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During a survey for grasshopper pathogens in Argentina in 2005-2006, individual Covasacris pallidinota from halophylous grasslands in Laprida, Buenos Aires province were found to be infected with a microsporidium. Infection was restricted to the salivary gland epithelial cells. The microsporidium produced ovocylindrical spores averaging 2.6+/-0.28 x 1.4+/-0.12 microm (range 2.2-3.4 x 1.1-1.7 microm), which resembled in size and shape the spores of Liebermannia patagonica and L. dichroplusae, two recently described species that also parasitize Argentine grasshoppers. The life cycle of the microsporidium included the formation of polynucleate, diplokaryotic, moniliform, merogonial plasmodia wrapped in flattened cisterns of the host endoplasmic reticulum (ER). Plasmodia divided to produce diplokaryotic cells. The latter underwent elongation, dissociation of diplokarya counterparts, vacuolization, dismantling of the host ER envelope, and deposition of electron-dense material outside the plasma membrane. The resultant binucleate sporogonial plasmodia divided into two uninucleate sporoblasts, which eventually transformed into spores. Uninucleate spores contained a lamellar polaroplast, embraced by an elongated polar sac, anchoring disc, 3-5 polar filament coils, and a cluster of anastomizing tubules (sporoblast trans-Golgi, posterosome) at the posterior end. Sequence similarity of the SSU rDNA of the newly discovered microsporidium (Genbank accession no. EU709818) to L. patagonica and L. dichroplusae was 99% and 97%, respectively, suggesting that the three species belong to one genus. All three species fell into one clade in SSU rDNA-based phylogenetic trees produced by neighbor joining, maximum parsimony, and maximum likelihood analyses with 100% statistical support. We assign the name Liebermannia covasacrae to this microsporidium. It can be easily differentiated from both congeners by host species, tissue tropism, type of sporogony, and several features of morphology. Comparison of the three Liebermannia spp. demonstrates that the nuclear phase (dikaryotic versus monokaryotic spores) and type of sporogony (polysporous versus disporous) may vary in closely related species.

The microsporidium Heterovesicula cowani, discovered in 1985, was initially identified as Vairimorpha sp. because it produces two types of spores: Nosema-like diplokaryotic spores and Thelohania-like mononuclear meiospores. However, light and electron microscopy studies revealed characters that did not fit any known microsporidian genera, and a new monotypic genus Heterovesicula was erected. The goal of this study was to test the validity of the genus Heterovesicula by molecular characterization of H. cowani and to assess its phylogenetic relationships to other microsporidia from insects. DNA from spores stored at -32 degrees C since 1992 was isolated and PCR-amplified with V1-1492 primers to obtain a partial small subunit ribosomal RNA gene sequence of 1165bp, which was submitted to GenBank (Accession No. EU275200). Neighbor joining, maximum parsimony and maximum likelihood analyses performed against 18 microsporidia sequences, placed H. cowani as a sister taxon to the Nosema-Vairimorpha clade. The consensus of these analyses suggests that the Heterovesicula-Nosema-Vairimorpha group forms a dichotomy with the Encephalitozoon spp. branch. Other microsporidia parasitizing Orthoptera fell into two unrelated (or distantly related) lineages of terrestrial microsporidia: the Liebermannia spp. branch forms a dichotomy with Orthosomella operophterae within the Endoreticulatus-Orthosomella-Liebermannia group; and the Paranosema spp. branch clusters together with the Tubulinosema-Systenostrema lineage. The minimum pairwise distance in Kimura-2-Parameter analysis among 18 analyzed sequences was 0.37, which supports well the generic status for Heterovesicula. The obtained phylogenetic trees suggest that H. cowani is related to the Vairimorpha necatrix group, but not to other insect microsporidia producing octospores.

