Progress 10/01/06 to 09/30/11
Outputs
OUTPUTS: Chitin is the second most abundant biological polymer after cellulose. It is widely distributed in arthropods, fungi and nematodes. In arthropods, chitin is a vital component of the cuticular exoskeleton and thus is crucial for growth and development. Chitin is also found in internal structures of insects and other arthropods, including the cuticular linings of trachea and in the peritrophic matrix (PM) lining the gut epithelium. Chitin synthase is a crucial enzyme catalyzing the transfer of sugar moieties from activated sugar donors to specific acceptors. However, current knowledge on chitin synthase in insects, especially their structures, functions, and regulations, is still limited. This study is expected to better understand the molecular properties of insect chitin synthases and potentially help the development of new insecticides targeting on chitin metabolic pathways in insects. Results from this research have been published in two scientific journals including PLoS ONE (volume 6, article e19899) and Insect Biochemistry and Molecular Biology (volume 41, pages 521-528). Another manuscript has also been submitted to Insect Biochemistry and Molecular Biology. The results were also presented at the Third International Symposium on Insect Physiology, Biochemistry and Molecular Biology (Shanghai, China), the Institute of Zoology, Chinese Academy of Sciences (Beijing, China), the Chinese Center for Disease Control and Prevention (China CDC, Beijing, China), and the College of Agriculture and Biotechnology at Zhejiang University (Hangzhou, China). PARTICIPANTS: Xin Zhang, Graduate Student, Kansas State University, Manhattan, KS Sharon R. Starkey, Lab Technician, Kansas State University, Manhattan, KS TARGET AUDIENCES: The primary target audiences of this study are the researchers interested in chitin biosynthesis and the mode of action of insecticides. Since chitin is a vital component of the arthropod exoskeleton and peritrophic matrix, and is not present in vertebrates, our research may lead to new ideas for developing novel insecticides specifically targeting the chitin biosynthetic system for controlling mosquitoes and other arthropod pest species. Therefore, pesticide industries may be interested in our findings. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We characterized the enzymatic properties of the chitin synthase and its inhibition in the African malaria mosquito (Anopheles gambiae) by using a nonradioactive method. Our results showed that this method could be effectively used for high throughput assay of chitin synthesis in the crude enzymes from insects. Optimal conditions for the enzyme assays were determined. The optimal pH was 6.5-7.0, and the highest activity was detected at 37 and 44 degrees Celsius. Dithithreitol (DTT) was required for the activity to prevent melanization of the enzyme extract. Enzyme activity was enhanced at low concentration of the substrate, but inhibited at high concentrations. Proteolytic activation of the activity was significant both in the 500g supernatant and the 40,000g pellet. High concentration of diflubenzuron and nikkomycin Z showed marginal in vitro inhibitory effects on chitin synthase activity, whereas no in vivo inhibitory effects were observed in our experiments. We further identified and characterized two chitin synthase genes, AgCHS1 and AgCHS2, in African malaria mosquito (Anopheles gambiae). Transcriptional analysis indicated that AgCHS1 was expressed in egg, larval, pupal and adult stages whereas AgCHS2 appeared to be expressed at relatively low levels, particularly during the larval stages as examined by reverse transcription (RT)-PCR and real-time quantitative PCR. Relatively high expression was detected in the carcass followed by the foregut and hindgut for AgCHS1, and the foregut (cardia included) followed by the midgut for AgCHS2. Fluorescence in situ hybridization (FISH) and immunohistochemical analysis revealed new information including the localization of the two enzymes in the ommatidia of the compound eyes, and AgCHS2 in the thoracic and abdominal inter-segmental regions of pupal integument. This study has provided new information on chitin synthetic system in the mosquitoes, which is expected to help understanding the mechanism of chitin synthesis inhibition and regulation, and facilitate identifying vulnerable points of chitin biosynthesis and regulation for developing novel insecticides.
