Brief Summary of Minutes of Annual Meeting

9:15 am Call to order by Ed Lewis. Announcement of officers: Ed Lewis - Chair, David Shapiro-Ilan Vice Chair Robert Behle - Secretary taking notes, Denny Bruck member at large (absent). S. Jaronski made the motion to approval of notes for the 2008 meeting, the motion was seconded, no discussion, and the motion was passed. Individual introductions and research updates: David Shipro-Ilan, (ARS-Byron, GA) Pecan weevil and peachtree borer control promising with entomopathogenic nematodes (EPNs), found EPNs respond directionally to electrical current. Applications of nematodes to peach trees for borers can use targeted application equipment to apply specifically to tree trunks. For pecan weevil, trunk applications of fungal conidia and soil applications of EPNs are effective. Lerry Lacey, (ARS-Yakama, WA): Apple clearwing moth is an invasive pest that bores and kills trees. Evaluating control by applications of gel spray of EPNs and foam-type formulations. Research continues on the use of granuloviruses for control of codling moth and leaf rollers, still looking for UV protection. Pear ester extract looks promising as a kairomone for control of codling moth; fumigant for cherry fruit fly applied by tilling, cover & kill. Potato tuber moth virus being studied for use in the field and during potato storage. Wireworms control with Metarhizium spp. is being evaluated. New pests are expected to include, cherry bark tortix, and European chafer. A potato disease, zebra chip, is caused by a bacterium spread by a psyllid and is similar to citris greening. Entomopathogenic fungi will be evaluated for efficacy against the psyllid. Robin Stuart (University of Florida, Lake Alfred, FL): Researching control of Diaprepes, why treatments work at some locations, but not at others. Observing soil differences such as heavy clay soils known as Flatwood soils, H. indica are present but do not provide strong control, while Ridge soil plantings, where the soil is much sandier have S. diaprepesi, H. indica, and H. zealandica, that provide good control. Also studying nematophagous fungi impact on nematodes, attraction and repulsion, coarse sand microcosms for nematode control. Overall, citrus greening is the #1 concern in Florida & interest/concern of Diaprepes is more back burner. Jarrod Leland (Novozome Biologicals Inc., Salem VA): F52 has markets in Holland for control of black vine weevil, and expects to move into the NW US next, F52 products also target ticks, white flies, thrips, mites. Company efforts are becoming more focused, toward vegetables and berries, and may market as a rotation with Spinosad. Ed Lewis, (UC Davis, Davis, CA): Along with Harry Kaya, currently studying control of BVW in Ukraine by identifying native isolates of EPNs, and modeling EPN infection dynamics  herding. Soledad Villamil, (Student, UC Davis): Studying olive fruit fly behavior. Amanda Hodson, (Student, UC Davis): Currently studying applications of EPNs through irrigation in pistachios for control of navel orange worm. Using EPNs in commercial vs nut mummies. Looking at the field ecology and the impact of EPNs on collembolan, and mite populations. Reports very little persistence of EPNs (2 wks) and predators of EPNs go way up. Anne Nielson (Postdoc, UC Davis): Studying Diaprepes preventative and curative control with EPNs and Metarhizium when applied to soilless media. Also, evaluating switchgrass (biofuel) for invasion of pests (this grass is not native to California), with respect to water and nutrient needs. Mike Blackburn, (ARS Beltsville): Conducting an evaluation of the Bt collection, organizing the Bts based on different biotypes and bio/eco differences in order to develop a useful phylogenetic association. Make Bt a biological control agent rather than bioinsecticide. Harry Kaya, (UC Davis, Davis, CA): Discovery work in Turkey, mite associated with white grubs that when the grub dies, the mites eat the EPNs. Bob Nowierski, (USDA-CSREES-PAS, Washington DC): Microbial Control workshop, non-target impact on endangered spp. Symposium in 2010. Preparing a position paper on regulation of microbial control agents. NPL bio-based Pest Management. Ken Spence, (Postdoc, UC Davis): Studying herbivore impact and ecology by using a mini rhizotron camera system, photographing roots over time. Also mode of action on plant parasitic nematodes in pasteurized soil is not good model because community effects are often important. Hanayo Arimoto, (Postdoc, UC Davis): Studying host-parasite relationships of face-flies and