Thelohania solenopsae is a unique microsporidium with a life-cycle finely tuned to parasitizing fire ant colonies. Unlike other microsporidia of social hymenopterans, T. solenopsae infects all castes and stages of the host. Four distinctive spore types are produced: diplokaryotic spores, which develop only in brood (Type 1 DK spores); octets of octospores within sporophorous vesicles, the most prominent spore type in adults but never occurring in brood; Nosema-like diplokaryotic spores (Type 2 DK spores) developing in adults; and megaspores, which occur occasionally in larvae 4, pupae, and adults of all castes but predominantly infect gonads of alates and germinate in inseminated ovaries of queens. Type 2 DK spores function in autoinfection of adipocytes. Proliferation of diplokaryotic meronts in some cells is followed by karyogamy of diplokarya counterparts and meiosis, thereby switching the diplokaryotic sequence to octospore or megaspore development. Megaspores transmit the pathogen transovarially. From the egg to larvae 4, infection is inapparent and can be detected only by PCR. Type 1 DK spore and megaspore sequences are abruptly triggered in larvae 4, the key stage in intra-colony food distribution via trophallaxis, and presumably the central player in horizontal transmission of spores. Molecular, morphological, ultrastructural and life-cycle data indicate that T. solenopsae must be assigned to a new genus. We propose a new combination, Kneallhazia solenopsae.

The use of mulch in urban landscapes has increased in the United States for the past decade. Tree-based organic mulches can supply Coptotermes formosanus Shiraki with food, moisture, and shelter. The current research contributes to mulch management technology in termite control. A choice test arena was designed to determine the repellence and mortality caused by commercial mulches treated with different concentrations of the fungus Metarhizium anisopliae (Metschnikoff) against C. formosanus. Each of six tree-based mulches (pine bark, pine straw, bald cypress, eucalyptus, water oak, and melaleuca) was coated with six conidial concentrations ranging from 1 x 10(3) to 1 x 10(8) conidia/ml. The foragers of C. formosanus were repelled significantly by the fungal-treated mulch substrates; the proportion of termites on fungal-treated mulch was usually <20% during the 28-d test. By day 28, >99% of the termites were killed in test arenas containing a chamber with mulch treated with 10(7) or 10(8) conidia/ml. M. anisopliae significantly reduced mulch consumption by 34-71%. Mulch consumption by the termites was negatively correlated with fungal concentration, and the type of mulch also affected consumption. The differences in termite foraging activities, mortality, and food consumption among mulches were usually confounded by differences in fungal concentrations of M. anisopliae. The results indicate that repellence and virulence of M. anisopliae conidia should significantly reduce the suitability of these six mulches as a habitat for C. formosanus.

The purpose of the research was to determine threshold concentrations of nucleopolyhedrovirus (NPV) in soil for abiotic transport to cotton plants in the field and under conducive and nonconducive conditions in the greenhouse. Under the assumption that 2% mortality would suffice to initiate foci of infection in Heliothis virescens larvae on the plants, thresholds ranged from 25 to 2,311 viral occlusion bodies (OB)/g soil in the greenhouse. Thresholds generally were smaller for rain on sandy soil and wind on clay soil than for wind on sand or rain on clay. Thresholds generally increased with height of the plant above the soil surface. In field plots, percentage mortality in bioassays of cotton plants was greatest on leaves versus other tissues, and mortality increased with soil dosage and decreased with plant height and over time. Season-long soil-NPV-transport thresholds for 2% plant-bioassay mortality of larvae ranged from 1 OB/g to 7.4 x 10(8) OB/g soil based on the amounts of NPV applied to the soil at planting time, and they ranged from 8 OB/g to 1.2 x 10(5) OB/g soil based on bioassays of soil samples collected concurrently with plant samples throughout the growing season. These results should contribute to NPV epizootiology, biological control, and risk assessment through better understanding of viral soil-to-plant transport.

We surveyed 165 sites to determine the ecological factors that might influence the distribution and prevalence of Thelohania solenopsae, and its effect on the demography of the red imported fire ant (Solenopsis invicta) in Louisiana. The microsporidium was found in 9.9% of colonies and at 16% of sites. Its distribution was clumped within the state with the majority of infected colonies and sites occurring in two infection patches. The proportion of polygyne colonies was a strong (positive) predictor of the proportion of infected colonies at a site. Infected monogyne colonies, however, still accounted for nearly 20% of infected colonies, a much higher proportion than anticipated. Several other factors, including the numbers of colonies at a site, precipitation, proximity to commercial waterways and ports, and type of habitat were also retained in the multiple logistic regression model describing T. solenopsae prevalence. The microsporidium appears to adversely affect the occurrence of brood, and possibly the size of S. invicta colonies and the mass of workers. It, however, was not included in the multiple regression model of the number of colonies or the density of ants at a site. Although our findings do not imply causation, they have identified several variables that might influence the epizootiology of T. solenopsae. Future work should concentrate on experimentally manipulating these variables to confirm these relationships.