Publications
- Zhang J., Zhang X., Arakane Y., Muthukrishnan S., Kramer K. J., Ma E., Zhu K. Y. 2011. Comparative genomic analysis of chitinase and chitinase-like genes in the African malaria mosquito (Anopheles gambiae). PLoS ONE 6(5): e19899. doi:10.1371/journal.pone.0019899
- Zhang J., Zhang X., Arakane Y., Muthukrishnan S., Kramer K. J., Ma E., Zhu K. Y. 2011. Identification and characterization of a novel chitinase-like gene cluster (AgCht5) possibly derived from tandem duplications in the African malaria mosquito, Anopheles gambiae. Insect Biochem. Mol. Biol. 41: 521-528.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Chitin is the second most abundant biological polymer after cellulose, and is widely distributed in arthropods, fungi and nematodes. It is a vital component of the cuticular exoskeleton and inner linings of peritrophic matrixes (PM) of the gut epithelium and trachea in all arthropods. Arthropods depend on chitin biosynthesis for their growth and development. Chitinases belong to a large and diverse family of hydrolytic enzymes that break down glycosidic bonds of chitin. However, very little is known about the function of chitinase genes in regulating the chitin content in the PM of the midgut in insects. Our long-term goal is to understand chitin synthetic system, regulation and inhibition in arthropods. Our specific aims are to: 1) characterize chitin metabolic enzymes, including chitin synthases involved in chitin biosynthesis and chitinases involved in degradation of chitin, in insects; and 2) functionally analyze the genes encoding chitin metabolic enzymes in cuticular and PM chitin biosynthesis. The outputs of this project in 2010 include: 1) characterization of a gut-specific chitinase gene essential for regulation of chitin content of the PM and growth in the European corn borer (Ostrinia nubilalis) larvae, and 2) identification and characterization of a novel chitinase gene cluster (AgCht5) possibly derived from tandem duplications in the African malaria mosquito (Anopheles gambiae). Results of the research were presented as an oral presentation at the general meeting of K-State Arthropod Genomics Center at Kansas State University (Manhattan, KS) and presented as three different posters at the 15th K-State Research Forum at Kansas State University (Manhattan, KS), the K-State 4th Annual Arthropod Genomics Symposium (Kansas City, MO); and the Annual National Meeting of the Entomological Society of America (San Diego, CA). The results were also presented as six invited presentations in the Department of Entomology, Kansas State University (Manhattan, KS), at the Institute of Plant Physiology and Ecology, Chinese Academy of Sciences (Shanghai, China), the Research Institute of Applied Biology at Shanxi University (Taiyuan, China), the College of Life Sciences at Shaanxi Normal University (Xi'an, China), the College of Agriculture and Biotechnology at Zhejiang University (Hangzhou, China), and at the Annual National Meeting of the Entomological Society of America (San Diego, CA). In addition, the research resulted in four manuscripts including two published in Insect Biochemistry and Molecular Biology, one published in Insect Molecular Biology, and another submitted to Insect Biochemistry and Molecular Biology. PARTICIPANTS: Xin Zhang, Graduate Student, Kansas State University, Manhattan, KS; Chitvan Khajuria, Graduate Student, Kansas State University, Manhattan, KS; Sharon R. Starkey, Lab technician, Kansas State University, Manhattan, KS TARGET AUDIENCES: The primary target audiences of this study are the researchers interested in chitin biosynthesis and the mode of action of insecticides. Since chitin is a vital component of the arthropod exoskeleton and peritrophic matrix, and is not present in vertebrates, our research may lead to new ideas for developing novel insecticides specifically targeting the chitin biosynthetic system for controlling mosquitoes and other arthropod pest species. Therefore, pesticide industries may be interested in our findings. PROJECT MODIFICATIONS: Since 2008, we have included the African malaria mosquito species in our study due to: 1) the availability of its genome sequence; 2) the availability of commercial genechips which may be used in our future studies; and 3) its significant importance as a human disease vector.