nematodes. Host preference, spatial patterns of infected hosts, physical effects of nematodes and influence on distribution of parasites. Roy Kaspi, (Postdoc, UC Davis): Studying control of Diaprepres with EPNs, specifically on impact of different soils found in CA, relating physical and chemical soil characteristics with efficacy. Stefan Jaronski, (ARS, Sidney, MT): Recently contributed to APHIS-PPQ serving on a board of advisors on regulation of biological control agents. For control of Orthoptera on rangeland, continues efforts to use green muscle (M. anisopleae acridum) for large scale field trials. Cage trials vs. mormon cricket have been successful. Field research requires minimum plot size is 20 A due to hopper movement. Continues (unsuccessfully) looking for M. anisopleae acridum in US, but has identified new and potentially useful M. anisopleae and Beauvaria bassiana; 342 isolates can use 20X less. Also studying Bts for control of hoppers. For sugar beets, F52 best for control of sugar beet root maggot when maggot pressure is low, but does not work in moderate to high pressure. Microsclerotia of Ma are being studied and can be formulated in different granule sizes. Mark Boetel, (NDSU, Fargo, ND): Continues cooperative work with Stefan on sugar beet pests, including root maggots, collembolan and wireworms. Would like to evaluate fungi and nematodes for soil application, looking for formulations. Have identified Fusarium (solani?) infected root maggot pupae. Robert Behle, (ARS, Peoria, IL): Working under Mark Jacksons CRIS (fermentation), along with Chris Dunap. Recently has worked on imported cabbageworm GV. UV protection formulations have included, lignin encapsulation for viruses and soyscreen for hydrophobic fungal conidia. Procedures have been developed to encapsulate oil to form a dry powder. Peoria ARS will likely be going to go thru re-organization. Drion Boucias, (University of Florida, Gainesville, FL): Looking at chronic viral pathogens that do not kill but sterilize house flies. These are a new family extremely widespread and spies specific. Also looking at thripinema nematodes that sterilizes female thrips. For the asian citrus psyllid, has identified a pheromone that is proded by Hirsutella fungus to attract healthy insects. Has identified a Bt active against cactoblastis (sp?). Ellie Groden, (University of Maine, Orono, ME): Currently studying invasive stingin ants, Northern sp, that takes over yard along the coast. Fungi found include Bb and Met. Ants have defense against infection. Assorted baits are being evaluated for attraction and transmission (what gets to queen  appears this can be exploited). EPNs dont cause colony movement, and a new EPN has been found. Bob Nowierski, CSREES presentation: Update on organizational changes in CSREES, funding opportunities, and coordination of invasive species efforts at the national level Organization changes resulting from the farm bill CSREES will be re-named: National Institute of Food and Agriculature (NIFA) Break for lunch Shirley Luckhart presentation: Systems biology: an overview and applications to complex host-parasite systems Afternoon Session: Individual Subproject Breakout Meetings or one whole group meeting (depends on what we decide) to discuss group projects and ideas for next years project renewal Note: This session was an open discussion simultaneously covering the up-coming project re-write and potential cooperative projects. The notes recorded by Shipro-Ilan and Behle were edited between these two topics for the minutes presented below. PROJECT RE-WRITE: Due September 2010 A. do we want/need change the organization? - Bob Nowierski says to make sure impact statements and publications are strong. If these are OK then it is probably OK to not change objectives. There may be no advantage for changing objectives. Objectives must be real objectives that are doable. So, we probably need to re-write within discovery and implementation. - One option: put primary/best impacts up front, with an appendix to provide more information about topics to show breadth of the work actually completed. o Subproject leaders pool impact from their group & highlight potential best ones. o Web site for impact statements is: http://www.cals.ncsu.edu/saaesd/infobook/Multistate%20project%20impact%20statements.htm o Perhaps make table on breadth of what we doing. B. Also, we all need develop goals for next round and can organize by subproject as well. - Look at W2185 (http://lgu.umd.edu/lgu_v2/homepages/home.cfm?trackID=9596) as model. NE1034 (http://lgu.umd.edu/lgu_v2/homepages/home.cfm?trackID=7456). C. Can list participants broadly (industry & Gov), but essentially the university hatch people are the actual members. - Need to try get the University people who should be here to come back  how? o Admin force. o Or also make meeting more attractive, § eg location, § or what we doing together, § or hold our meeting with/near another big meeting. - Need to make clear how our project impacts industry eg Jarrod comes to see what future projects might be for his company. - [meet with national biocontrol producers meeting, and biopesticide alliance (Stefan)] D. Look at NIMS to see some tips on rewrite. - Bob N is happy to look at draft proposal. GROUP PROJECTS Management of insect pests in biofuels, e.g., switchgrass (Lewis). Look at conservation. Could intercrop biofuels with pecan and peach trees (Shipro-Ilan). Nowierski says RAMP grants would be good outlet for above or other multi-regional pathogen projects, but would probably want weed and plant pathology components in it too. Groden says invasive pests would be good topic for study. Behle suggested a safety paper, perhaps published on the web could be a nice joint project. Cuts across pathogen and commodity groups. Jaronski: We need to get at base question of why microbials have not gone further and how to expand their use. Social, Efficacy, Price, Production Groden identified another common theme as how to reduce costs, especially by focusing on targeted application of pathogens. Lewis offered to lead a biofuel grant. Groden said, after July 1, she would be willing to lead either invasive grant or one on targeted delivery systems. Nowierski says preliminary grants may be submitted to PMAP, as projects progress then submit for RAMP grants, Having education and extension components are helpful for successful grant funding. Monday, March 2, 2009 Discussion of potential group cooperative projects Objectives for the re-write can be circulated via e-mail Stefan will spear head grant concerning target specific improved delivery. Ellie will spear head grant concerning invasives, AFRI. (Native pathogens vs Exotic Pests) Ed will spear head biofuel grant. Discussion of upcoming renewal of the project; we are scheduled to end in September of 2010 Stefan: put the objectives under model systems umbrella. Ellie & Ed: delivery systems would be good to put into it. Ed: but keep in commodity headings. David: Break down under commodities: improve organism, improve production/application, efficacy testing. Seems most agree; Stefan adds overall Obj is how to get MCAs into use. Include control of invasives under each commodity. Ellie Groden: suggests do away with commodity headings. Ed Lewis: Discovery (invasive pests), efficacy, prod/app. Arm-twisting session to determine next years meeting location and final closing of meeting Location: Orlando Host: Drion Boucias will organize with help from Lake Alfred group. Theme: Invasive pests, (e.g., psyllid) or biofuels. (tentative) Date: Similar time frame Funds: S-1024 currently has around $400, held by Lee Solter Illinois Natural History Survey account. This meeting will registration fees to cover costs and will send extra funds to Lee to deposit.

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) Research reports received for this subproject covers crops (cotton, corn, peanut, sugarbeet) and identifies the cooperation of four ARS locations (Ames IA; Corvallis, OR; Peoria, IL, Sidney, MT) and five universities (Auburn University; Iowa State University, North Dakota State University, Penn State University, University of Florida). Research is reported for Field Ecology (spread and survival of beneficial agents relative to common cropping systems), Resistance Development (for insects exposed to transgenic crops), Efficacy (specifically for parasites of thrips capable of transmitting viral diseases and fungal pathogens of sugarbeet root maggot), and Formulation (to prevent degradation of the beneficial pathogen by sunlight exposure). The most significant information may be monitoring of Helicoverpa zea and Heliothis virescens in the field show no detectable level resistance to Bt-transgenic crops. Field Ecology: Over the 3-year transition period from conventional to certified organic production of field crops, detection of Metarhizium anisopliae declined. There was a non-significant trend for detection of M. anisopliae to be greater in full-till (moldboard plow) systems compared with minimum till (chisel plow) systems. We hypothesize that greater spread and mixing of the soil in full-till