Two aspects of abiotic transport of nucleopolyhedrovirus from soil to cotton plants were examined in greenhouse experiments: the distance from the plants and depth in soil from which the virus could be transported under controlled conditions of soil type and moisture, wind, and precipitation. Transport distance and depth were tested separately under relatively conducive (precipitation/sandy soil and wind/clay soil) and non-conducive (precipitation/clay soil and wind/sandy soil) conditions, as determined in previous research. The amount of virus transported by precipitation generally decreased as distance from the plant increased, but in wind the amounts of virus transported were best described by polynomial models, with transport efficiency usually peaking at a distance of 60 cm. Depending on plant height and tissue, the farthest distances that virus was transported ranged from 30 to 60 cm by precipitation from clay soil, 60-75 cm in precipitation/sand, 60-80 cm in wind/clay, and 60-80 cm in wind/sand. In the depth experiments, transport by precipitation and wind generally decreased as the depth of virus in soil increased. The greatest depth from which NPV was transported ranged from 0 to 0.5 cm by precipitation from clay soil, 0.5-1.0 cm in precipitation/sand, 1.0-2.0 cm in wind/clay, and 0.5-1.0 cm in wind/sand. All of the experimental parameters (distance or depth, soil type, plant height, plant tissue) and all two-way interactions significantly (P<0.05) affected transport in all four experiments, except for the "soilxplant tissue" interaction in the depth/wind experiment. In all of the experiments, transport was significantly greater (P<0.05) to lower than to upper portions of plants and to leaves than to buds and squares. Transport was significantly greater from sandy soil than from clay in precipitation, and it was greater from clay than from sandy soil in wind. The results will contribute to NPV epizootiology, microbial control, and risk assessment.

Perezia dichroplusae Lange, 1987 is a parasite of the Malpighian tubules of an Argentine grasshopper, Dichroplus elongatus (Orthoptera, Acrididae, Melanoplinae). In order to determine relationships of this microsporidium with Perezia nelsoni and with other microsporidia, we sequenced its small subunit ribosomal RNA gene (SSU rDNA) (GenBank Accession No. EF016249) and performed phylogenetic analysis of the novel sequence against 17 microsporidian SSU rDNA sequences from GenBank, using neighbor-joining (NJ), maximum-parsimony (MP), and maximum-likelihood (ML) methods. This analysis revealed the highest similarity (96%) of the new sequence to Liebermannia patagonica, a parasite of gut epithelium cells of another grasshopper from Argentina, versus only 65% similarity to P. nelsoni, a parasite of muscles of paenaeid shrimps. In phylogenetic trees inferred from SSU rDNA sequences, the microsporidium from D. elongatus is sister taxon to L. patagonica and both cluster with Orthosomella operophterae. At the higher hierarchical level, the Liebermania-Orthosomella branch forms a clade with the Endoreticulatus-Cystosporogenus-Vittaforma group and with Enterocytozoon bieneusi. Perezia nelsoni falls into another large clade together with Nosema and Ameson species. We propose transferring P. dichroplusae to the genus Liebermannia and creating a new combination Liebermannia dichroplusae n. comb., based both on SSU rDNA sequence analysis and on common characters between P. dichroplusae and L. patagonica, which include the presence of elongated multinuclear sporonts, sporoblastogenesis by a similar process of sequentially splitting off sporoblasts, ovocylindrical spores of variable size, tissue tropism limited to epithelial cells, Orthoptera as hosts, and geographical distribution of hosts in the southern temperate region of Argentina. We argue that the condition of the nuclei in spores (i.e. diplokaryotic in L. patagonica or monokaryotic in L. dichroplusae) cannot be used to distinguish genera. Therefore, we remove the statement about the presence of diplokaryotic spores from the revised diagnosis of the genus Liebermannia.

