Brief Summary of Minutes of Annual Meeting

February 21-22, 2010, Orlando, FL Notes for the meeting recorded by Bob Behle and David Shapiro-Ilan, compiled by Bob Behle: " Seventeen Participants convened at 8:30 AM. " Participants were welcomed by Ed Lewis, Chair for the meeting " Local Arrangements: Drion updated on the meeting arrangements. Registration = $40 " Meeting minutes: Bob behle will update 09 minutes and they can be posted on the website. " Need to update the mailing list with current E-mail addresses, list was passed around. " Other names that may be interested in participating in S-1024 were added. " Major focus of this meeting is to set up plan for the re-write of the project plan. Bob Nowierski - National Institute of Food and Agriculture (NIFA) An extramural funding agency undergoing major overhaul of funding structure and specific information was provided as a handout of a Power Point presentation. New programs should be listed in March 2010. AFRI will provide funding up to $25 Million per project. CAR, RAMP, MBT are being phased out. Fate of other grant programs are unclear although Organic Ag Research and Specialty Crop Research are mandates in the Farm Bill. Project Renewal Discussions Final report of current project " Identify Benefits from current project in the final report : note successes  eg. Bt transgenic crops and Bti use for mosquitoes " Previous section Chairs should write their respective summaries. Project Rewrite " Ag Exp Stat Directors require good impact statements (not a laundry list)as an important portion of the write up. Bob N. has a power point to help with this part of the write-up. " Write-up will need to be filed by September. Based on subprojects, chairs will be responsible for section " Discussion focuses determining the basic structure of the write up, proposals include commodity based, vs organism based, etc. o Primary Objectives § Annual crops = row crops, vegetables etc. § Perennial crops = orchards, small fruits, forage, etc § Natural Landscapes = Med/vet, turf, ornamental, urban, forests " Additional discussions on sub-objectives were tabled for later in the meeting Subproject 1 Large Acreage crops Mark Boetel, NDSU: Working with sugar beet root maggots to integrate biocontrol and management (cover crops) for control of SBRM, previous years. Currently working on adoptable technologies, applied Ma granules (F52 microsclerotia) with or without neonicoitinoid insecticides for control. Last season was wet and delayed planting of sugar beets. Thus the plants were small when infested by SBRM and Ma did not protect the plants from damage. Chemical seed treatments did not work well either, but normal chemical controls provided the best control. Work done in cooperation with S. Jaronski (ARS). Collembola are a pest of sugar beets by killing seedlings. Flour granules seemed to attract collembola. Also have some wireworms problems, but have little effort for control. Lerry Lacey =Potatoes used to have a large project with potato tuber moth. Evaluated the virus in the field and were encouraged and also tested with dipped potatoes in cold storage. Results were good in that worms did not transfer to other potatoes during storage. Brian Federici. For support of Bt corn for rootworm. Supported the use of Bt for CRW control because it would reduce the application of chemicals (chlropyrifos) for control. There remain concerns for the development of HPR to Bt toxin. Movement of the CRW to Europe may help to change perception toward acceptance of transgenic crops in Europe. He also reported that about 98% of cotton grown was Bt cotton. David Shapiro noted that research is being conducted on the use of nematodes for CRW control. Larry Duncan (UofF) Nematode treatments per acre are priced at about $25 acre in Florida. (Lunch Break) Todd Ugine  Bt cotton reduced the use of pesticides and the Tarnished plant bug is considered the primary pest of cotton. J. Leland selected a Beauveria isolates to target lygus (previously). Controlling wild weed hosts reduces the initial population of lygus. Thus, evaluating temperatures vs development and reproduction showed that treating field margins in early spring (January) is the best control strategy. Subproject 2. Ornamental, vegetable, fruit and nut crops. Robin Stewart - For citrus, control of Diaprepes is difficult because of continual emergence that requires multiple sprays. Nematode applications have been a good biocontrol success in most soils, especially in ridge soils but not in flatwood soils. Diaprepes are not often a big problem for ridge soils where there is a diversity of nematode species. Current research is directed at the ecology