treatments resulted in greater detection of M. anisopliae compared with minimum till treatments. Other biological and physical differences in soil conditions in the two tillage treatments may have also resulted in differential survival of the fungus or its hosts. We compared the effect of simple (crop) Vs. more complex (crop plus refuge) habitats on the persistence and movement of the entomopathogenic nematode Steinernema carpocapsae applied to soil in an organic maize field. We detected S. carpocapsae as far as 3m away from the application site after 30 days. S. carpocapsae movement does not necessarily depend on plant diversity, but may respond to variation in plant density, and subsequently, soil moisture. Complex habitats may serve as movement corridors for S. carpocapsae, depending on the contrast between the refuge and crop area. (Penn State University; State College, PA) Nosema pyrausta, an entomopathogenic microsporidium, is an important population regulator of the European corn borer, Ostrinia nubilalis. A review of research on the relationships between N. pyrausta and O. nubilalis has been submitted for publication. (USDA-ARS; Ames, IA and Corvallis, OR and Iowa State University, Ames, IA) Resistance Development: Cotton bollworm, Helicoverpa zea was selected for Bt Cry1Ac toxin resistance. Current resistance levels exceed 200-fold although larvae still cannot survive until pupation on Bt cotton squares suggesting plant-Bt toxin interactions. Resistance is not binding based, and appears to be at least partly protease-based. Inheritance of resistance is not sex-linked and incompletely dominant. Monitoring of H. zea and Heliothis virescens shows no detectable field resistance. (Auburn University, Auburn, AL) Field Efficacy: Frankliniella fusca (Hinds) is the predominant thrips species found inhabiting and reproducing in peanut (Arachis hypogaea L.) and is one of at least seven thrips species reported to transmit Tomato spotted wilt virus (TSWV). The entomogenous nematode Thripinema fuscum Tipping & Nguyen, a natural enemy of F. fusca, parasitizes larval and adult populations under field conditions. All known Thripinema species render female thrips sterile and potentially suppress pest populations to near extinction. Thus, secondary spread of TSWV in peanut is reduced. Reduction of the virus in field conditions may also be due to lower transmission rates caused by parasite-induced alterations in host feeding behavior. The feeding rates of healthy and parasitized F. fusca male and female cohorts on leaf discs were recorded daily and analyzed. Viral transmission rates were compared daily using a local lesion assay and ELISA. Parasitism by T. fuscum significantly reduced male longevity, but not female longevity. Thripinema fuscum reduced the feeding frequency of both male and female F. fusca, and the persistence of females to transmit TSWV. These results provide evidence that T. fuscum aids in regulating viruliferous F. fusca populations and suggests its potential as a biological control agent for inoculative release in peanut. (University of Florida, Gainesville, FL) In sugarbeet root maggot (SBRM), Tetanops myopaeformis (Röder), management research, two granular formulations (i.e., corn meal-based, and mycelial) of M. anisopliae (F52) conidia were tested alone and in combination with an insecticidal seed treatment (clothianidin+betacyfluthrin). Unlike previous findings, neither formulation provided significant levels of SBRM control or associated yield benefits, and there were no apparent impacts of application rate or formulation. The recently discovered isolate (ARSEF7382) of Fusarium solani (Martius) Saccardo was found to have strong virulence against SBRM pupae in two bioassays. Current research aims to determine the potential plant pathogenicity to the sugarbeet crop and other potential host crops. (North Dakota State University, Fargo, ND and USDA-ARS, Sidney, MT) Formulation: Efforts continue to provide UV protection for beneficial microbial agents. Concern about costs of registering spray-dried lignin formulations has prompted efforts to develop an adjuvant formulation based on similar lignin technology. Experiments comparing spray dried formulation with adjuvant treatments to protect baculovirus show some promise for the adjuvant to extend residual activity for field applications. Work continues on using soyscreen in oil-based formulations for hydrophobic fungal spores. Field applications of treatments containing soyscreen have been disappointing apparently because the oils are absorbed by the substrate