2006 Annual Report
Agriculture will benefit from biological control in the broad sense of the use of natural enemies, SIT / autocidal strains and techniques to manipulate pest behavior. Even with the heavy use of chemical pesticides, insect pests inflict enormous damage on the agricultural productivity of most countries. Treatment strategies using pesticides as the only control tactic present serious economic and ecological costs as well. Control failures due to insecticide resistance are reducing the efficacy of agricultural chemicals, while the safety of farm workers, our food supply, and the environment is threatened by the volume of toxins currently used. Consequently, many chemicals have been or are being removed from the market either by State or Federal agencies. The loss of pesticides, combined with a lack of suitable substitute chemicals or alternative methods of pest control, threaten the continued existence of many small and medium farms in the USA, and the export of agricultural products. For example, the annual damage caused by New World fruit flies, due to quarantines and lost exports, damaged crops, loss of jobs, and pesticide use, exceeds hundreds of millions of dollars per year. These amounts would be dwarfed by the losses that would occur should insects such as the Mediterranean fruit fly become established in the USA. Such an invasion would cost an estimated $1.1 billion per year to California agriculture alone.
Work by this CRIS impacts the Research by this CRIS project relates to National Program 304 (Crop and Commodity Pest Biology, Control and Quarantine), and addresses the following goals: The following goals are addressed: Develop an understanding of the organismal biology, behavior, chemical ecology, genetics and bionomics of major, minor, occasional and potential pests and their natural enemies (Component II, Part IIb); Increase basic knowledge of plant-pest-natural enemy interactions among principal biotic components of agricultural production systems in a variety of open field environments (Component III, Part IIa); Conduct research on exotic pests in their natural habitats before they become established in the United States (Component IV, Part IIa); Improve existing and develop new genetic and autocidal control technologies for exotic insect pests (Component IV, Part IIc); Develop new and existing traps and lures to detect and monitor injurious stages or both sexes of pests and to monitor beneficial organisms, including biological control agents (Component VI, Part IIa). The reduction of chemical pesticide usage on crops is an important national priority and fits the intent of the USDA IPM initiative.
Develop techniques and strategies that utilize molecular gene transfer methods to create transgenic strains of Diptera, Lepidoptera, and Coleoptera that will facilitate genetic-sexing or have novel autocidal properties for use in IPM programs.
Subobjective 1.1: Develop transgenic strains of fruit flies and moths for improved and novel biological control strategies.
12 MONTHS: • Create piggyBac vectors with new fluorescent protein markers and conditional lethality constructs- initiate transformation experiments. • Construct plasmids that contain expression cassettes for the production of double stranded RNA to target GFP expression. • Incorporate GFP-dsRNA cassettes into JcDNV somatic and piggyBac germline vectors.
24 MONTHS: • For genetically marked insects, initiate fitness and reproductive competitiveness studies. • Perform molecular analysis of conditional lethal transgenic strains, and initiate phenotypic analysis. • Test efficiency of gene silencing of GFP expression in transformed cell lines and germline transformed flies. • Construct plasmids that contain expression cassettes for the production of double stranded RNA to target vasa expression in flies and moths.
36 MONTHS: • Initiate in-lab large scale rearing of transgenic strains to assess strain and transgene stability. • Continue phenotypic analysis of conditional lethal strains and expand populations of efficient strains. • Test efficiency of gene silencing of vasa expression in flies using both somatic and germline transformation vectors. • Construct plasmids that contain conditional promoters in expression cassettes for the production of double stranded RNA.
48 MONTHS: • Initiate contained green-house studies for reproductive competitiveness of transgenic strains relative to wild type. • Test efficiency of gene silencing of vasa expression in moths using both somatic and germline transformation vectors. • Test efficiency of gene silencing under conditional promoters in flies using both somatic and germline transformation vectors.
60 MONTHS: • Initiate risk assessment analysis for environmental impact statement required for contained release studies of transgenic tephritids and/or moths. • Test efficiency of gene silencing of vasa expression in beetles using both somatic and germline transformation vectors. • Test efficiency of gene silencing under conditional promoters in flies using both somatic and germline transformation vectors.
Subobjective 1.2 To identify and isolate genes from tephritid and moth species with subsequent functional analysis for use in transgenic strains for biological control.