of the soil. Multiple applications for controlling greening (psyllids) has helped to reduce diaprepres infestations. Larry Duncan - Starting trees in a Sand plug for the orchard tend to favor the development of nematodes and detrimental to Diaprepes. Also weevil control is helped by adding a landscape fabric (lumite) to break the lifecycle of the pest (neonates going in and adult emergences out of the soil.) Fewer EPNs being applied for weevil control. Raquel Campos Herrera - Has developed selective qpcr primers for distinguishing introduced vs natural nematodes. Using the information to correlate nematode populations with physical environmental conditions. New irrigation practices may be having a negative impact on EPNs. Ed suggested the reduced EPN density may result from increased nematophagous fungi. David Shapiro-Ilan - Pecan Weevil  previously reported on treating emerging weevils, but that success was limited (60%). Now trying to control soil stage (2 year diapauses). Also applying fungus to tree trunks has some success. This misses individuals that fly to the tree and the delayed kill allows some damage before mycosis. For peach tree borer, EPN applications gave similar control as insecticide applications. This application has support of Becker Underwood. For Plum curculio that attacks the fruit, attract to sentinel trees treated with pheromone. Then treat the adults with chemical and clean up larvae with nematodes in the soil. Have also documented the attraction/repulsion of nematodes to electrical current. Also, have developed a taped cadaver to make a more user friendly version for the cadaver formulations. Trait deterioration during sub-culture remains an issue. Lerry Lacey. Zebra stripe potatoes are the result of starch build-up in the tuber that is caused by the potato psyllid, and will report more next year. For citrus, treatments of baracade over nematodes to protect the nematodes when applied to trees. Application is made with a texture gun from home improvement. A hard to mix foam performed better than the gel application. Cadaver formulations applied with mulch provided some improved control when the soil remains moist. Worried about a new pest, clearwing apple moth, borer that will kill young apple trees. Have found some control with Beauveria. Also work with the Gassing Fungi is showing control of possible storage pests for potato and apples. Ed Lewis Pistachio with Joe Segal. S. carpocapsae put out through irrigation will control moths from dropped nuts. After applications, free living populations have a disrupted environment for several weeks. European Earwig is killed by Sc and serve as a host for the nematode. Some species of mites and collembolan increase after applications, maybe by feeding on applied nematodes. Root growth dynamics in walnut orchard treated with S riobrave, there is a flush of root growth. For examining nematode uses to satisfy quarantine requirements, soil potting mixes have influence on nematode efficacy. The result is to develop customized soil mixes to target potential target/beneficial agent control tactics. Subproject 3 From Ann Hajek (see report Gypsy moth, E. miamiaga) Todd using fungal bands for long horn borer control. He has not seen any data that demonstrates that imidacloprid injections actually kill beetles. Imidacloprid may have synergy with Ma fungi. Many additional studies are planned. Adjourned for the day at 5:00 pm Monday 8:00 (16 attended) Subproject 3 (continued) Lee Solter  Reported about a microsporidia found by D. Bruck that is highly infective/toxic to black vine weevil. Lee is doing the identification. For forestry, two of three microsporidian pathogens were released in 2008 for gypsy moth. Apparently E. miamiaga has competed successfully against the microsporidia. Hemlock wooly adlegid has microsporidia in the colonies used to develop biological controls that are released. The result is the identification of 2-3 new species of microsporidia that may be a problem for biological control efforts. Also working with bumble bees to determine if bumble bee populations are declining because of bottle necks in populations, diseases, or natural fluctuations. Have found a European microspridia in the population, but it is found everywhere. Has joined honey bee project for nosema problem to evaluate interactions among virus and microsporidia. Subproject 4  Veterinary and Structural David Oi - read report submitted by Jimmy Becnel. (He has joined the mosquito project NE1043). He believes that problems with fire ant colonies in the lab is a result of a virus. No such decline has been observed in the field. Fire ant microsporidia was found in decapitating