Impacts
We identified a cDNA putatively encoding a chitinase (OnCht) in the European corn borer. The OnCht transcript was predominately found in larval midgut but undetectable in eggs, pupae, or adults. When the larvae were fed on an artificial diet, the OnCht transcript level increased by 4.4-fold but the transcript level of a gut-specific chitin synthase (OnCHS2) gene decreased by 2.5-fold as compared with those of unfed larvae. In contrast, when the larvae were fed with the food and then starved for 24h, the OnCht transcript level decreased by 1.8-fold but the transcript level of OnCHS2 increased by 1.8-fold. Furthermore, there was a negative relationship between OnCht transcript level and chitin content in the midgut. By using a feeding-based RNA interference (RNAi) technique, we were able to reduce the OnCht transcript level by 63-64% in the larval midgut. Consequently, these larvae showed significantly increased chitin content (26%) in the PM but decreased larval body weight (54 %) as compared with the control larvae fed on the diet containing GFP dsRNA. Therefore, for the first time, we provide strong evidence that OnCht plays an important role in regulating chitin content of the PM and subsequently affecting the growth and development of the European corn borer larvae. We also identified a chitinase 5 (Cht5) gene cluster consisting of five different genes (AgCht5-1, AgCht5-2, AgCht5-3, AgCht5-4 and AgCht5-5) by a bioinformatics search of the genome of Anopheles gambiae. Cht5 is usually encoded by a single gene in other insect species. Our five-gene models were confirmed by cloning and sequencing of the corresponding cDNAs and gene expression profiling during insect development. All of these genes are found in a single cluster on chromosome 2R. Their open reading frames (ORF) range from 1227 to 1713 bp capable of encoding proteins ranging in size from 409 to 571 amino acids. The identities of their cDNA sequences range from 52 to 66%, and the identities of their deduced amino acid sequences range from 38 to 53%. There are four introns for AgCht5-1, two for AgCht5-2 and AgCht5-3, only one for AgCht5-4, but none for AgCht5-5 in the genome. All five of the predicted chitinases possess a catalytic domain with all of the conserved sequence motifs, but only AgCht5-1 has a chitin-binding domain. Phylogenetic analysis of these chitinases along with those from other insect species suggests that AgCht5-1 is orthologous to the Cht5 proteins identified in other insect species. The differences in expression patterns of these genes at different developmental stages further support that these genes may have distinct functions. Additional searching of the genomes of two other mosquito species led to the discovery of four Cht5 genes in Aedes aegypti and three in Culex quinquefasciatus. Thus, the presence of a Cht5 gene cluster appears to be unique to mosquito species and these genes may have resulted from gene tandem duplications.
Publications
- Khajuria C., Buschman L. L., Chen M.-S., Muthukrishnan S. and Zhu K. Y. 2010. A gut-specific chitinase gene essential for regulation of chitin content of peritrophic membrane and growth of Ostrinia nubilalis larvae. Insect Biochem. Mol. Biol. 40: 621-629.
- Zhang X., Zhang J. and Zhu K. Y. 2010. Chitosan/double-stranded RNA nanoparticle-mediated RNA interference to silence chitin synthase genes through larval feeding in the African malaria mosquito (Anopheles gambiae). Insect Mol. Biol. 19: 683-693.
- Zhang J., Liu X., Zhang J., Li D., Sun Y., Guo Y., Ma E. and Zhu K. Y. 2010. Silencing of two alternative splicing-derived mRNA variants of chitin synthase 1 gene by RNAi is lethal to the oriental migratory locust, Locusta migratoria manilensis (Meyen). Insect Biochem. Mol. Biol. 40: 824-833.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Chitin is the second most abundant biological polymer after cellulose, and is widely distributed in arthropods, fungi and nematodes. It is a vital component of the cuticular exoskeleton and inner linings of peritrophic matrixes of the gut epithelium and trachea in all arthropods. Arthropods depend on chitin biosynthesis for their growth and development. Insect chitin synthesis inhibitors, such as diflubenzuron and lufenuron, are insecticides that specifically interfere with the formation of chitin. Despite the widespread use of insect chitin synthesis inhibitors for controlling various agricultural and public health pests, the exact mechanism of action of these insecticides is still not known. Our long-term goal is to understand chitin synthetic system, regulation and inhibition in arthropods. Our specific aims are to: 1) characterize chitin metabolic enzymes, including chitin synthases involved in chitin biosynthesis and chitinases involved in degradation of chitin, from malaria mosquitoes; and 2) functionally analyze the genes encoding chitin metabolic enzymes in cuticular and peritrophic matrix chitin biosynthesis. The outputs of this project in 2009 include: 1) the identification