A new microsporidium, Liebermannia patagonica n. gen., n. sp., is described from midgut and gastric caecum epithelial cells of Tristira magellanica, an apterous grasshopper species of southern Patagonia, Argentina. L.patagonica is diplokaryotic, apansporoblastic, homosporous, and polysporoblastic. Transitional (from merogony to sporogony) stages and sporonts of L. patagonica were surrounded by host rough endoplasmic reticulum. The ovocylindrical spores measured 2.9 +/- 0.09 x 1.2 +/- 0.04 microm (fresh, n = 50), and they had an isofilar polar filament of only three coils and a cluster of tubules instead of a classical posterior vacuole. Prevalence was high (up to 80.6%) at the type locality for the four years sampled . Maximum likelihood , neighbor joining, maximum parismony analyses of the small submit rDNA all placed L.patagonica(Accession No. DQ 239917) in one with Orthosomella operophterae.

Two Steinernema isolates found in Louisiana and Mississippi were later identified as isolates of S. rarum. DNA sequences of ITS regions of the United States isolates are identical with sequences of Argentinean S. rarum strains Samiento and Noetinger and differ by two bases from the Arroyo Cabral isolate from Córdoba, Argentina. SEM observations revealed several new structures in the isolates from the US: female face views have a hexagonal-star perioral disc and eye-shaped lips; some females do not have cephalic papillae; lateral fields of infective juveniles are variable; there are two openings observed close to the posterior edge of the cloaca. Virulence of the US isolates to Anthonomus grandis, Diaprepes abbreviatus, Solenopsis invicta, Coptotermes formosanus, Agrotis ipsilon, Spodoptera frugiperda, and Trichoplusia ni and reproductive potential were evaluated in comparison with other heterorhabditid and steinernematid nematodes. Results such as particularly high virulence to S. frugiperda indicate that the biocontrol potential of the new S. rarum strains merits further study.

An octospore microsporidium was found in the nymphs of Aeshna viridis, collected in intermittent streams near Novosibirsk, Siberia, Russia in 2003. Spores were uninucleate and measured 6.1+/-0.07 x 3.0+/-0.04 microm on fresh smears. The polar filament was anisofilar having 10-11 anterior coils (thicker filament diam.) and 10-11 posterior (thinner filament diam.) coils. Sporophorous vesicles were persistent and measured 12.3+/-0.23 x 11.9+/-0.20 microm. The infection was restricted to the adipose tissue and caused the formation of whitish "cysts" containing mature octospores. Based on ultrastructural similarity we consider this Siberian isolate to be Systenostrema alba, a species described from Aeshna grandis collected in Sweden (Larsson 1988). Maximum likelihood, neighbor joining, and maximum parsimony analyses of the small subunit rDNA all placed Systenostrema alba (Accession no. AY953292) as the sister taxon to a clade consisting of Thelohania solenopsae, Tubulinosema ratisbonensis, and Tubulinosema acridophagus.

Sixty four percent of Solenopsis invicta workers infected with Thelohania solenopsis contained 1-6 "cysts" ranging from 70 to 260 microm in diameter. Light and electron microscope analyses showed that cysts are hypertrophied adipocytes transformed by the parasites, each cyst presumably forming from a single cell. In the first step of the pathogenesis, Nosema-like spores functioning in autoinfection are produced; a diplokaryotic sequence leading to their formation causes fat body hypertrophy. When meiosis occurs, it switches parasite development to production of octospores and/or megaspores. Adipocytes become 2-4xlarger than normal in conjunction with intensive parasite multiplication and octospore maturation. Infected cells eventually lose their cellular organization and are converted into reservoirs for spores. There were no manifestations of cellular immunity, such as encapsulation or nodule formation. Similarly, there were no signs of specialized host-parasite interaction that might be interpreted as xenoma-like complexes. The role of the cysts in the parasite's life cycle is unclear. They may represent a defensive reaction of the host sacrificing the infected cells to segregate the infection. Alternatively, the cyst may help protect spores from environmental hazards and provide a concentrated infectious dose to aid horizontal transmission of the microsporidium. We propose to refer to hypertrophied adipocytes filled with T. solenospsae spores as "sporocytosacs", not "cysts."