12 MONTHS: • Initiate PCR studies for specific genes of interest.
24 MONTHS: • Initiate functional analysis of genes of interest (GOI) by germ-line transformation. • Begin cDNA library construction and characterization for embryos, males and females, reproductive tissue and induced cell death.
36 MONTHS: • For putative GOIs cloned by PCR, complete functional analysis and isolate complete genomic sequence by inverse PCR or library screening
48 MONTHS: • Begin subtractive hybridization of cDNA libraries and sequencing of putative GOIs.
60 MONTHS: • Isolate geneomic sequences for cDNA GOIs. Integrate regulatory or coding sequences of new GOIs into conditional lethal transgene constructs. • Begin integration of constructs by transformation and transgenic analysis.
Subobjective 1.3: Develop a means of generating somatic transformations to test the phenotypes and efficiencies of foreign gene constructs that lead to genetic control of pest insects.
12 MONTHS: • Continue PCR and hybridization surveys for piggyBac in tephritid and moth strains. • Continue testing target and stabilization vectors in Drosophila.
24 MONTHS: • Complete sequencing of new piggyBac elements. • Create new acceptor and donor vectors for use in moths and tephritids
36 MONTHS: • Complete genomic comparative analysis of piggyBac sequences. • Begin creation of new acceptor site target strains.
48 MONTHS: • Initiate excision/ transposition assays for piggyBac mobility in species harboring piggyBac. • Complete genomic and strain analysis of new acceptor site target strains with determination of optimal target site strains.
60 MONTHS: • Isolate complete piggyBac elements in species harboring functional elements. • Assess stability of transgene vectors in species harboring piggyBac. Begin RMCE donor vector transformations into target strains for functional analysis of GOIs and conditional lethal constructs. • Remobilize embedded piggyBac vector after donor integration - test for stabilization of remaining genomic transgenes.
Subobjective 1.4 Assess the potential for vector re-mobilization in released transgenic strains and develop new vectors that allow increased stability and targeted integration for greater efficacy and ecological safety.
12 MONTHS: • Conduct deletion analyses of JcDNV vectors to identify DNA sequences critical for vector activity. • Test efficiency of lepidopteran viral promoters in somatic and germline vectors in moths.
24 MONTHS: • Construct plasmids with germ cell specific promoters and assess the fidelity of the expression patterns in transformed flies and moths.
36 MONTHS: • Isolate insertional sites formed following insertion of JcDNV somatic transformation vectors into host cell chromosomes and determine conserved sequences.
48 MONTHS: Conduct isolation of recombinational complexes between somatic and germline transformation vectors that are formed in insect cell lines.
60 MONTHS: • Construct plasmids with enhanced recombinational activities that are specific to germ cells and test for higher efficiency as gene vectors in flies and moths.
Objective 2 - Population Dynamics:
Identify strains of Lepidoptera pests, describe differences in behaviors, and isolate the genetic factors controlling these differences in order to understand how species adapt to new ecological niches and to better target biologically-based control strategies in area-wide IPM programs.
Sub-Objective 2.1: Define the seasonal distributions of genetically defined subpopulations of fall armyworm in order to investigate strain-specific behaviors related to plant host usage, migration, and mating.
12 MONTHS: • Determine the temporal and spatial distribution of fall armyworm strains in central and southern Florida.
24 MONTHS: • Initiate a three year field survey of fall armyworm populations in Florida and adjacent states to examine strain-specific behaviors related to migration, mating, and habitat preference. • Initiate field studies examining interstrain mating and the plant host preferences of the two strains
36 MONTHS: • Field survey data for FY2005 will be assessed and the survey methodology modified as necessary
48 MONTHS: • Continue field surveys and modify or expand as necessary. • Estimate the degree of interstrain mating occurring in the wild and define the habitat and mating preferences of the resulting hybrids.
60 MONTHS: • Complete the field survey and analysis of data. • Develop a comprehensive explanation for the distribution of the host strains in various habitats in overwintering and migratory areas. • Identify habitats and locations in overwintering areas that should be targeted to reduce migratory populations.