and characterization of 20 chitinase genes from the Africa malaria mosquito (Anopheles gambiae), and 2) the development of a novel feeding-based RNAi method for effectively repressing two chitin synthase genes (AgCHS1 and AgCHS2) by using nanoparticles in the malaria mosquito larvae. Results of the research were presented as two posters at the 85th Annual Meeting of the Kansas (Central States) Entomological Society in Manhattan, KS; an invited seminar in the Institute of Insect Sciences at Zhejiang University in Hangzhou, China; an invited presentation at the Second International Symposium on Insect Physiology, Biochemistry and Molecular Biology in Chengde, China; and as two posters at the Annual Meeting of the Entomological Society of America in Indianapolis in IN. In addition, a manuscript on the nanoparticle-based gene silencing to mediate systemic RNAi of the two chitin synthase genes (AgCHS1 and AgCHS2) in the mosquito was submitted to a scientific journal for publication. PARTICIPANTS: Xin Zhang, Ph.D. student, Kansas State University, Manhattan, KS 66506. Jianzhen Zhang, Visiting scientist, Kansas State University, Manhattan, KS 66506. Sharon R. Starkey, Lab technician, Kansas State University, Manhattan, KS 66506. TARGET AUDIENCES: The primary target audiences of this study are the researchers interested in chitin biosynthesis and the mode of action of insecticides. Since chitin is a vital component of the arthropod exoskeleton and peritrophic matrix, and is not present in vertebrates, our research may lead to new ideas for developing novel insecticides specifically targeting the chitin biosynthetic system for controlling mosquitoes and other arthropod pest species. Therefore, pesticide industries may be interested in our findings. In addition, industries may also be interested in our nanoparticle-based RNAi technology because there is a great potential of using this technology for high-throughput screening of effective genes for developing novel strategies for insect pest control. PROJECT MODIFICATIONS: Since 2008, we have included the African malaria mosquito species in our study due to: 1) the availability of its genome sequence; 2) the availability of commercial genechips which may be used in our future studies; and 3) its significant importance as a human disease vector.
Impacts
We identified 20 putative chitinase genes from the African malaria mosquito through genome-wide search, and assigned these genes into eight different chitinase classes (I-VIII). Domain analysis of chitinase and chitinase-like proteins showed that all the proteins contained at least one catalytic domain. However, only seven chitinases (AgCht4, AgCht5-1, AgCht6, AgCht7, AgCht8, AgCht10 and AgCht23) displayed chitin binding domain(s). Our analyses of stage- and tissue-specific gene expression revealed that most of these genes were expressed in several developmental stages. However, AgCht8 was mainly expressed in pupal and adult stages. AgCht2 and AgCht12 were specifically expressed in foregut, whereas AgCht13 appeared to be expressed only in the midgut. Immunohistochemistry of selected chitinases in cryosections of A. gambiae indicated high expression of these proteins in certain body parts. This study is expected to provide new insights into the functions of diverse chitinase genes in insects. We also developed a novel feeding-based RNA interference (RNAi) method for effectively repressing two chitin synthase genes (AgCHS1 and AgCHS2) by using nanoparticles that were self-assembled from chitosan and dsRNA for malaria mosquito larvae. RNAi is a phenomenon of which double-stranded RNA (dsRNA) or small interfering RNA (siRNA) triggers the degradation of its homogenous mRNA in eukaryotic organisms. Although it is a conserved mechanism, RNAi in certain organisms or certain stages of an organism (e.g., mosquito larvae) has been a great challenge due to the lack of delivery methods and/or possible lack of cellular uptake for dsRNA or siRNA. On the other hand, AgCHS1 is responsible for biosynthesis of the chitin associated with cuticular exoskeleton and related ectodermal tissues such as trachea, whereas AgCHS2 is responsible for biosynthesis of the chitin associated with peritrophic membrane in the midgut. Our nanoparticle-based RNAi repressed the transcript level of AgCHS1 by 62.8% and ultimately reduced the chitin content by 33.8%. Because such feeding-based RNAi apparently initiated in gut epithelial cells but its effect extended to the epidermal cells where AgCHS1 was exclusively expressed, our study revealed for the first time that RNAi in mosquito larvae was systemic. Our further studies demonstrated the increased larval susceptibilities to diflubenzuron, and calcofluor white or dithiothreitol after the larvae fed on the nanoparticles assembled from AgCHS1 and AgCHS2 dsRNA, respectively. All these findings suggest great potentials of using nanoparticle-based RNAi for high-throughput screening of gene functions and for developing novel strategies for mosquito control.