phorids. Efforts continue to import Varimorpha invicta, but are having regulatory problems (from Argentina). Brian Federici - Main focus is to make new recombinants of Bti (B. sphaericus has not been successfully genetically modified). Cost of materials to produce Bt is about 15 cents per acre treatment for production. Best recombinant is Bti bsben. Result has been the increase in the ability to produce more cry toxins per unit of fermentation. B. sphaericus (recombinant) is highly effective against many mosquitioes and has been field tested. Site A is beneficial by masking resistance development of the larvae to the toxin. Ascoviruses are widely distributed in nature, are transmitted by parasitic Hymenoptera, and are being studied in terms of evolution and relations with other viruses. Drion  working on novel group of viruses (family Hytrosaviridae) that are active against Muscid flies. Determined that the Musca domestica salivary gland hypertrophy virus (or MdSGHV) is orally transmitted causing both gland hypertrophy and sterility in female flies within 48-72 h post-exposure. In nature, this relatively homogenous virus is distributed worldwide in housefly populations. Technology for the mass production of infected flies has been developed allowing for dissemination via release of infected flies or spray applications of fly homogenates. Ellie - working with invasive European fire ant that has been found in the northeast. Looking at ecology and management including pathogens. How it defends itself against pathogens. Behavior to orient to high light and heat for disposing cadavers. Have found pathogens and nematodes apparently related to crashing colonies. Also found a new fungus that has caused mortality of ants and needs to be identified (looks like Hirsutella). Nematodes have been found as well and will require additional work. Also trying to get money to study a microsporidia found in dead ants in England. Primary Objectives for Project Rewrite = Subproject Sections " Annual crops = row crops, vegetables etc. " Perennial crops = orchards, small fruits, forage, etc " Natural Landscapes = Med/vet, turf, ornamental, urban, forests Subproject Section Chairs " Annuals  Mark Boetel and Bob Behle " Perennial  Ed Lewis " Landscapes  David Oi and Steven Arthurs Sub Objectives: to address under each subproject " Discovery and characterization, which includes biology and ecology " Production application and integration (product (materials and information) development) Cooperative Projects: Two topics discussed for cooperative research, one proposed with no discussion. " Invasive insects  impact of native pathogens vs emerald ash borer. o Had support of Ellie, Lee, and possibly Ann H. and John V (not present) " Biofuels  utilize DOE grants to determine microbial epizootics. o Tabled till next meeting due to many unresolved issues " Improved delivery eg baiting, fungal bands, etc. (proposed by S. Jaronski via E-mail) no discussion New Business: Election for Member at large: Steve Arthurs, nominated, second and votes yes. Theme for 2011 meeting? Molecular applications in microbial control, as a topic or theme In Tucson, AZ Hosted by Dawn Gouge, and Patricia Stock Adjourned approximately 1 pm.

Accomplishments (2009) SUBPROJECT 1 Discovery of entomopathogens and their integration and safety in pest management programs for major acreage crops. [Co-Chairs: Bob Behle and Mark Boetel] Two research reports received for this subproject that covers large acreage crops (sugarbeet) and identify the cooperation of three ARS locations (Peoria, IL; Sidney, MT) and one university (North Dakota State University). Research is reported for efficacy control of sugar beet root maggot with Metarhizium anisopliae granule, and production of Metarhizium anisopliae microsclerotia by liquid fermentation. Field Efficacy: The sugarbeet root maggot (SBRM), Tetanops myopaeformis (Röder), is the key insect pest of sugarbeet in the Red River Valley of North Dakota and Minnesota. This pest is also a significant threat to sugarbeet production in several western states and the Canadian province of Alberta. In 2009, a granular formulation containing Metarhizium anisopliae (F52) conidia was tested alone and in combination with the chemical seed treatment insecticides Poncho Beta (i.e., clothianidin+betacyfluthrin) and Cruiser (i.e., thiamethoxam) for control of SBRM larvae. Granules were formulated as a novel microsclerotial composition developed by ARS, and applied at either 22.5 or 33.6 kg formulated product/ha. Granule placement was achieved by using three conventional methods (i.e., band, modified in-furrow, and direct in-furrow) that are commonly