Sub-Objective 2.2: Characterize new strain-specific genetic polymorphisms and identify genes controlling strain-defining phenotypes that can be used as more accurate genetic markers of strain identity and in genetics-based biological control strategies
12 MONTHS: • Develop more accurate and efficient methods for detecting genetic markers from fall armyworm collected in the field. • Develop methods for estimating interstrain mating behavior from the distribution of different genetic markers in field-collected fall armyworms.
24 MONTHS: • Develop laboratory bioassays to test for and characterize strain-specific behaviors and traits, including those related to mating, migration, and host plant choice. • Identify strain-specific genetic polymorphisms by ISSR and RAMP methods.
36 MONTHS: • Screen for genes associated with strain-specific behaviors and traits by differential display, subtractive hybridization, and other genomic screening methods. • Select and maintain fall armyworm lines with distinctive behaviors.
48 MONTHS: • Genetically characterize strain-specific behaviors identified by bioassays. • Clone and characterize genes identified by differential expression assays. • Test for genetic differences between geographically distant populations.
60 MONTHS: • Identify by genetics and transgenics the functions of differentially expressed genes. • Determine the degree of genetic introgression between the two strains in overwintering areas. Objective 3 - Detection:
Describe acoustic and other signals and cues produced by pest arthropods, including Mediterranean fruit fly, Diaprepes root weevil and other cryptic/hidden insects, and develop detection technologies and attractive devices that can be used in IPM projects to target, monitor and control pests.
Subobjective 3.1: Identify hidden/cryptic pests and precisely target control measures to limited areas where they will be most effective
12 MONTHS: • Describe acoustic and other signals and cues for pest detection.
24 MONTHS: • Test acoustic devices developed with AEC Inc. • Analyze Termatrac microwave radar signals.
36 MONTHS: • Develop and test novel acoustic sensors and interfaces. • Analyze signals of Rite Trak radar system. • Field-test Termatrac radar devices.
48 MONTHS: • Develop library of microwave radar signals collected in different environments. • Field-test Rite Trak radar system. • Initiate acoustic and microwave technology transfer to end users.
60 MONTHS: • Identify promising aspects and problematic areas in transfer of acoustic and microwave technology and modify efforts to optimize transfer process.
Subobjective 3.2: Design a highly effective trapping system to delimit and control Mediterranean fruit fly.
12 MONTHS: • Develop detection technologies and attractive devices.
24 MONTHS: • Develop and analyze library of sounds collected from different environments. • Conduct bioassays with ASRC automated medfly traps and modify software and hardware as needed.
36 MONTHS: • Combine visual acoustic, and pheromone cues in female-selective medfly traps. • Field-test ASRC automated medfly traps
48 MONTHS: • Field test female medfly traps. • Analyze results of ASRC-trap testing.
60 MONTHS: • Explore incorporation of insect acoustic and microwave signals into RFID technology
Objective 4 – Biological Control:
Develop strategies for effective use of parasitoids and predators in IPM of Lepidoptera and tephritid fruit fly pests, such as Mediterranean fruit fly, potentially invasive species of Anastrepha fruit flies and fall armyworm, through behavioral and ecological studies of their feeding, mating, dispersal and oviposition.
Subobjective 4.1: Develop economical all-female strains of fruit fly parasitoids for mass-rearing and augmentative release.
12 MONTHS: • Collect parasitoids and determine progeny sex-ratio of individual females. • Identify endosymbionts in known sex-ratio distorted strains.
24 MONTHS: • Examine the role of temperature in sex-ratio distortion in known distorted strains. • Begin experiments to horizontally transmit endosymbionts. • Continue parasitoid collections and offspring sex-ratio monitoring.
36 MONTHS: • Continue temperature experiments with additional species. • Continue attempts at horizontal transmission. • Compare fitness characteristics between infected and uninfected strains.
48 MONTHS: • Continue temperature experiments with additional species. • Continue attempts at horizontal transmission with addition of egg-injection. • Continue to compare fitness characteristics.
60 MONTHS: • Develop mass-rearing procedures for candidate sex-ratio distorted strains.
Subobjective 4.2: Determine the most efficacious fruit fly parasitoids for mass-rearing and augmentative release.