Publications
- Zhang, X., Zhang, J. and Zhu, K. Y. 2009. Chapter 20. Advances and prospects of RNAi technologies in insect pest management, pp. 202-208. In: T.-X. Liu & L. Kang [eds.], Recent Advances in Entomological Research: From Molecular Biology to Pest Management. Higher Education Press, Beijing (in press).
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Chitin is the second most abundant biological polymer after cellulose, and is widely distributed in arthropods, fungi and nematodes. All arthropods depend on chitin biosynthesis for their growth and development. Chitin is a vital component of the cuticular exoskeleton and inner linings of peritrophic matrixes of the gut epithelium and trachea in arthropods. Insect chitin synthesis inhibitors, such as diflubenzuron and lufenuron, are insecticides that specifically interfere with the formation of chitin. Despite the widespread use of insect chitin synthesis inhibitors for controlling various agricultural and public health pests, the exact mechanism of action of these insecticides is still not known. Our long-term goal is to understand chitin synthetic system, regulation and inhibition in arthropods. Our specific aims are to: 1) characterize chitin metabolic enzymes, including chitin synthases involved in chitin biosynthesis and chitinases involved in degradation of chitin, from malaria mosquitoes; and 2) functionally analyze the genes encoding chitin metabolic enzymes in cuticular and peritrophic matrix chitin biosynthesis. In 2008, we performed a genome-wide search of both chitin synthase and chitinase gene families from the Africa malaria mosquito (Anopheles gambiae) and molecularly characterized these genes including their gene structures, phylogenetic analysis, and stage- and tissue-specific expressions in the mosquito. PARTICIPANTS: Xin Zhang, Ph.D. student, Kansas State University, Manhattan, KS 66506; Jianzhen Zhang, Visiting scientist, Kansas State University, Manhattan, KS 66506; Sharon R. Starkey, Lab technician, Kansas State University, Manhattan, KS 66506 TARGET AUDIENCES: The primary target audiences of this study are the researchers interested in chitin biosynthesis and the mode of action of insecticides. Since chitin is a vital component of the arthropod exoskeleton and peritrophic matrix, and is not present in vertebrates, our research may lead to new ideas for developing novel insecticides specifically targeting the chitin biosynthetic system for controlling mosquitoes and other arthropod pest species. Therefore, pesticide industries may also be interested in our findings. PROJECT MODIFICATIONS: We have included the African malaria mosquito species in our study due to the availabilities of its genome sequence and commercial genechips which may be used in our future studies. In addition, the African malaria mosquito is more important than the common malaria mosquito as a human disease vector.
Impacts
Chitin synthases (CHS) are important enzymes for chitin biosynthesis in insects and other chitin-containing organisms. In An. gambiae, we identified two chitin synthase (AgCHS1 and AgCHS2) genes. AgCHS1 and AgCHS2 were predicted to encode the proteins of 1,566 and 1,610 amino acid residues, respectively. These genes showed high similarities to those of other insect species in both the genome organization and deduced amino acid sequences. AgCHS1 was expressed in egg, larval, pupal and adult stages whereas AgCHS2 appeared to be expressed at relatively low levels, particularly during the larval stages as examined by reverse transcription polymerase chain reaction (RT-PCR). Relatively high gene expression levels were detected in the carcass followed by the foregut and hindgut for AgCHS1, and the foregut followed by the midgut for AgCHS2, respectively. Functional analysis of these genes is currently being pursued using RNAi. Through a manual genome-wide search, we identified 17 putative chitinase genes from An. gambiae and assigned these genes into five different chitinase classes (I-V). The total number of chitinase genes in A. gambiae is similar to those identified in other insect species. Both chitinase-like genes (AgChtIDGF4 and AgChtIDGFz) appeared to be constitutively expressed in all developmental stages as determined by RT-PCR, whereas other genes were only expressed in certain developmental stages. Specifically, AgCht2z, AgCht3z, AgCht4, AgCht5, AgCht9, AgCht12 and AgCht13 appeared to be expressed only in the late egg stage and larval stages, whereas AgCht8 was only expressed in the pupal and adult stages. Our tissue-specific gene expression analysis revealed that AgChtIDGFz, AgCht1z and AgCht4z were constitutively expressed in all four tissues examined, including foregut, midgut, hindgut and carcass. In contrast, AgCht2 and AgCht12 were mainly expressed in the foregut, AgCht13 in the midgut, and AgCht5 in the whole gut, whereas AgCht6 was only expressed in the carcass. Other chitinase genes were expressed in both the certain regions of the gut and the carcass with variable expression levels. Functional analysis of the selected genes is also currently being pursued by using RNAi. Our study is expected to: 1) shed new light on chitin synthetic system in arthropods; 2) help understanding the mechanism of chitin synthesis inhibition and regulation; and 3) facilitate identifying vulnerable points of chitin biosynthesis and regulation for developing novel insecticides.