used by growers to apply conventional granular chemical insecticides. Spring flooding resulted in delayed planting for this trial. Fungus granules did not provide significant levels of root maggot control or associated yield benefits, and there were no apparent impacts associated with application rate. Moreover, F52 granules did not appear to provide an additive benefit in relation to SBRM control or yield when combined with seed treatment insecticides. Efficacy could have been compromised by the extremely late planting date which resulted in plants that were atypically vulnerable to insect attack during the SBRM peak larval feeding period. The resulting short interval between application and larval activity also probably minimized or precluded optimal timing of fungus sporulation for SBRM control. (North Dakota State University, Fargo, ND and USDA-ARS, Sidney, MT) Production A process has been developed to produce sclerotia with a liquid fermentation process. This propagule is likely to provide new uses for entomopathogenic fungi. These sclerotia are adapted to soil environments and were initially tested for control of sugar beet root maggot larvae in sugar beet fields. Sclerotia are applied with the intent that the sclerotia germinates to produce conidia to infect the target insect. Initial applications will be targeted at sugar beet root maggot control. (USDA-ARS, Peoria, IL; USDA-ARS Sidney, MT) SUBPROJECT 2 Discovery of entomopathogens and their integration and safety in pest management programs for ornamental, vegetable, fruit and nut crops. [Co-Chairs: Robin Stuart & Patricia Stock] Research Accomplishments A number of successful efficacy trials indicate substantial potential for microbial control agents in peach and pecan orchards. In a pecan orchard, multiple applications of S. carpocapsae made to the pecan weevil, Curculio caryae, during the insects 2-yr life-cycle in the soil, reduced the pests survival to less than 1% (81% control relative to the untreated plots) [USDA-ARS, Byron, GA]. In a peach orchard, a two-year study indicated that a sprayable gel formulation (Barricade®) applied as a post-application covering to Steinernema carpocapsae, significantly enhanced control of the lesser peachtree borer, Synanthedon pictipes compared with nematodes applied alone; the combination resulted in 0 to 30% S. pictipes survival. The sprayable gel (normally used for fire protection) may have broad applicability in improving aboveground applications of entomopathogenic nematodes [USDA-ARS Byron, GA; University of Florida; University of Georgia]. A new method of formulating and packaging entomopathogenic nematode-infected cadavers was developed. The infected hosts (e.g., Tenebrio molitor) are automatically wrapped in tape using a specialized packaging machine. The tape formulation protects the cadavers from rupturing and provides ease-of-handling. The formulated cadavers provided high levels of control versus the small hive beetle Aethina tumida, and the Diaprepes root weevil, Diaprepes abbreviatus [USDA-ARS, Byron, GA; USDA-ARS Stoneville, MS; Southeastern Insectaries, Inc.]. The biofumigant fungus, Muscodor albus, was efficacious for control of codling moth (CM) larvae and adults in and on stored apples [USDA-ARS, Yakima, WA]. The fungus also produced deleterious effects in the western cherry fruit fly. The potato tubermoth (PTM) granulovirus was shown to effectively control PTM in stored tubers by killing larvae in infested tubers and limiting their dispersal in refrigerated warehouse storage [USDA-ARS, Yakima, WA]. Preliminary research on the insecticidal activity of entomopathogenic fungi for control of potato psyllid, Bactericera cockerelli, demonstrated sufficient insecticidal activity to encourage field trials for 2010 [USDA-ARS, Yakima, WA]. The research included development of bioassay techniques, effect of fungal species and stage of the psyllid on insecticidal activity. Infective juveniles of Steinernema feltiae and S. carpocapsae formulated in wood flour foam or fire retardant gel were more effective for control of overwintering CM than aqueous suspensions of IJs increasing the utility of nematodes by orchardists [USDA-ARS, Yakima, WA]. Laboratory experiments tested the pathogenicity of Metarhizium anisopliae on adult Japanese beetles and adult Oriental beetles. The F52 strain was obtained from Novozymes Biologicals, Inc., Virginia. The results indicated pathogenic activity against Japanese beetle adults. Percent mortality of Japanese beetles at 11 days after exposure was 67, 81, 47, 86 and 83% for the 0.5, 1, 2.5, 5, and 10mg/100 beetles dose respectively. Oriental beetles did not exhibit similar