Insect Biochem. Mol. Biol. (In press)); and we have, for first time, identified several genes that are only expressed when flies were treated by insecticides and propose that these genes may have a role in resistance (Zhang and Liu, in preparation). 3. We have identified several resistance mechanisms that exist in an Alabama German cockroach strain (Wei et al. Pest Manag. Sci. 57, 1055-1059). We have recently found several genes are expressed differentially between susceptible and resistant strains. We proposed that these genes might be involved in resistance and are now isolating and characterizing the
gene and their functions in resistance. 4. My research has been concentrated on identification and characterization gene(s) involved in the regulation of development, reproduction, defense, and pheromone production in the red imported fire ant, Solenopsis invicta (B.). Cytochrome P450 genes are involved in the synthesis and degradation of many insect hormones and pheromones, and play an important role in growth and development. We have successfully cloned and sequenced the first two P450 genes from the fire ant (Liu. in preparation). The role of these P450 genes in development, reproduction and pheromone production is under investigation. We know that substantial physiological differences between queen and worker fire ants must reflect differential expression of genes present in both castes. It is reasonable to hypothesize that some differentially expressed genes may play roles in the regulation of pheromone production in fire ant queens and/or in the regulation of the defense system
in workers. Thus, we are conducting the study on identification and characterization of the gene(s) involved in the regulation of pheromone production the regulation of the defense system.
Impacts
Our studies will greatly improve our understanding of insecticide resistance and provide very important information for designing strategies to control insect pests.
Publications
- Liu, N. and L. Zhang. 2002. Identification of two cytochrome P450 genes and their 5' Flanking regions from the house fly, Musca domestica (L). Insect Biochem. Mol.Biol. (In press)
- Liu, N. and X. Yue. 2001. Genetics of pyrethroid resistance in a strain (ALHF) of house fly (Diptera: Muscidae). Pestic. Biochem. Physiol. 70, 151-158.
- Wei, Y., A. G. Appel, W. J. Moar, and N. Liu. 2001. Pyrethroid resistance and cross-resistance in the German cockroach, Blattella germanica (L). Pest Manag. Sci. 57,1055-1059.
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Progress 01/01/00 to 12/31/00
Outputs
1. Investigation of Pyrethroid Resistance and PotentialCross-Resistance in Cotton Bollworm, Helicoverpa zea. The tests were conducted from May to September using Hercon H. zea pheromone traps. Results showed that 14 to 26 % field population of H. zea in the area of Lee County, 3 % in Limestone County and 6 % in Headland have developed resistance to pyrethroid insecticides. According to these three years' investigation, we propose that the potential of the pyrethroid resistance in H. zea is exited. 2. Identifying Differentially Expressed Genes between Queens and Workers of the Imported Fire Ant, Solenopsis Invicta B. We have compared mRNA populations in queens and workers to analyze genes that are differentially expressed between both castes using differential display. 12 cDNA bands were apparently unique to samples from queens and 7 cDNA bands were detected solely in samples from workers. Six of the twelve cDNA fragments showed amino acid sequences significantly
matched with protein sequences in the GenBank database. Another six cDNA fragments from the sample of queens failed to return significant matches with any existing sequences from GenBank databases either because the fragments was too short or because they might be unique sequences that had not been published. 3. Pyrethroid Resistance and Mechanisms in the German Cockroach, Blattella germanica (L.) A German cockroach strain, Apyr-R, was collected from Opelika, Alabama after control failures with pyrethroid insecticides. Levels of resistance to permethrin and deltamethrin in Apyr-R (97- and 480-fold, respectively, compared with a susceptible strain, ACY) were partially or mostly suppressed by PBO and DEF, suggesting that P450 monooxygenases and hydrolases are involved in resistance. Injection did not change levels of resistance to either permethrin or deltamethrin, suggesting that decreased cuticular penetration (pen) may not play an important role in pyrethroid resistance in Apyr-R. 4.
Insecticide Resistance and genetics of ressitance in the House Fly, Musca domestica. A house fly strain, ALHF, was collected from a poultry farm in Alabama after a control failure with permethrin and further selected in the laboratory with permethrin for five generations. The level of resistance to permethrin in ALHF was 1800-fold after selection. Incomplete suppression of permethrin resistance by PBO and DEF reveals that P450 monooxygenase- and hydrolase-mediated detoxication, and one or more additional mechanisms are involved in resistance to permethrin. Five house fly lines were derived from crosses of the pyrethroid resistant ALHF and susceptible aabys strains. The level of resistance to permethrin was measured for each line to determine the genetic linkage of pyrethroid resistance in ALHF. Our study has demonstrated that factors on autosomes 3 and 5 play very important roles in pyrethroid resistance while factors on autosomes 1 and 2 may have relatively small roles in resistance.
Study also suggests that the factor(s) on autosome 5 plays the most important role in P450 monooxygenase-mediated resistance. Injection study indicated that pendoes not play an role in ALHF resistance.
Impacts
Studies will provide very important information for designing strategies to control insect pests.
