Source: NORTH CAROLINA STATE UNIV submitted to NRP
MOLECULAR ECOLOGY AND PATHOLOGY OF A COCKROACH-SPECIFIC DENSONUCLEOSIS VIRUS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0217021
Grant No.
2009-35302-05303
Cumulative Award Amt.
(N/A)
Proposal No.
2008-04063
Multistate No.
(N/A)
Project Start Date
Mar 1, 2009
Project End Date
Feb 28, 2013
Grant Year
2009
Program Code
[51.2B]- Arthropod and Nematode Biology and Management (B): Suborganismal Biology
Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
ENTOMOLOGY
Non Technical Summary
The prevalence and incidence of asthma have been rising worldwide at alarming rates and cockroaches have been recognized as a major etiological agent of allergic disease and asthma, especially among inner-city children, the elderly, and workers in the confined animal industry, particularly swine farms. In the course of molecular ecology studies of cockroach populations we discovered a new entomopathogenic densovirus in a cockroach infested swine farm. Infected individuals display several symptoms of pathology, including lethargy, flaccidity, poorly coordinated movements, and partial or complete paralysis of the hind legs. The long-term goal of this project is to gain a comprehensive understanding of the pathology and molecular ecology of the newly discovered densovirus (BgDNV). We aim to develop BgDNV as a highly species-specific biological control agent to mitigate the veterinary and medical impacts of the cockroach. Toward this end, it is critical first to gain an understanding of fundamental aspects of DNV biology, including its life cycle, mechanisms of virus replication, regulation of gene expression, splicing strategies, formation of infectious virus, transduction range, molecular ecology of the cockroach virus, and molecular evolution of the Densovirus genus. A major thrust of this project is investigate the pathology of the infection cycle and its species-specificity. Results of this study will provide critical insights into an alternative, safe, effective, and environmentally compatible insect control technique that can readily be incorporated into sustainable IPM programs in structural agricultural and urban settings. This research fits with Program 51.2 FY2008 priorities 2, 3: "Understanding the cellular, biochemical, and molecular level interactions of arthropods or nematodes with associated organisms (e.g. host plants, livestock, microbes, or beneficial organisms). Elucidation of the mechanism of action of novel targets for pest control, including semiochemicals and fundamental pesticide resistance studies."
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113599113010%
3115330113010%
3116010113010%
7213599113010%
7215330113010%
7215320113010%
8045320113010%
8045399113010%
7235320113010%
7235399113010%
Knowledge Area
311 - Animal Diseases; 804 - Human Environmental Issues Concerning Apparel, Textiles, and Residential and Commercial Structures; 723 - Hazards to Human Health and Safety; 721 - Insects and Other Pests Affecting Humans;

Subject Of Investigation
5330 - Farm structures and related facilities; 5399 - Structures, facilities, and equipment, general/other; 5320 - Houses (human residences), furniture, household equipment, non-textile furnishings; 6010 - Individuals; 3599 - Swine, general/other;