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Chitin is the second most abundant biological polymer after cellulose, and is widely distributed in arthropods, fungi and nematodes. All arthropods depend on chitin biosynthesis for their growth and development. Chitin is a vital component of the cuticular exoskeleton and inner linings of peritrophic matrixes of the gut epithelium and trachea in arthropods. Insect chitin synthesis inhibitors, such as diflubenzuron and lufenuron, are insecticides that specifically interfere with the formation of chitin. Despite the widespread use of insect chitin synthesis inhibitors for controlling various agricultural and public health pests, the exact mechanism of action of these insecticides is still not known. Our long-term goal is to understand chitin synthetic system, regulation and inhibition in arthropods. Our specific aims are to: 1) determine specific effects of the chitin synthesis inhibitor diflubenzuron on chitin biosynthesis and deposition; 2) characterize each of two distinct
chitin synthase cDNAs from malaria mosquitoes; and 3) functionally analyze each of two chitin synthase genes in cuticular and peritrophic matrix chitin biosynthesis. In 2007, we: 1) expressed and purified two polypeptides (one from chitin synthase 1 and other from chitin synthase 2) from the Africa malaria mosquito (Anopheles gambiae); 2) initiated the project to produce polyclonal antibodies using each of the purified polypeptides; 3) tested the first bleeds for their immunological activities against the crude protein preparations from the mosquito; and 4) examined possible effect of diflubenzuron on the chitin synthase activity.
PARTICIPANTS: Xin Zhang, Ph.D. student, Kansas State University, Manhattan, KS 66506 Sharon R. Starkey, Lab technician, Kansas State University, Manhattan, KS 66506
TARGET AUDIENCES: The primary target audiences of this study are the researchers interested in chitin biosynthesis and the mode of action of insecticides. Since chitin is a vital component of the arthropod exoskeleton and peritrophic matrix, and is not present in vertebrates, our research may lead to new ideas for developing novel insecticides specifically targeting the chitin biosynthetic system for controlling mosquitoes and other arthropod pest species. Therefore, pesticide industries may also be interested in our findings.
PROJECT MODIFICATIONS: We have included the African malaria mosquito species in our study due to the availabilities of its genome sequence and commercial genechips which may be used in our future studies. In addition, the African malaria mosquito is more important than the common malaria mosquito as a human disease vector.
Impacts
We successfully expressed each of two African malaria mosquito chitin synthase polypeptides in bacterial cells and purified them to homogenous. The purified polypeptides were sent to the Hybridoma Core Facility of the Interdisciplinary Center for Biotechnology Research at the University of Florida to produce polyclonal antibodies for each polypeptide. We examined two first test bleeds sent back from the University of Florida for their immunological activities against the crude protein preparations from the mosquitoes by using western blotting. Preliminary analyses indicated that both test bleeds were able to recognize their corresponding recombinant polypeptides. However, each bleed showed a cross interaction with a different polypeptide although the two polypeptides were expressed from the less conserved regions of the two mosquito chitin synthase genes. Furthermore, the test bleeds did not seem to show detectable signals when the mosquito crude preparations were used
in western blotting. It is most likely that we need to improve our extraction methods and increase the amount of total proteins for our western blotting analyses. We have been working on the extraction methods and will examine the second and third test bleeds once they become available to us. In addition, we have successfully adapted a method for assaying chitin synthase activity by using wheat germ agglutinin (WGA) conjugated horseradish peroxidase. We have optimized the assay conditions and have now been able to measure the chitin synthase activity in the mosquito larvae and pupae. Such a method will be used to examine the effect of diflubenzuron and other compounds on chitin synthase activity both in vivo and in vitro. Our study is expected to: 1) shed new light on chitin synthetic system in arthropods; 2) help understanding the mechanism of chitin synthesis inhibition and regulation; and 3) help identifying vulnerable points of chitin biosynthesis and regulation for developing
novel insecticides.