12 MONTHS: • Initiate field cage behavioral observations of foraging behaviors at different densities with individual braconid species. • Initiate field cage studies of microhabitat preference with individual braconid species.
24 MONTHS: • Initiate foraging behavior observations with multiple braconid species.
36 MONTHS: • Initiate foraging behavior and microhabitat observations with single and multiple species of Figitidae.
48 MONTHS: • Formulate a program for the most effective parasitoid(s) for augmentative release.
60 MONTHS: • Conduct field-plot tests of various parasitoids under different environmental conditions.
Subobjective 4.3: Determine larval and adult food sources to be incorporated into agroecosystems in order to conserve, amplify and prevent the dispersal of natural enemies of Lepidoptera and fruit fly pests.
12 MONTHS: • Begin field observations and trap sampling of natural enemies attracted to various flowering plants. • Begin morphological correlations between insect and flower morphology. • Identify non-floral food sources. • Locate disturbed and undisturbed site for sampling of fruit flies and parasitoids. • Observe potential tephritid food sources in the field in Florida and Mexico. • Initiate feeding experiments in the laboratory.
24 MONTHS: • Expose sentinel larvae in patches of candidate flowering plants. • Continue field observations and trap sampling. • Predict additional floral food sources from morphological studies. • Begin field sampling. • Continue field observations. • Continue feeding experiments. • Initiate nutritional state analyses.
36 MONTHS: • Compare field plots with and without adult natural enemy food resources and larval alternative-host plants. • Continue with small plot units of varying sizes of weedy borders around field corn. • Continue fruit fly field sampling. • Continue nutritional state analyses.
48 MONTHS: • Continue field plot comparisons and expand to include new food sources and alternative host plants. • Begin fruit fly habitat manipulations.
60 MONTHS: • Formulate procedures for amplifying the numbers of hymenopterous natural enemies in various agro-ecosystems. • Continue fruit fly habitat manipulations.
Virus-based genetic transfer (NP 304 Crop Protection and Quarantine, Components II and IV): The development of stable gene transfer systems is central to genetic modification of insects and for utilization in genetic control methods for pest insects. A small DNA sequence derived from an insect virus was found to efficiently promote the insertion of foreign DNA into the chromosomes of insects. The viral DNA sequence also contains a highly active “promoter” that results in high levels of protein expression. This system provides and excellent tool for new genetic modifications for the control of pest insects.
All-female parasitoid strain (NP 304 Crop Protection and Quarantine, Components II and IV): Mass-reared female parasitoids are a useful means of area-wide pest fruit suppression. However, the expense of rearing prevents their use in some situations. An all female strain of one fruit fly parasitoid obtained from collaborators at the Instituto de Ecologia (Xalapa, Veracruz, Mexico) and placed in quarantine at the Center for Medical, Agricultural, and Veterinary Entomology (Gainesville, Florida) was determined to be infected with a bacterium that changes males into females. If this bacterium can be successfully transferred to other species then it may be possible to employ diverse all-female colonies at half the cost of male and female colonies.
“Smart” fruit fly trap (NP 304 Crop protection and Quarantine, Component VI): Being able to monitor the spread of Mediterranean fruit flies and the effects of control measures is critical to their area-wide management In cooperation with Robert Jones and Roman Machan of SSRC, Inc., an automated acoustic trapping system was developed at the Center for Medical, Agricultural and Veterinary Entomology, Gainesville, Florida. The novel trap includes new digital signal processing techniques that successfully identified and discriminated female and male Mediterranean fruit flies in the laboratory. Field-cage studies of a prototype detection system have been initiated. Adaptations based on the prototype are expected to be deployable in niche applications where trap servicing is difficult and result in substantial reductions in cost and increases in accuracy.
Novel parasitoid attractant (NP 304 Crop Protection and Quarantine, Components II, III and VI): While parasitoids are regularly mass-reared and released for the area-wide control of pest fruit flies there are no simple means of trapping released insects to determine their dispersal and survival. A widely used parasitoid was found to be highly attracted to one species of flower, alyssum, but not to another, false buttonweed. The odors of the two flowers differed by a single compound, acetophenone, and certain doses of this chemical were attracted female, but not male, parasitoids. This compound is one of the first floral attractants identified for parasitic insects and may form the basis of a new trapping system.