Field Of Science
1130 - Entomology and acarology;
Goals / Objectives
The prevalence and incidence of asthma have been rising worldwide at alarming rates and cockroaches have been recognized as a major etiological agent of allergic disease and asthma, especially among inner-city children and the elderly. Cockroach infestations, mainly of the German cockroach, Blattella germanica, are generally controlled with scheduled applications of broad-spectrum neurotoxic insecticides, most commonly pyrethroid insecticides, which also expose people (in residences, schools, etc.) and animals (in infested structures, such as confinement swine production farms) to unnecessary health and environmental risks. Moreover, the efficacy of most insecticides becomes limited because resistance to them develops rapidly in pest populations. In the course of molecular ecology studies of cockroach populations we discovered a new entomopathogenic densovirus that has the potential to represent a major new direction in global pest control of cockroaches. This virus was isolated from a cockroach infestation in North Carolina, and infected individuals display a broad array of pathology symptoms, including lethargy, flaccidity, poorly coordinated movements, and partial or complete paralysis of the hind legs. We have maintained the virus in the laboratory for several years with no apparent loss of virulence. Our long-term goal is to gain a comprehensive understanding of the pathology and molecular ecology of this newly discovered B. germanica densovirus (BgDNV). We aim to develop BgDNV as a highly species-specific biological control agent to mitigate the medical and veterinary impacts of B. germanica. Toward this end, it is critical first to gain an understanding of fundamental aspects of DNV biology, including its life cycle, mechanisms of virus replication, regulation of gene expression, splicing strategies, formation of infectious virus, transduction range, molecular ecology of the cockroach virus, and molecular evolution of the Densovirus genus. A second major thrust of our collaboration is to develop the BgDNV as a promising cockroach-specific pest control agent by investigating the pathology of the infection cycle and its species-specificity. Finally, in the long-term we will use the BgDNV as a tool for genetic engineering of cockroaches by expressing target genes, namely arthropod toxins, and RNAi sequences using DNV promoters and delivering genes in densoviral virions. The specific objectives of this proposal are: 1. BgDNV molecular ecology: Determine its distribution in nature and search for new DNV variants. 2. BgDNV infectivity: Analyze the dynamics of host pathology and BgDNV efficacy.
Project Methods
A. Analysis of BgDNV distribution in nature. We will collect cockroaches from various global localities, with emphasis on the U.S. and Russia, including collections from pig farms in NC. DNA will be extracted from one half of each individual cockroach, while the other half will be stored at -80C for other analyses. Each DNA sample will be PCR amplified with virus-specific primers. Presence of the 500 bp amplification product will verify that virus DNA is present in a given DNA sample. B. Search for new BgDNV variants in nature. Stored frozen material corresponding to DNA samples which, based on the PCR analysis described in (A), contain viruses will be homogenized in the cell line mediumm and used to infect lines and cockroaches. Virus strains demonstrating pathological effects more severe than those caused by the P6 virus will be selected and maintained in vitro and/or in vivo. C. Analysis of species-specificity of the virus. Healthy cockroaches of different families and species, as well as insects of other Orders and environmentally important crustaceans will be infected with virus isolated from infected B. germanica or cell culture. After several weeks, DNA will be extracted from injected animals and checked for the presence of the additional 5 kb band corresponding to BgDNV on agarose gels. The results will be checked by blot hybridization of total DNA samples with a BgDNV probe. The physiological and behavioral status of the infected animals will be followed in order to detect manifestations of virus pathology. D. Identification of early steps of DNV infection and distribution of DNV in cockroach tissues. Adult virgin females will be infected with virus and after specific times cockroaches will be removed from the colony and divided equally for (a) real-time qPCR which will follow changes of BgDNV titers; (b) TEM which will describe cytopathological effects in infected tissues, the digestive system in particular; and (c) in situ PCR followed by in situ hybridization analysis which will describe the distribution of virus particles inside the cockroach. E. Efficacy and mode of transmission: Infectivity of BgDNV to various developmental stages of the German cockroach, transmission of BgDNV through cockroach feces, vertical transmission of virus.