Publications
- Liu, N. and X. Yue. 2000. Insecticide resistance and cross-resistance in the house fly (Diptera: Muscidae). J. Econ. Entomol. 93, 1269-1275. _____________________________________________________________________ ___ Liu, N. and X. Yue. 2001. Genetics of pyrethroid resistance in the house fly (Diptera: Muscidae). Pestic. Biochem. Physiol. (Accepted). _____________________________________________________________________ ___
- Wei, Y., A. G. Appel, W. J. Moar, and N. Liu. 2000. Pyrethroid resistance and mechanisms in the German cockroach, Blattella germanica (L). Pest Manag. Sci. (Submitted). _____________________________________________________________________ ___
- Martin, S. H., Bacheler, J. S., Bagwell, R. D., Boyd, M. L., Freeman, B. L., Herzog, G. A., Johnson, D. R., Layton, M. B., Leonard, B. R., Liu, N., Payne, G. T., Pietrantonio, P. V., Roof, W. E., Seward, R., Sprenkel, R. K., Sullivan, J. W., Duyn, V. and Weeks, J. R. 2000. Status of bollworm, Helicoverpa zea, susceptibility to pyrethroids in the mid-south and southeast: IRAC-US 1999 Update. Proc. Beltwide Cotton Conf. Vol. 2, 1359-1364.
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Progress 01/01/99 to 12/31/99
Outputs
1. Investigation of pyrethroid resistance in Helicoverpa zea. We tested susceptibility of field population of H. zea to pyrethroid insecticides in Alabama. Our results showed that 15 to 20% field population of H. zea in the area of Lee county, 4 % in Limestone Count, and 9 % in Headland have developed resistance to pyrethroid insecticides in Alabama. These results are warning us that the pyrethroid resistance in H. zea. may start to be spread in the Alabama State. 2. Investigate the Level of Pyrethroid Resistance in House Fly from Alabama The toxicity of different insecticides (permethrin and deltamethrin (pyrethroid), baygon (carbamate), and fipronil (phenylpyrazole insecticide) to ALHF has been studied by topical application in the presence and absence of piperonyl butoxide (PBO), a P450 monooxygenase inhibitor. The level of resistance in ALHF was 1,750-fold higher compared to the aabys susceptible strain and was decreased to 51.8-fold following the treatment of
PBO. ALHF also shown cross-resistance to deltamethrin, baygon, and fipronil, respectively. Cross-resistance to deltamethrin, baygon, and fipronil can be suppressed to very low levels by PBO. Our results suggest that increased P450 monooxygenase mediated detoxification is one of the major mechanisms responsible for pyrethroid resistance and cross-resistance in ALHF. 3. Examine the Level of Total Cytochrome P450 in ALHF To better understand the role of P450 monooxygenase-mediated detoxification in pyrethroid resistance of ALHF and to determine whether its resistance is correlated with an increased level of cytochrome P450, the level of total cytochrome P450 in both ALHF and aabys strains was measured. Compared with the susceptible aabys strain, the ALHF strain was found to have 3.5-fold higher total cytochrome P450. This further indicates that pyrethroid resistance in ALHF is associated with an elevated level of total cytochrome P450. 4. Clone and Sequence the P450 Gene(s) from Both
ALHF and aabys Strains To better understand the molecular basis and regulation of P450 mediated resistance in ALHF, two new cytochrome P450 genes, CYP28B1 and CYP4G13v2 from ALHF and aabys strains had been cloned and sequenced by RT-PCR (reverse transcription-mediated polymerase chain reaction). 5' promoter regions of the two P450 genes from ALHF have been cloned and sequenced. 5. Investigate the Expression of the P450 in Both ALHF and aabys To determine the expression of CYP28B1 in both ALHF and aabys, mRNA levels of the new P450 in both strains were determined by northern hybridization. Our data indicated that the expression of the P450 gene in ALHF was higher compare to the aabys strain suggesting that overexpression of the new P450 may involved in resistance in ALHF. 6. Genetically manipulate the resistance gene in ALHF To do this, we have genetic transferred a modified mariner transposable element from Drosophila mauritiana into the germ line of the house fly, Musca domestica, by
electroporation.
Impacts
Our study will greatly improve our understanding of mechanisms involved in resistance and molecular basis of insecticide resistance mediated by cytochrome P450 monooxygenases. It will provide extremely important information to design resistance management tools to overcome or delay the development of resistance. This study will also provide a useful framework for the design of future experiments.
Publications
- Liu, N. and X. Yue. 2000. Permethrin resistance and cross-resistance to various insecticides in the house fly, Musca domestica (L) (Diptera: Muscidae). J. Econ. Entomol. (Accepted). ________ Martin et al. 2000. Status of bollworm, Helicoverpa zea, susceptibility to pyrethroids in the mid-south and southeast. Proc. Beltwide. Cotton. Conf. ________ Liu, N. 1999. Pyrethroid resistance in cotton bollworm, Helicoverpa zea. Annual report of cotton insecticide data, Alabama Cooperative Extension System. ________
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