results but mycosis was confirmed in a few Oriental beetle specimens [University of Connecticut]. Laboratory experiments were conducted in sand columns and on water agar in Petri dishes to study the attraction or repellency of three species of nematophagous fungi (NF; Gamsylella gephyropaga, Myzocytium sp. and Catenaria sp.) to five species of entomopathogenic nematodes (EPNs; Steinernema diaprepesi, Steinernema sp. glaserigroup, S. riobrave, Heterorhabditis zealandica and H. indica). EPN species were generally repelled by the trapping NF G. gephyropaga in sand, whereas on agar only S. riobrave was repelled while S. diaprepesi, Steinernema sp. glaserigroup, H. zealandica and H. indica were attracted to the fungus. With the exception of S. riobrave, the endoparasitic NF Myzocytium sp. consistently attracted EPN in sand and on agar. A second endoparasite, Catenaria sp., also tended to attract EPN in either medium. There was no evidence that EPN species which differ in susceptibility to a given fungal species also differ in whether they are attracted or repelled by the fungus. Furthermore, the different results in sand column versus agar plate assays indicate the potential importance of conducting such assays under the most naturalistic conditions possible [University of Florida]. Quantitative realtime PCR (qPCR) assays were developed to detect and quantify 6 species of EPN that are naturally distributed in Florida citrus orchards (Steinernema diaprepesi, S. riobrave, Heterorhabditis indica, H. zealandica, H. floridensis, and an undescribed species in the S. glaserigroup) and an exotic species, S. glaseri. A survey of an 8ha orchard in April 2009 compared the EPN spatial patterns derived from qPCR to that obtained by baiting soil samples with Galleria mellonella larvae and to data obtained from the same site in 20002001 by baiting soil in situ with caged larvae of the root weevil Diaprepes abbreviatus. The spatial patterns of EPN in the orchard were remarkably stable over nearly a decade. The qPCR assay was more effective than the Galleria baiting method for detecting EPN species composition in population mixtures and offers great potential for studying the ecology of EPN [University of Florida]. Greenhouse experiments were conducted to measure the persistence and efficacy of three species of entomopathogenic nematodes (EPN) against the citrus root weevil, Diaprepes abbreviatus, in soils of different textures. Analysis of plant root and top weights showed highly significant main effects of soil and nematode species, and a highly significant interaction between soil type and nematode species. Soil porosity was inversely related to plant damage by weevils, whether or not EPN were present; and porosity was directly related to plant protection by EPN. Steinernema riobrave afforded protection to citrus root systems in all 3 soils and the plants often had significantly higher root weights than other EPN treatments. Root weights were greater than positive controls in pots treated with S. diaprepesi in coarse and fine sand but not in sandy loam; and Heterorhabditis indica protected root systems significantly only in coarse sand. A second experiment produced similar results except that plant protection by S. diaprepesi was greater than that by S. riobrave. The results of these experiments confirmed the hypothesis that EPN would provide greater protection of seedlings against DRW in coarse textured soil than in soils of finer texture, a pattern previously observed in nature but the mechanism for this interaction was unresolved because there was no evidence that EPN killed weevils more effectively in any soil type. Rather, less vigorous growth of the negative control seedlings in the two finer textured soils suggests that unidentified factors stressing seedlings in those soils also impaired the ability of seedlings to tolerate weevil herbivory [University of Florida. In a study dealing with soils from citrus orchards, a multiple linear regression model that explains a significant amount of the variation in efficacy among soil types was developed. Much of the variation in insect mortality may be due to the combined action of naturally occurring dissolved solids (salts) and other soil amendments along with particle size. The drop off in nematode efficacy in soils with high sand content is due to the amount of dissolved solids in the soil, as measure by electrical conductivity (EC). SUBPROJECT 3 Discovery of entomopathogens and their integration and safety in pest management programs for urban and natural landscapes. [Chair: Parwinder Grewal] Studies of the spatial ecology of the gypsy moth pathogen Entomophaga maimaiga in central