Progress 03/01/09 to 02/28/13

Outputs
Target Audience: Academic scientists, pest control operators, pest management professionals, municipalities, livestock production managers, health care workers, allergy clinicians. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Several scientists in the US and Russia collaborated on this project. Senior scientists: Dr. Schal at North Carolina State University Dr. Dmitry Mukha at the Vavilov Institute, Russian Academy of Sciences Post-doctoral researchers: Dr. Ayako Wada-katsumata Dr. Warren Booth Dr. Katalin Boroczky Graduate students: Adrienn Uzsak Tatiana Kapelinskaya Elena Martynova Research Specialist: Mr. Rick Santangelo This project also provided professional development opportunities for two undergraduates to engage in scientific research projects. How have the results been disseminated to communities of interest? Results of this project have been widely disseminated through: 1. Refereed publications (listed above) 2. Press releases 3. Professional presentations at conferences 4. Talks to professional pest management specialists at pest control conferences 5. Talks at Healthy Homes conferences to clinicians and end-users. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In the course of molecular ecology studies of cockroach populations we discovered a new entomopathogenic densovirus that has the potential to represent a major new direction in global pest control of cockroaches. This virus was isolated from a cockroach infestation in North Carolina, and infected individuals display a broad array of pathology symptoms, including lethargy, flaccidity, poorly coordinated movements, and partial or complete paralysis of the hind legs. Our long-term goal is to gain a comprehensive understanding of the pathology and molecular ecology of this newly discovered B. germanica densovirus (BgDNV). The swine production system in the United States is characterized by a small number of management companies, each controlling a large number of swine producers. Individual producers are linked to a management company through the supply of animals and husbandry products from central company-specific warehouses. Thus, alliance to a management company may represent a channel, as well as a barrier, to gene flow and pathogen transmission from pests. Within the last 15 to 20 years, the German cockroach has emerged as a major pest within this system, posing considerable health risks to both humans and animals. Cockroaches are also recognized as a major cause of asthma morbidity and they have been implicated as vectors of pathogenic microbes. Our ultimate goal, in addition to understanding the basic population genetics of cockroaches, is to apply this information towards developing new strategies and tactics of cockroach control and mitigation of the harmful effects of insecticides. The findings from these studies will be used to develop insights into new pest management approaches. Results were presented at annual meetings of the NC Pest Management Association and National Pest Management Association. Additional presentations were made at annual meetings of the Entomological Society of America and the National Urban Entomology Conference. We are aiming to develop B. germanica densovirus (BgDNV) as a highly species-specific biological control agent to mitigate the medical and veterinary impacts of B. germanica. Toward this end, it is critical first to gain an understanding of fundamental aspects of DNV biology, including its life cycle, mechanisms of virus replication, regulation of gene expression, splicing strategies, formation of infectious virus, transduction range, molecular ecology of the cockroach virus, and molecular evolution of the Densovirus genus. A second major thrust of our collaboration is to develop the BgDNV as a promising cockroach-specific pest control agent by investigating the pathology of the infection cycle and its species-specificity. Finally, in the long-term we will use the BgDNV as a tool for genetic engineering of cockroaches by expressing target genes, namely arthropod toxins, and RNAi sequences using DNV promoters and delivering genes in densoviral virions. We have found BgDNV in several swine farms in NC, but not in any residential settings in the U.S. or elsewhere. BgDNV is highly genus-specific, affecting other Blattella species, but not other closely related blattellids. In work on the BgDNV genome we sequenced its entire genome and characterized 2 promoters, polyadenylation signals, transcription end sites, splice donor and acceptor sites, and ITRs. BgDNV possesses three regulatory proteins: NS1, NS2 and NS3. NS1 is the major noncapsid protein with presumable helicase and endonuclease activities, which plays role in regulation of viral genome replication, regulation of gene expression, and encapsidation. NS2 and NS3 are two minor noncapsid proteins, whose functions and roles during viral life cycle have not been studied yet. BgDNV capsid consists of three proteins as well: VP1, VP2 and VP3. All these molecules possess the same C- termini and unique N-termini and the VP3 amino acid sequence is identical to the C- terminus of VP1. mRNAs for VP and NS proteins are transcribed in opposite directions. We conducted mass-spectrometric identification to confirm these proteins in BgDNV. The expression strategy of BgDNV differs from that of all other parvoviruses analyzed thus far. Splicing events are involved both in production of NS and VP transcripts. VP2 protein is translated according the ribosome shunting mechanism while VP3 according to the leaky scanning mechanism. For the first time we have shown monoubiquitination as a densovirus capsid protein and unequal distribution of the NS-1 proteins inside the nucleus of the virus in infected cells. The proper localization of viral-encoded proteins in cellular compartments is important for completion of the viral life cycle and production of viral progeny, and therefore for viral pathogenesis. We obtained polyclonal antibodies and using infected BGE-2 cells, we investigated the intracellular distribution of structural and nonstructural virus proteins. NS1 is localized in the nucleus, VP1 and VP3 proteins are localized mainly in nuclei, while VP2 is detected both in the nucleus and the cytoplasm.