leaving the pathogens exposed to degradation by sunlight. (USDA-ARS, Peoria, IL: USDA-ARS, Byron, GA) Subproject 2. Discovery of entomopathogens and their integration and safety in pest management programs for ornamental, vegetable, fruit, and nut crops. (Robin Stuart & Patricia Stock, co-chairs) Several successful efficacy trials indicate substantial potential for microbial control of peach and pecan orchard pests. In peach orchards, during 2007, tree-limb damage from lesser peachtree borer (Synanthedon exitiosa) was reduced by 80-100% following applications of Steinernema carpocapsae; these high levels of efficacy were only achieved when the nematodes were applied using a protective gel or bandage [USDA-ARS Byron, GA; University of Florida; University of GA]. In a pecan orchard, up to 90% suppression of adult pecan weevil (Curculio caryae) was observed following trunk applications with B. bassiana or Metarhizium anisopliae [USDA-ARS Byron, GA; University of GA; USDA-ARS Peoria, IL; Novozymes, Inc.]. In laboratory research, metabolites from Xenorhabdus and Photorhabdus bacteria were found to suppress key plant diseases of pecan and peach [USDA-ARS Byron, GA]. Additionally, a new nematode species, Heterorhabditis georgiana was isolated in Georgia and found to have microbial control potential [University of Florida; USDA-ARS Byron, GA; Fort Valley State University, GA]. In Florida, the invasive Asian citrus phyllid vectors greening disease to citrus and presents a serious threat to the industry. Adult psyllids killed by Hirsutella citriformis (ARSEF 8315) were found attached to the underside of citrus foliage in Polk, Hendry and Marion counties, and those killed by Paecilomyces fumosoroseus (ARSEF8316) were collected in Polk county. Hundreds of psyllids were collected that were killed by H. citriformis, but only a few psyllids killed by P. fumosoroseus were found. This difference in abundance is probably due to the fact that psyllids killed by H. citriformis are tightly attached to the citrus foliage by the synnemata, but those killed by P. fumosoroseus are loosely attached and may be removed from the tree by wind and rain. Two pilot field trials using this strain of P. fumosoroseus as a microbial insecticide resulted in reduced populations of psyllid nymphs. However, economic and technical issues, coupled with the frequent use of copper applications to control citrus canker and other plant pathogens, likely will prevent the commercial development of this beneficial fungus for use in Floridas citrus pest management programs. We do not yet understand the role of these two fungi on Asian citrus psyllids in Floridas citrus groves. So far, we have identified two new natural enemies in Florida that can kill Asian citrus psyllids and gained knowledge about the basic biology of each pathogen in our laboratory studies. (University of Florida; Gainesville, FL) Several enteric bacteria isolated from the Colorado potato beetle inhibited the growth of the entomopathogens Photorhabdus temperata and Beauveria bassiana in vitro. In New Jersey, cranberry bogs are damaged by the cranberry white grub, Phyllophaga georgiana. In a series of laboratory and greenhouse experiments evaluating three entomopathogenic nematode species for control of P. georgiana, S. scarabaei was the most effective species with 76-100% control at a rate of 2.5 billion IJs/ha in the greenhouse, whereas H. zealandica and H. bacteriophora were less effective requiring 5 billion IJs/ha for acceptable control. Larval stage had no effect on H. zealandica and H. bacteriophora performance; S. scarabaei tended to be more effective against third than second instars. SUBPROJECT 3: Discovery of entomopathogens and their integration and safety in pest management programs for urban and natural habitats. (Parwinder Grewal, chair) Sampling continued in long-term plots in central New York to document the activity of natural enemies attacking gypsy moth larvae and to document associated gypsy moth densities. In particular, the fungal pathogen Entomophaga maimaiga continues to cause infection in very low density gypsy moth populations although its now been 16 years since the last epizootic caused by E. maimaiga in that area (Cornell University) Studies of the spread of fungal and viral pathogens into newly established gypsy moth populations also continued. Field work in central Wisconsin was completed in 2007 and samples were processed toward analysis and preliminary conclusions in 2008. In central Wisconsin weve found that the gypsy moth fungus and virus keep up with