Publications
- Zhu, K. Y., Heise, S., Zhang, J., Anderson, T. D. and Starkey, S. R. 2007. Comparative studies on effects of three chitin synthesis inhibitors on common malaria mosquito (Anopheles quadrimaculatus). J. Med. Entomol. 44: 1047-1053.
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Progress 10/01/06 to 12/31/06
Outputs
Chitin is a vital component of the cuticular exoskeleton and inner linings of peritrophic matrixes of the gut epithelium and trachea in insects and other arthropods. Insect chitin synthesis inhibitors, such as diflubenzuron and lufenuron, are insecticides that specifically interfere with the formation of chitin. Despite the widespread use of insect chitin synthesis inhibitors for controlling various agricultural and public health pests, the exact mechanism of action of these insecticides is still not known. In collaboration with visiting scholar Jianzhen Zhang from Shanxi University in China and technician Sharon R. Starkey, we sequenced a complete cDNA encoding chitin synthase 1 (AqCHS1) from the common malaria mosquito (Anopheles quadrimaculatus). Northern blot and real-time quantitative PCR analyses revealed a significant increase of AqCHS1 mRNA level in the larvae exposed to diflubenzuron at 100 and 500 micrograms per liter of water (i.e., parts per billion or
ppb). As confirmed by real-time quantitative PCR, AqCHS1 mRNA level was enhanced by 2-fold in the larvae exposed to diflubenzuron at 500 ppb for 24 hours. In contrast, exposures of the larvae to diflubenzuron at 4.0, 20, 100 and 500 ppb for 48 hours resulted in decreases of chitin content by 9.0, 43, 58 and 76%, respectively. Significantly increased AqCHS1 mRNA level associated with decreased chitin synthesis may imply possible inhibition of chitin synthase, or abnormal chitin synthase translocation or chitin microfibril assembly conferred by diflubenzuron. Increased AqCHS1 expression due to increased transcription and/or increased mRNA stability may serve as a feedback mechanism to compensate such an effect in the mosquitoes. In collaboration with veterinary research scholar Stephanie Heise from Germany, we examined the effect of diflubenzuron on chitin synthesis in different developmental stages and body parts of the mosquito, we assayed the chitin contents in the guts and in the
carcasses after third-instar mosquito larvae were exposed to diflubenzuron at 4.0, 20, 100 and 500 ppb for 48 hours. Diflubenzuron seemed to mainly affect chitin synthesis in the epidermis underlying the cuticular exoskeleton and related to the ectodermal cells since the chitin content in the carcasses showed a concentration-dependant decrease. Such an effect was not found in the total chitin content in pupae and adults. Thus, diflubenzuron may only affect chitin synthesis in the cuticular exoskeleton of the mosquito larvae, which may be directly resulted from the contact of the chemical to the insect cuticle in water. Diflubenzuron may confer its toxicity to mosquito larvae mainly by inhibiting cuticular chitin synthesis rather than the chitin synthesis in gut epithelial cells.
Impacts
This study is expected to shed new light on the chitin synthesis system in arthropods and set the stage for developing a functional model of the mechanism of action for insect chitin synthesis inhibitors. Because chitin is an extremely important component of exoskeleton and cuticular linings of certain gut tissues in insects and other arthropods, and is not present in vertebrates, results from this research are expected to help researchers develop novel and pest-specific insecticides for pest management.
Publications
- Heise S. and Zhu K. Y. Effect of diflubenzuron on chitin synthesis in different stages and body parts of the common malaria mosquito (Anopheles quadrimaculatus). Abstracts, 2006 Merck/Merial National Veterinary Scholar Symposium: Greating the Gumbo of Progress, Baton Rouge, LA. P. 144.
- Zhang J. and Zhu K. Y. 2006. Characterization of a chitin synthase cDNA and its increased mRNA level associated with decreased chitin synthesis in Anopheles quadrimaculatus exposed to diflubenzuron. Insect Biochemistry and Molecular Biology 36: 712-725.
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