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Boroczky K., A. Wada-Katsumata, D. Batchelor, M. Zhukovskaya and C. Schal. 2013. Insects groom their antennae to enhance olfactory acuity. Proceedings of the National Academy of Sciences, USA 110: 36153620.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Carr A. L., R. M. Roe, C. Arellano, D. E. Sonenshine, C. Schal and C. S. Apperson. 2013. Responses of Amblyomma americanum and Dermacentor variabilis (Acari: Ixodidae) to odorants that attract haematophagous insects. Medical and Veterinary Entomology 27: 8695.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Groot A.T., H. Staudacher, A. Barthel, O. Inglis, G. Sch�fl, R.G. Santangelo, S. Gebauer-Jung S, H. Vogel, J. Emerson, C. Schal C, D.G. Heckel and F. Gould. 2013. One quantitative trait locus for intra- and interspecific variation in a sex pheromone. Molecular Ecology 22: 10651080.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Uzsak A. and C. Schal. 2013. Sensory cues involved in social facilitation of reproduction in Blattella germanica females. PLoS ONE 8(2): e55678. doi:10.1371/journal.pone.0055678.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Uzsak A. and C. Schal. 2013. Social interaction facilitates reproduction in male German cockroaches, Blattella germanica. Animal Behaviour 85: 15011509.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Wada-Katsumata A., J. Silverman and C. Schal. 2013. Changes in taste neurons support the emergence of an adaptive behavior in cockroaches. Science 340: 972975.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Eliyahu D., S. Nojima, R. G. Santangelo, S. Carpenter, F. X. Webster, D. J. Kiemle, C. Gemeno, W. S. Leal and C. Schal. 2012. An unusual macrocyclic lactone sex pheromone of Parcoblatta lata, a primary food source of the endangered red-cockaded woodpecker. Proceedings of the National Academy of Sciences, USA 109: E490E496.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Uzsak A and C Schal. 2012. Differential physiological responses of the German cockroach to social interactions during the ovarian cycle. Journal of Experimental Biology 215: 3037-3044.
  • Type: Journal Articles Status: Published Year Published: 2011 Citation: Kapelinskaya T, E Martynova, C Schal and D Mukha. 2011. Expression strategy of densonucleosis virus from the German cockroach, Blattella germanica. Journal of Virology 85: 11855-11870.
  • Type: Journal Articles Status: Published Year Published: 2011 Citation: Nojima S, DJ Kiemle, FX Webster, CS Apperson and C Schal. 2011. Nanogram-scale preparation and NMR analysis for mass-limited small volatile compounds. PLoS ONE 6(3): e18178. doi:10.1371/journal.pone.0018178.
  • Type: Journal Articles Status: Published Year Published: 2011 Citation: Wada-Katsumata A, J Silverman and C Schal. 2011. Differential inputs from chemosensory appendages mediate feeding responses to glucose in wild-type and glucose-averse German cockroaches, Blattella germanica. Chemical Senses 36: 589-600.
  • Type: Journal Articles Status: Published Year Published: 2010 Citation: Mankin R. W., R. D. Hodges, H. T. Nagle, C. Schal, R. M. Pereira and P. G. Koehler. 2010. Acoustic indicators for targeted detection of stored product and urban insect pests by inexpensive infrared, acoustic, and vibrational detection of movement. Journal of Economic Entomology 103: 1636-1646.
  • Type: Journal Articles Status: Published Year Published: 2009 Citation: Nalyanya G., J. C. Gore, H. M. Linker and C. Schal. 2009. German cockroach allergen levels in North Carolina schools: Comparison of integrated pest management and conventional cockroach control. Journal of Medical Entomology, Forum 46: 420-427.
  • Type: Journal Articles Status: Published Year Published: 2011 Citation: Ahmad A., A. Ghosh, C. Schal and L. Zurek. 2011. Insects in confined swine operations carry a large antibiotic resistant and potentially virulent enterococcal community. BMC Microbiology 11: 23.
  • Type: Book Chapters Status: Published Year Published: 2011 Citation: Blomquist G. J., R. Jurenka, C. Schal and C. Tittiger. 2011. Pheromone production: Biochemistry and molecular biology. In: Insect Endocrinology (L. I. Gilbert, ed.), Chapter 12, pp. 523567, Academic Press.
  • Type: Journal Articles Status: Published Year Published: 2011 Citation: Booth W., R. G. Santangelo, E. L. Vargo, D. V. Mukha and C. Schal. 2011. Population genetic structure in German cockroaches (Blattella germanica): Differentiated islands in an agricultural landscape. Journal of Heredity 102: 175-183.
  • Type: Journal Articles Status: Published Year Published: 2011 Citation: Gemeno C., G. M. Williams and C. Schal. 2011. Effect of shelter on reproduction, growth and longevity of the German cockroach, Blattella germanica (Dictyoptera: Blattellidae). European Journal of Entomology 108: 205-210.
  • Type: Journal Articles Status: Published Year Published: 2011 Citation: Mukha D. V., V. Mysina, V. Mavropulo and C. Schal. 2011. Structure and molecular evolution of the ribosomal DNA external transcribed spacer in the cockroach genus Blattella. Genome 54: 222-234.
  • Type: Book Chapters Status: Published Year Published: 2011 Citation: Schal C. 2011. Cockroaches. In: Handbook of Pest Control (S. Hedges and D. Moreland, eds.), pp. 150291, GIE Media.
  • Type: Journal Articles Status: Published Year Published: 2010 Citation: Crissman J. R., W. Booth, R. G. Santangelo, D. V. Mukha, E. L. Vargo and C. Schal. 2010. Population genetic structure of the German cockroach (Blattodea: Blattellidae) in apartment buildings. Journal of Medical Entomology 47: 553-564.
  • Type: Book Chapters Status: Published Year Published: 2009 Citation: Schal C. 2009. The ABCs of indoor health: Allergens, Baits, and Cockroach mitigation strategies. In: Advances in Human Vector Control, ACS Symposium Series, Volume 1014, Chapter 7, pp. 89-106.