1) incorporation of fluorescent protein markers into the medfly temperature sensetive lethal genetic sexing strain for efficient field identification; (Components II and IV) 2) the discovery of the new piggybac transposable elements throughout the Bactrocera fruit fly complex and several lepidopteran species will facilitate transformation and lead to autocidal controls (Components II and IV)
3) analysis of the hopper transposable element in the oriental and melon fruit flies for development into new transformation vectors will facilitate transformations and lead to autocidal controls (Components II and IV)
4) a system was devised that stabilizes certain forms of genetic transformations. The danger of genes from modified organisms being transferred to other animals or plants has hindered or prevented the application of transformation technology in insect control programs (Components II and IV)
5) discovery in Africa and Latin America of new and efficacious natural enemies of pest fruit flies will contribute to suppression of medfly populations and add to eradication programs (Component III and IV)
6) construction of a new Central American quarantine facility managed by the international organization MOSCAMED with support of APHIS-CPHST will further biological control efforts in Central America (Component II)
7) improvements in parasitoid mass-rearing through host irradiation and sexual separation of hosts have lowered costs and made augmentative releases more financially feasible;(Component II)
8) development of aerial release techniques that allow parasitoids to be chill-processed and released over difficult and mountainous terrain have allowed the expansion of augmentative releases (Component II)
9) demonstrations of synergistic increases in fruit fly suppression through combinations of sterile males and various parasitoids will provide a new tool for fruit fly suppression in areas where insecticide applications are impractical (Component II and IV)
10) augmentation of parasitoid numbers through habitat manipulation for control of beet armyworm and diamondback moth have provided growers with cost saving, non-chemical means of control in row crops (Components II and III)
11) identification of different fall armyworm host strains with different propensities for migration through genetic techniques will allow controls to be precisely targeted during area-wide control programs (Components II and IV)
12) development of techniques for the injection of DNA constructs into fall armyworm and diamondback moth embryos with transient expression of transgenic genes in host embryos will contribute to genetic modifications and ultimately to autocidal control techniques (Component II and IV)
13) development of transgenic constructs in which an inducible heat shock promoter causes female flies to become sterile, thereby providing a novel method for biological control through creation of fly strains that are fertile under laboratory conditions, but sterile in the field (Component IV)
14) demonstration that acoustic methods could be used to detect and map hidden insect infestations in grain, packaged goods, wood and soil and so improve quality and pest control (Component VI)
15) development of improved interfaces between acoustic sensors and automated recognition / quantification programs will allow more precise identification of pests and improve targeting of controls (Component VI)
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Meagher, R. & R. Nagoshi. 2005. Mating behavior of fall armyworm host strains in Florida. Ann. Meeting, Entomol. Soc. Am., 15-18 Dec., Ft. Lauderdale, FL.
Shirk, P., R.B. Furlong, J.L. Gillett and H. Bossin; Functionality of JcDNV-Derived Somatic Transformation Vectors in Insects and the Role of Viral Enhancer Sequences. The 5th International Symposium on Molecular Insect Science, Tucson AZ May 20-25, 2006.
Shirk, P., R.B. Furlong, J.L. Gillett and H. Bossin; Functionality of JcDNV-Derived Somatic Transformation Vectors in Insects and the Role of Viral Enhancer Sequences. The 5th International Symposium on Molecular Insect Science, Workshop on Development and applications of insect transgenesis, Tucson AZ May 20-25, 2006.
Shirk, P., R.B. Furlong, J.L. Gillett and H. Bossin; Functionality of JcDNV-Derived Somatic Transformation Vectors in Insects and the Role of Viral Enhancer Sequences. 89th Annual Meeting of the Florida Entomological Society Jupiter Beach, FL, July 23 – 26, 2006.
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Murakami, M., Gallo-Meagher, M., Gorbet, D.W., Meagher Jr, R.L. 2006. Utilizing immunoassays to determine systemic tomato spotted wilt virus infection for elucidating field resistance in peanut. Crop Protection Journal. 25:235-243.
Rendon, P., Sivinski, J.M., Holler, T., Bloem, K., Lopez, M., Martinez, A., Aluja, M. 2006. The effects of sterile males and two braconid parasitoids, Fopius arisanus and Diachasmimorpha krausii, on caged populations of Mediterranean fruit flies, Ceratitus capitata at various sites in Guatemala.Biological Control. 36:224-231.