Pennsylvania demonstrated that the fungus was present and active at all sites but a cool wet spring resulted in synchronous epizootics in outbreak host populations across the area. Larger scale studies involving 64 sites across four mid-Atlantic states will work toward investigating factors that could help to predict whether E. maimaiga epizootics will occur. We found synergistic interactions between M. anisopliae and imidacloprid killing Asian longhorned beetle adults although imidacloprid also has some minor negative impacts on sporulation and growth of this fungus. Applied and basic research was conducted on the use of entomopathogenic nematodes. Fundamental aspects of the Heterorhabditis bacteriophora genome have been elucidated, and the susceptibility of different instars of Japanese beetle to H. bacteriophora was determined. SUBPROJECT 4 Discovery of entomopathogens and their integration and safety in pest management programs for medical, veterinary, and structural pests. [Co-Chairs: Jimmy Becnel & David Oi] Entomopathogens of veterinary pest included viruses, bacteria and microsporidia. Molecular and biological characteristics of the Musca domestica salivary gland hypertrophy virus (MdSGHV) was compared to the tsetse fly virus from Glossina pallidipes (GpSGHV) and used to establish the taxonomic position of this unique virus group. A project to expand the current collection of MdSGHVs was initiated to examine the distribution and biological activity of the MdSGHV. Protocols were established to improve the detection of virus in field collected flies and for the mass production of viremic flies suitable for introducing virus into populations lacking this biocontrol agent. A recombinant larvicidal stain of B. thuringiensis subsp. israelensis (Bti) and Bacillus sphaericus (Bs) referred to here as Bti/BsBin was evaluated against Anopheles gambiae. Evaluations against fourth instar larvae of An. gambiae found that Bti/BsBin was nine-fold more effective at the LC95 level than the strains of Bti and Bs used in current commercial products. Also, preliminary selection studies with the Bti/BsBin recombinant show no resistance development in An. gambiae after nine generations of selection. Complete genome sequencing for 3 diverse species of microsporidia from mosquitoes is in progress and part of a larger project to sequence a total of 12 microsporidian species with implications for human health. This information will provide a unique insight into the core set of genes that are responsible for the specialized intracellular lifestyle and development of these pathogens in these important disease vectors. For structural arthropod pests, a third virus (SINV-3) from the red imported fire ant, Solenopsis invicta was characterized. The virus is virulent and causes significant colony mortality. The microsporidian fire ant pathogen Kneallhazia (=Thelohania) solenopsae was detected in 12% of field collected fire ant parasitic flies. Thus far, there is no evidence that the flies are vectors of K. solenopsae. Pathogenicity was confirmed for a newly isolated Hirsutella sp. and insect parasitic nematodes found infecting the invasive ant Myrmica rubra in coastal Maine. Developmental studies were conducted on entomopathogenic nematodes infecting desert subterranean termites (Heterotermes aureus). Veterinary Pests: The genome size and gene content, tissue tropism beyond the salivary gland, virus transmission mode and virulence characteristics of MdSGHV has been compared to the GpSGHV. In addition, the general genome organization of SGHVs has been compared to other circular dsDNA insect viruses and to obtain phylogenetic relationships. The molecular and biological data has been compiled to describe the taxonomic position of this unique virus group. A series of experiments were conducted to quantify the levels and assess the infectivity of MdSGHV released by individual infected flies via salivation and excretion. Our experimental approach was to orally challenge individual, newly emerged healthy flies with filter-sterilized homogenates of viremic salivary glands or crops, with saliva collected from individual infected flies during one feeding event, or with excreta collected from individual viremic flies over night. In addition, crops were examined by transmission electron microscopy, and saliva and excreta samples were subjected to quantitative real-time PCR analysis to estimate the viral load in these organs. We initiated a program to expand the current collection of MdSGHVs. One goal of this research was to examine the distribution and biological activity of the MdSGHV; until recently, the only research on MdSGHV has been conducted