Progress 03/01/10 to 02/28/11

Outputs
OUTPUTS: We are aiming to develop B. germanica densovirus (BgDNV) as a highly species-specific biological control agent to mitigate the medical and veterinary impacts of B. germanica. Toward this end, it is critical first to gain an understanding of fundamental aspects of DNV biology, including its life cycle, mechanisms of virus replication, regulation of gene expression, splicing strategies, formation of infectious virus, transduction range, molecular ecology of the cockroach virus, and molecular evolution of the Densovirus genus. A second major thrust of our collaboration is to develop the BgDNV as a promising cockroach-specific pest control agent by investigating the pathology of the infection cycle and its species-specificity. Finally, in the long-term we will use the BgDNV as a tool for genetic engineering of cockroaches by expressing target genes, namely arthropod toxins, and RNAi sequences using DNV promoters and delivering genes in densoviral virions. We have found BgDNV in several swine farms in NC, but not in any residential settings in the U.S. or elsewhere. BgDNV is highly genus-specific, affecting other Blattella species, but not other closely related blattellids. In work on the BgDNV genome we sequenced its entire genome and characterized 2 promoters, polyadenylation signals, transcription end sites, splice donor and acceptor sites, and ITRs. BgDNV possesses three regulatory proteins: NS1, NS2 and NS3. NS1 is the major noncapsid protein with presumable helicase and endonuclease activities, which plays role in regulation of viral genome replication, regulation of gene expression, and encapsidation. NS2 and NS3 are two minor noncapsid proteins, whose functions and roles during viral life cycle have not been studied yet. BgDNV capsid consists of three proteins as well: VP1, VP2 and VP3. All these molecules possess the same C- termini and unique N-termini and the VP3 amino acid sequence is identical to the C- terminus of VP1. mRNAs for VP and NS proteins are transcribed in opposite directions. We conducted mass-spectrometric identification to confirm these proteins in BgDNV. The expression strategy of BgDNV differs from that of all other parvoviruses analyzed thus far. Splicing events are involved both in production of NS and VP transcripts. VP2 protein is translated according the ribosome shunting mechanism while VP3 according to the leaky scanning mechanism. For the first time we have shown monoubiquitination as a densovirus capsid protein and unequal distribution of the NS-1 proteins inside the nucleus of the virus in infected cells. The proper localization of viral-encoded proteins in cellular compartments is important for completion of the viral life cycle and production of viral progeny, and therefore for viral pathogenesis. We obtained polyclonal antibodies and using infected BGE-2 cells, we investigated the intracellular distribution of structural and nonstructural virus proteins. NS1 is localized in the nucleus, VP1 and VP3 proteins are localized mainly in nuclei, while VP2 is detected both in the nucleus and the cytoplasm. PARTICIPANTS: Coby Schal (PI, Professor, NCSU), Dmitry Mukha (Collaborator, Vavilov Institute, Russia), Rick Santangelo (research specialist, NCSU), Ayako Katsumata (postdoc, NCSU), Warren Booth (postdoc, NCSU). This project provided professional development opportunities for undergraduates to engage in scientific research projects. TARGET AUDIENCES: Academic scientists, pest control operators, municipalities, livestock production managers, allergy clinicians. PROJECT MODIFICATIONS: A no-cost extension was requested and granted.