gypsy moth spread; as soon as gypsy moth populations are abundant enough that larvae can be collected both pathogens are present, although low in prevalence (USDA, Forest Service; Wisconsin Dept. Natl. Res.; Univ. Wisconsin, Cornell University). After 2 years of preliminaty surveys (that were negative), E. maimaiga was released on two of the Apostle Islands late in the gypsy moth field season in 2007; the gypsy moth virus had been sprayed on these islands previously. Gypsy moth larvae were caged and field-collected during 2008. Our results have demonstrated that E. maimaiga persisted at release sites and spread to a limited extent. The virus, which was sprayed from an airplane, persisted very well and on one island, percent viral infection was very high. A graduate student in my laboratory completed his doctoral dissertation on the gypsy moth virus: effect of gypsy moth density on disease resistance, differential spread of the virus by differences in prey-processing behavior and the potential for vertical transmission of the gypsy moth virus (Cornell University). We continued a study to investigate the spatial and temporal dynamics of Entomophaga maimaiga activity. We are investigating the contributions of variability in site conditions, weather conditions and biotic conditions toward infection prevalence. At present, we have conducted two very intensive field seasons in central Pennsylvania (USDA Forest Service and Agricultural Research Service, Ithaca, NY). Our studies have continued on non-woven bands containing M. anisopliae cultures for control of Asian longhorned beetle (ALB). We conducted studies evaluating interactions between imidacloprid, the major method used for ALB control by APHIS (besides detection and destruction of infested trees), and M. anisopliae. We have also continued our studies that demonstrate that M. anisopliae can be vectored from bands to ALB not walking on the bands; studies along these lines during 2008 focused on transfer of fungal conidia by mates (USDA, Agricultural Research Service, Ithaca, NY and Stoneville, MS and Newark, DE). Multi-year field studies were concluded testing the long-term effects of application of the entomopathogenic nematode Steinernema scarabaei on scarab populations and the nematodes persistence. S. scarabaei provided excellent control (77-100%) within 1 month of application at 0.25 to 2.5 billion IJs/ha and particularly in the following spring at 0.1 to 2.5 billion IJs/ha (86-100%). In the next oriental beetle generation (i.e., 13 months after application) still provided good control (average of 65%) with again improved control in the following spring. Thereafter, control was unreliable. In a quarantine study with Indian hawthorn bushes, the nematode, S. riobrave, provided good but not complete control of the citrus root weevil, Diaprepes abbreviatus, using standard methods of application. The fungus, M. anisopliae, was not effective in controlling D. abbreviatus. Though there was not a significant difference between the nematode treatments and the combination treatment of the nematode and fungus, there is a suggestion that the two biological control agents do not work well when combined. The root mass data agree with the efficacy data in that the plants treated with S. riobrave had a greater mass than any of the other treatments. (UC Davis) From a study dealing with soils from citrus orchards in California, we developed a multiple linear regression model that explains a significant amount of the variation in efficacy of entomopathogenic nematodes among soil types. We noted that much of the variation in nematode mortality may be due to the action of naturally occurring dissolved solids (salts) and other soil amendments. Further analyses suggest that in soils where sand content is low, sand content actually explains much of the variability. However, where sand content is more than about 40%, other soil factors come into play. The efficacy of nematodes increases in soils up to about 60% sand, but then drops. We believe that the drop off in nematode efficacy in soils with high sand content is due to the amount of dissolved solids in the soil, as measured by electrical conductivity (EC). If the dissolved solids are washed with water from soil that has 72% sand, nematode efficacy is greatly improved compared to native soil with the high EC level. When the citrus soil was baited with G. mellonella, we found that H. bacteriophora was isolated from a 3 orchards and an as-yet unknown species of Heterorhabditis was isolated from a fourth. SUBPROJECT 4: Development, evaluation