in our laboratory on a strain isolated in 2005 from a dairy in North Florida. The house fly, a cosmopolitan and synanthropic insect, can be readily collected on all continents in areas of human activity. Protocols have been established to improve the detection of virus in field collected flies; in cohorts (one thousand flies) a single infected fly can be readily detected and amplified for analysis. Secondly protocols have been established for the mass production of viremic flies suitable for introducing virus into populations lacking this biocontrol agent. Large-scale tent studies have been conducted to examine the transmission and intergenerational persistence of this virus. (FL.) Previous studies have shown that several species of anophelines are sensitive to the Bacillus sphaericus (Bs) binary toxin (Bin toxin). Thus, we initiated studies on the efficacy and resistance management properties of our two best recombinant larvicidal stains of B. thuringiensis subsp. israelensis (Bti) against Anopheles gambiae. Both of these strains have been genetically engineered to produce large amounts of the Bs Bin toxin. The best strain, referred to here as Bti/BsBin, produces the Bs Bin in combination with the four major endotoxins of Bti, which are Cry4A, Cry4B, Cry11A and Cyt1A. When tested against fourth instars of An. gambiae, this strain was nine-fold more effective at the LC95 level than the strains of Bti and Bs used in current commercial products. Interestingly and importantly, preliminary selection studies with the Bti/BsBin recombinant show no resistance development in An. gambiae after nine generations of selection. These results are similar to what we have observed in selection studies with the same Bti/BsBin strain against Cx. quinquefasciatus, which is known to develop high levels of resistance quickly under field conditions when only treated with B. sphaericus formulations (CA). A white paper for microbial genome sequencing was prepared by a consortium of researchers and presented to the National Institute of Allergy and Infectious Diseases titled Comparative genomics of the phylum Microsporidia: defining shared and specific genes in a phylum of emerging human pathogens. This proposal proposed to sequence the complete genome of approximately 12 species of microsporidia. This proposal was approved, August 2009 and sequencing has begun with 3 species from mosquitoes selected for sequencing, specifically Anncallia algerae originally isolated from Anopheles stephensi, Vavraia culicis floridensis from Aedes albopictus and Edhazardia aedis from Aedes aegypti. Complete genome sequences for these 3 diverse species from mosquitoes will provide a unique insight into the core set of genes that are responsible for their specialized intracellular lifestyle and development in these important disease vectors (ARS-FL). Structural Pests: A multiplex PCR method was developed and used to determine the infection rate of the microsporidian pathogen of fire ants Kneallhazia solenopsae in individual parasitoid flies of fire ants, Pseudacteon curvatus, in north-central Florida. The mean K. solenopsae infection rate of P. curvatus from 4 sites in Florida, was 12.3 ± 5.0%. The K. solenopsae infection rate for P. curvatus was independent of the K. solenopsae infection rate observed among red importedfire ant, Solenopsis invicta, nests from where the fly collections took place. Of P. curvatus flies that developed in K. solenopsae -infected fire ants 24% (12/50) were positive for K. solenopsae upon eclosion. Thus far, flies have not been shown to vector K. solenopsae to other fire ants in laboratory studies. (ARS-FL) A new positive-strand, RNA virus was discovered from the red imported fire ant, Solenopsis invicta, using the metagenomics approach. This virus (Solenopsis invicta virus 3) represents the third virus discovered from this invasive ant. The virus is virulent and causes significant colony mortality. (ARS-FL) Research continues on the isolation and identification of pathogens of the aggressive invasive ant, Myrmica rubra, in both its native range in Europe and introduced range in northeastern U.S. Pathogenicity was confirmed for a newly isolated Hirsutella sp. and insect parasitic nematodes found infecting M. rubra colonies in coastal Maine. Identification of these species is currently being address in collaboration with project colleagues [fungus  Humber, ARS-Ithaca; nematodes  Stock, Univ. of Arizonia]. (ME) Developmental studies were conducted on entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) infecting desert subterranean termites (Heterotermes aureus). (AZ)

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