Impacts
In the course of molecular ecology studies of cockroach populations we discovered a new entomopathogenic densovirus that has the potential to represent a major new direction in global pest control of cockroaches. This virus was isolated from a cockroach infestation in North Carolina, and infected individuals display a broad array of pathology symptoms, including lethargy, flaccidity, poorly coordinated movements, and partial or complete paralysis of the hind legs. Our long-term goal is to gain a comprehensive understanding of the pathology and molecular ecology of this newly discovered B. germanica densovirus (BgDNV). The swine production system in the United States is characterized by a small number of management companies, each controlling a large number of swine producers. Individual producers are linked to a management company through the supply of animals and husbandry products from central company-specific warehouses. Thus, alliance to a management company may represent a channel, as well as a barrier, to gene flow and pathogen transmission from pests. Within the last 15 to 20 years, the German cockroach has emerged as a major pest within this system, posing considerable health risks to both humans and animals. Cockroaches are also recognized as a major cause of asthma morbidity and they have been implicated as vectors of pathogenic microbes. Our ultimate goal, in addition to understanding the basic population genetics of cockroaches, is to apply this information towards developing new strategies and tactics of cockroach control and mitigation of the harmful effects of insecticides. The findings from these studies will be used to develop insights into new pest management approaches. Results were presented at annual meetings of the NC Pest Management Association and National Pest Management Association. Additional presentations were made at annual meetings of the Entomological Society of America and the National Urban Entomology Conference. The prevalence and incidence of asthma have been rising worldwide at alarming rates and cockroaches have been recognized as a major etiological agent of allergic disease and asthma, especially among inner-city children and the elderly. Cockroach infestations -- mainly of the German cockroach, Blattella germanica -- are generally controlled with scheduled applications of broad-spectrum neurotoxic insecticides, most commonly pyrethroid insecticides, which also expose people (in residences, schools, etc.) and animals (in infested structures, such as confinement swine production farms) to unnecessary health and environmental risks. Moreover, the efficacy of most insecticides becomes limited because resistance to them develops rapidly in pest populations. Moreover, swine production is an important component of the agricultural economy of several States. Cockroaches have long been recognized as important pests in swine production and severe infestations may contribute significantly to disease maintenance and transmission. BgDNV has the potential to be highly species-specific biological control agent to mitigate the medical and veterinary impacts of B. germanica.

Publications

  • Uzsak A and C Schal. 2012. Differential physiological responses of the German cockroach to social interactions during the ovarian cycle. Journal of Experimental Biology 215: 3037-3044.
  • Kapelinskaya T, E Martynova, C Schal and D Mukha. 2011. Expression strategy of densonucleosis virus from the German cockroach, Blattella germanica. Journal of Virology 85: 11855-11870.
  • Nojima S, DJ Kiemle, FX Webster, CS Apperson and C Schal. 2011. Nanogram-scale preparation and NMR analysis for mass-limited small volatile compounds. PLoS ONE 6(3): e18178. doi:10.1371/journal.pone.0018178.
  • Wada-Katsumata A, J Silverman and C Schal. 2011. Differential inputs from chemosensory appendages mediate feeding responses to glucose in wild-type and glucose-averse German cockroaches, Blattella germanica. Chemical Senses 36: 589-600.