Sivinski, J.M., Aluja, M., Holler, T. 2006. Food sources for adult Diachasmimorpha longicaudata, a parasitoid of tephritid fruit flies: effects on longevity and fecundity. Entomologia Experimentalis et Applicata. 118:193-202.
Zimowska, G.J., Handler, A.M. 2006. HIGHLY CONSERVED PIGGYBAC ELEMENTS IN NOCTUID SPECIES OF LEPIDOPTERA. Insect Biochemistry and Molecular Biology. 3:421-428.
Pereira, R., Teal, P.E., Sivinski, J.M., Dueben, B.D. 2006. Influence of male presence on sexual maturation in female Caribbean fruit fly, Anastrepha suspensa (Diptera:Tephritidae). Journal of Insect Behavior. 19(1):31-43.
Nagoshi, R.N., Meagher Jr, R.L., Adamczyk Jr, J.J., Braman, K., Brandenburg, R.L., Nuessly, G. 2006. New restriction fragment length polymorphisms in the Cytochrome Oxidase I gene facilitate host strain identification of fall armyworm (Lepidoptera: Noctuidae) populations in the Southeastern United States. Journal of Economic Entomology. 99(3):671-677.
Nagoshi, R.N., Meagher Jr, R.L., Nuessley, G., Hall, D.G. 2006. Effects of fall armyworm (Lepidoptera: Noctuidae) interstrain mating in wild populations. Environmental Entomology. 35:561-568.
Lewter, J.A., Szalanski, A.L., Nagoshi, R.N., Meagher Jr, R.L., Owens, C.B., Luttrell, R.G. 2006. Genetic variation within and between strains of the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae). Florida Entomologist. 89:63-68.
Aluja, M., Sivinski, J.M., Rull, J., Hodgson, P.J. 2005. Behaviour and predation of fruit fly larvae (Anastrepha spp.) (Diptera: Tephritidae) after exiting fruit in four types of habitats in tropical Veracruz, Mexico. Environmental Entomology. 34(6):1507-1516.
Meagher Jr, R.L., Mislevy, P. 2005. Trapping Mocis spp. (Lepidoptera:Noctuidae) adults using different attractants. Florida Entomologist. 88(4):424-430.
Li, X., Harrell, R.A., Handler, A.M., Beam, T., Hennessy, K., Fraser, Jr., M.J. 2005. Terminal region proximal internal domain sequences of the piggyBac transposon are necessary for efficient transformation of target genomes. Insect Molecular Biology. 14:17-30.
Copeland, C.S., Mann, V.H., Morales, M.E., Kalinna, B.H., Brindley, P.J. 2005. The Sinbad retrotransposon from the genome of the human blood fluke, Schistosoma mansoni, and the distribution of related Pao-like elements. Journal of Evolutionary Biology. 5(1):20.
Morales, M.E., Ocampo, C., Cadena, H., Copeland, C.S., Termini, M., Wesson, D. 2005. Differential identification of Ascogregarina species (Apicomplexa: Lecudinidae) in Aedes aegypti and Aedes albopictus (Diptera: Culicidae) by the polymerase chain reaction. Journal of Parasitology. 91:1352-1356.
Holler, T., Sivinski, J.M., Jenkins, C., Fraser, S. 2006. A comparison of yeast hydrolosate with female synthetic food attractants for the capture of Caribbean fruit flies, Anastrepha suspensa (Diptera: Tephritidae). Florida Entomologist. 89:419-420.
Sivinski, J.M., Pereira, R. 2005. Do wing markings in fruit flies (Diptera: Tephritidae) have sexual significance? Florida Entomologist 88:321-324.
Nathan, S.S., Mankin, R.W., Kalaivani, K., Murugan, K. 2006. Effects of millet, wheat, rice, and sorghum diets on development of Corcyra cephalonica (Stainton) (Lepidoptera: Galleriidea) and its suitability as a host for Trichogramma chilonis Ishii (Hymenoptera:Trichogrammatidae). Journal of Environmental Entomology. 35:784-789.