and safety of entomopathogens for veterinary and structural arthropod pests. (Co-chairs: James J. Becnel, David Oi, ARS-Gainesville) Entomopathogens evaluated against veterinary pest included viruses and microsporidia. The genomes of two diverse microsporidia from mosquitoes, Edhazardia aedis and Brachiola algerae were examined. In both species, very large intergenic regions, many transposable elements, and a low gene-density were found all in contrast to the small, model microsporidian genomes. An EST survey of the mosquito parasite Edhazardia aedis was conducted. E. aedis seems to lack the multi-gene transcripts present in other microsporidia and the first documented case of transcription of a transposable element in microsporidia is presented. The Musca domestica salivary gland hypertrophy virus (MdSGHV), a large dsDNA virus that infects and sterilizes adult houseflies was characterized. MdSGHV tansmission within feral populations of M. domestica is believed to be mediated orally via deposition and consumption of saliva. For structural arthropod pests, seasonal and host infection effects of a second virus (SINV-2) from the red imported fire ant, Solenopsis invicta was characterized. Microsporidian fire ant pathogen Kneallhazia (=Thelohania) solenopsae was detected in fire ant parasitic flies that developed in K. solenopsae-infected S. invicta. Infected flies were also collected from the field and there was not any obvious impairment of infected flies. Entomopathogenic nematodes (Steinernema riobrave, S. carpocapsae, S. feltiae, and Heterorhabditis bacteriophora), killed the subterranean termite, Heterotermes aureus, in laboratory bioassays. Steinernema carpocapsae, S. riobrave and H. bacteriophora successfully reproduced in H. aureus. Veterinary Pests The Musca domestica salivary gland hypertrophy virus (MdSGHV) is a large dsDNA virus that infects and sterilizes adult houseflies. Rapid amplification of cDNA 3'-ends (3'-RACE) and reverse transcriptase polymerase chain reactions (RT-PCR) were used to validate the transcription of putative open-reading frames (ORFs) identified in silico. Direct sequencing of 3'-RACE products revealed 78 poly (A) transcripts containing 95 of the 108 putative ORFs. An additional six ORFs, not amplified by 3'-RACE were detected by RT-PCR. Only seven of the 108 putative ORFs were not amplified by either 3'-RACE or RT-PCR. A series of 5-RACE reactions were conducted on selected ORFs that were identified by 3-RACE to be transcribed in tandem (tandem transcripts). In the majority of cases, only the downstream ORFs were detected as single transcripts and as components of the tandem transcripts. The exception was the upstream ORF MdSGHV084; this transcript was differentially transcribed as a single transcript at one dpi and /or as a tandem transcript (MdSGHV084/085) at 2 dpi. Transcriptome analysis of MdSGHV also detected splicing in the 3' untranslated region (3'-UTR) of MdSGHV045, extensive heterogeneity in the polyadenylation signals and cleavage sites, and twenty overlapping sense-antisense transcripts. Transmission of this non-occluded, enveloped virus within feral populations of M. domestica is believed to be mediated orally via deposition and consumption of saliva, which is a composite of salivary gland secretions and crop contents. Oral treatments of newly emerged individual flies with viremic salivary gland homogenates, crop homogenates, or gradient-purified virus resulted in an average 44 ± 11% infection. Infection rates did not differ between females and males or between challenged flies that were kept individually and in groups. Using quantitative real-time PCR, MdSGHV DNA was quantified in salivary secretions and excreta obtained from viremic flies. Viral copy numbers increased exponentially until 4 dpi, and from 5-21 dpi each infected fly released an average 1.0 x 106 ± 0.2 x 106 MdSGHV copies per feeding event. Oral transmission experiments showed that the virus released via salivary secretion was infectious when ingested by newly emerged adult flies, resulting in an average 66 ± 10% infection rate. Excreta samples collected over night at 5 dpi from individual infected flies contained an average 6.5 x 105 ± 1.9 x 105 viral copies per sample but produced lw infection rates (2or 17 %) were produced when flies were challenged by oral treatments or injection, respectively. Additional studies have addressed: transcription of the virus in selected Musca domestica tissues; the genetic and phenotypic properties of MdSGHV collected from sites worldwide; the involvement of