Progress 03/01/09 to 02/28/10

Outputs
OUTPUTS: In the course of molecular ecology studies of cockroach populations we discovered a new entomopathogenic densovirus that has the potential to represent a major new direction in global pest control of cockroaches. This virus was isolated from a cockroach infestation in North Carolina, and infected individuals display a broad array of pathology symptoms, including lethargy, flaccidity, poorly coordinated movements, and partial or complete paralysis of the hind legs. Our long-term goal is to gain a comprehensive understanding of the pathology and molecular ecology of this newly discovered B. germanica densovirus (BgDNV). We aim to develop BgDNV as a highly species-specific biological control agent to mitigate the medical and veterinary impacts of B. germanica. Toward this end, it is critical first to gain an understanding of fundamental aspects of DNV biology, including its life cycle, mechanisms of virus replication, regulation of gene expression, splicing strategies, formation of infectious virus, transduction range, molecular ecology of the cockroach virus, and molecular evolution of the Densovirus genus. A second major thrust of our collaboration is to develop the BgDNV as a promising cockroach-specific pest control agent by investigating the pathology of the infection cycle and its species-specificity. Finally, in the long-term we will use the BgDNV as a tool for genetic engineering of cockroaches by expressing target genes, namely arthropod toxins, and RNAi sequences using DNV promoters and delivering genes in densoviral virions. We have found BgDNV in several swine farms in NC, but not in any residential settings in the U.S. or elsewhere. BgDNV is highly genus-specific, affecting other Blattella species, but not other closely related blattellids. In work on the BgDNV genome we sequenced its entire genome and characterized 2 promoters, polyadenylation signals, transcription end sites, splice donor and acceptor sites, and ITRs. mRNAs for VP and NS proteins are transcribed in opposite directions. We conducted mass-spectrometric identification to confirm the following proteins of BgDNV: VP1, VP2 (p80), VP2 (p85), and VP3. We obtained polyclonal antibodies against the VP and NS1 proteins. Using infected BGE-2 cells, we investigated the intracellular distribution of structural and nonstructural virus proteins and the expression strategy of BgDNV. The expression strategy of BgDNV differs from that of all other parvoviruses analyzed thus far. Splicing events are involved both in production of NS and VP transcripts. Splicing in VP mRNAs resulted in joining of two ORFs for capsid proteins into one with sufficiently elevated coding capacity to encode the largest VP1 protein. VP2 protein is translated according the "ribosome shunting" mechanism while VP3 according to the "leaky scanning" mechanism. For the first time we have shown monoubiquitination as a densovirus capsid protein and unequal distribution of the NS-1 proteins inside the nucleus of the virus in infected cells. PARTICIPANTS: Coby Schal (PI, Professor, NCSU), Dmitry Mukha (Collaborator, Vavilov Institute, Russia), Rick Santangelo (research specialist, NCSU), Ayako Katsumata (postdoc, NCSU), Warren Booth (postdoc, NCSU). This project provided professional development opportunities for undergraduates to engage in scientific research projects. TARGET AUDIENCES: Academic scientists, pest control operators, municipalities, livestock production managers, allergy clinicians PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The prevalence and incidence of asthma have been rising worldwide at alarming rates and cockroaches have been recognized as a major etiological agent of allergic disease and asthma, especially among inner-city children and the elderly. Cockroach infestations -- mainly of the German cockroach, Blattella germanica -- are generally controlled with scheduled applications of broad-spectrum neurotoxic insecticides, most commonly pyrethroid insecticides, which also expose people (in residences, schools, etc.) and animals (in infested structures, such as confinement swine production farms) to unnecessary health and environmental risks. Moreover, the efficacy of most insecticides becomes limited because resistance to them develops rapidly in pest populations. Moreover, swine production is an important component of the agricultural economy of several States. Cockroaches have long been recognized as important pests in swine production and severe infestations may contribute significantly to disease maintenance and transmission. BgDNV has the potential to be highly species-specific biological control agent to mitigate the medical and veterinary impacts of B. germanica.

Publications

  • Mankin R. W., R. D. Hodges, H. T. Nagle, C. Schal, R. M. Pereira and P. G. Koehler. 2010. Acoustic indicators for targeted detection of stored product and urban insect pests by inexpensive infrared, acoustic, and vibrational detection of movement. Journal of Economic Entomology 103: 1636-1646.
  • Nalyanya G., J. C. Gore, H. M. Linker and C. Schal. 2009. German cockroach allergen levels in North Carolina schools: Comparison of integrated pest management and conventional cockroach control. Journal of Medical Entomology, Forum 46: 420-427.
  • Ahmad A., A. Ghosh, C. Schal and L. Zurek. 2011. Insects in confined swine operations carry a large antibiotic resistant and potentially virulent enterococcal community. BMC Microbiology 11: 23.
  • Blomquist G. J., R. Jurenka, C. Schal and C. Tittiger. 2011. Biochemistry and molecular biology of pheromone production. In: Insect Endocrinology (L. I. Gilbert, ed.), Academic Press (in press).
  • Booth W., R. G. Santangelo, E. L. Vargo, D. V. Mukha and C. Schal. 2011. Population genetic structure in German cockroaches (Blattella germanica): Differentiated islands in an agricultural landscape. Journal of Heredity 102: 175-183.
  • Gemeno C., G. M. Williams and C. Schal. 2011. Effect of shelter on reproduction, growth and longevity of the German cockroach, Blattella germanica (Dictyoptera: Blattellidae). European Journal of Entomology 108: 205-210.
  • Mukha D. V., V. Mysina, V. Mavropulo and C. Schal. 2011. Structure and molecular evolution of the ribosomal DNA external transcribed spacer in the cockroach genus Blattella. Genome 54: 222-234.
  • Schal C. 2011. Cockroaches. In: The Mallis Handbook of Pest Control (S. Hedges and D. Moreland, eds.), GIE Media (in press).
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