the peritrophic membrane as a barrier to infection; the transmission of MdSGHV to healthy conspecifics in cage experiments, and the influence of infection on JH regulation of vitellogenesis. (FL) Genome sequence surveys were conducted on two diverse microsporidia from mosquitoes, Brachiola algerae and Edhazardia aedis. In both species we found very large intergenic regions, many transposable elements, and a low gene-density, all in contrast to the small, model microsporidian genomes. We also find no recognizable genes that are not also found in other surveyed or sequenced microsporidian genomes. These results demonstrate that microsporidian genome architecture varies greatly between microsporidia. Much of the genome size difference could be accounted for by non-coding material, such as intergenic spaces and retrotransposons, and this suggests that the forces dictating genome size may vary across the phylum. (ARS-FL) An EST survey of the mosquito parasite Edhazardia aedis was conducted. The only other microsporidian EST project is from A. locustae, and serves as a basis for comparison with E. aedis. The spore transcriptomes of A. locustae and E. aedis were compared and the numbers of unique transcripts that belong to each COG (Clusters of Orthologous Groups of proteins) category differ by at most 5%. The transcripts themselves have widely varying start sites and encode a number of proteins that have not been found in other microsporidia examined to date. However, E. aedis seems to lack the multi-gene transcripts present in A. locustae and E. cuniculi. We also present the first documented case of transcription of a transposable element in microsporidia. Although E. aedis and A. locustae are distantly related, have very disparate life cycles and contain genomes estimated to be vastly different sizes, their patterns of transcription are similar. The architecture of the ancestral microsporidian genome is unknown, but the presence of genes in E. aedis that have not been found in other microsporidia suggests that extreme genome reduction and compaction is lineage specific and not typical of all microsporidia. (ARS-FL) Structural Pests The fire ant pathogen Kneallhazia (=Thelohania) solenopsae was found in fire ant parasitic phorid flies, Pseudacteon obtusus and P. cultellatus, that developed in K. solenopsaeinfected fire ants reared in the laboratory. In addition, K. solenopsae was detected in field collected phorid flies (P. curvatus). Estimated infection rates ranged from 9-20% in the flies. Not all the flies that developed in infected ants acquired the pathogen, and there was no obvious impairment of infected flies. The ability of the flies to vector K. solenopsae to other fire ants is being investigated. (ARS-Gainesville). Solenopsis invicta virus 2 (SINV-2) is the second virus identified from the fire ant, S. invicta. Fire ant colonies testing positive for SINV-2 by RT-PCR did not exhibit any discernable symptoms. Prevalence of SINV-2 among 688 nests for two year ranged from 1.6% to 16.4%. SINV-2 was found in both monogyne and polygyne S. invicta ants. The SINV-2 infection was detected in all ant stages examined (eggs, larvae, pupae, workers, and queens). Tissue tropism studies indicated that the alimentary canal (specifically the midgut) is most likely the susceptible tissue. SINV-2 was successfully transmitted to uninfected S. invicta by feeding a partially purified homogenate of SINV-2-infected ants. The SINV-2 transmission rate ranged from 30% to 80%. (ARS-Gainesville). In laboratory bioassays Steinernema riobrave, S. carpocapsae, S. feltiae, and Heterorhabditis bacteriophora infected and killed the subterranean termite, Heterotermes aureus. Steinernema carpocapsae, S. riobrave and H. bacteriophora successfully reproduced in H. aureus and IJs exited the termite cadavers successfully. However, no progeny were produced by S. feltiae. IJs of S. carpocapsae formed two distinct size groups. Small S. carpocapsae IJs infect, reproduce and form normal size IJs after subsequent infection in Galleria mellonella. S. riobrave and H. bacteriophora showed a more gradual recovery in IJ size that needed 2 infection cycles in G. mellonella. In termite mortality tests, the progeny of small IJs of S. carpocapsae are comparably effective to the normal size IJs, under the conditions tested. (AZ). Participated in the construction of a pest management strategic plan (PMSP), for IPM in schools. The biocontrol section includes information on EPN use in school environments. (AZ).

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