This is the first report of Thelohania solenopsae infections in monogyne (single-queen) Solenopsis invicta colonies in the field. In a 0.2-ha plot near Baton Rouge, Louisiana, inter-colony prevalence was 63% infection in June, 1999, when the population was 100% monogyne. In February, 2000, 21% of 33 monogyne and 90% of 10 polygyne colonies were infected. By May, 2001, the polygyne colonies had disappeared and only one of 34 monogyne colonies was infected, the final detection of T. solenopsae in the plot. Colony size did not differ significantly among the four types (monogyne versus polygynexinfected versus uninfected).

Accurate definition and usage of terminology are critical to effective communication in science. In a recently published article, the clarity and consistency of the terms pathogenicity and virulence as used in invertebrate pathology were called into question, and a revision of these terms was proposed. Our objective was to examine definitions of pathogenicity and virulence and their use in invertebrate pathology, and respond to this article. Although usage of the terms pathogenicity and virulence varies, we found considerable consistency in the published definitions of these terms in the invertebrate pathology literature throughout the history of the discipline, as well as among related disciplines such as medicine and microbiology. We did not find the established definitions to be lacking in clarity or utility. Therefore, we recommend that the definition and use of these terms adhere to precedence. Specifically, pathogenicity is the quality or state of being pathogenic, the potential ability to produce disease, whereas virulence is the disease producing power of an organism, the degree of pathogenicity within a group or species. Pathogenicity is a qualitative term, an "all-or-none" concept, whereas virulence is a term that quantifies pathogenicity.

Development of Thelohania solenopsae, a parasite of the red imported fire ant (Solenopsis invicta), until recently was thought to include formation of two types of spores: unicellular meiospores, maturing inside sporophorous vesicles in sets of eight (octospores); and Nosema-like binuclear free spores. Megaspores, discovered in 2001, develop primarily in alates and are morphologically distinct from the two previously known types of spores. The role of megaspores in the T. solenopsae life cycle, as well as their existence, has been questioned. The current research includes light and electron microscopic descriptions of the three major spore morphotypes characteristic of T. solenopsae development. In addition, individual octospores and megaspores were isolated into groups of 8-20 from methanol-fixed and Calcofluor-stained smears of the infected ants for subsequent PCR analysis by the laser pressure catapulting function of a position ablative laser microbeam microscope, a technique applied for the first time to research of microsporidia. The PCR-amplified SSU rDNA nucleotide sequences from octospores and megaspores were identical. This, along with the consistency with which megaspores are detected in infected ants, demonstrates that megaspores are integral to the life cycle of T. solenopsae.

The main goal of this study was to compare the effectiveness of three staining techniques (calcofluor white M2R, Giemsa and modified trichrome), and the polymerase chain reaction (PCR) in detecting the microsporidium Thelohania solenopsae in red imported fire ants (Solenopsis invicta). The effect of the number of ants in a sample on the sensitivity of the staining techniques and the PCR, and the effect of three DNA extraction protocols on the sensitivity of PCR were also examined. In the first protocol, the ants were macerated and the crude homogenate was used immediately in the PCR. In the second protocol, the homogenate was placed on a special membrane (FTA card) that traps DNA, which is subsequently used in the PCR. In the third protocol, the DNA was purified from the homogenate by traditional phenol-chloroform extraction. Except for PCR using FTA cards, the sensitivity (number of samples positive for T. solenopsae) of all detection techniques increased with the number of ants in the sample. Overall, Giemsa was the least sensitive of all detection techniques. Calcofluor was more sensitive than modified trichrome with ants from one site and was equally as sensitive as PCR with crude DNA or a FTA card with ants from both sites. Trichrome staining was equally as sensitive as PCR with a FTA card at both sites, but it was less sensitive than PCR with crude DNA at one site. PCR on FTA cards was less sensitive than PCR with crude DNA for ants from one site but not the other. There was no difference whether crude or phenol-chloroform purified DNA was used as template. In summary, the results of this study show that PCR based on a crude DNA solution is equal to or more sensitive in detecting T. solenopsae than the other detection techniques investigated, and that it can be used as a reliable diagnostic tool for screening field samples of S. invicta for T. solenopsae. Nevertheless, ant smear stained with calcofluor or modified trichrome should be used to buttress findings from PCR.

Sporulation characteristics and virulence of Metarhizium anisopliae and Beauveria bassiana were examined in relation to laboratory transmission in Coptotermes formosanus. Fungal isolates significantly affected disease prevalence in termite populations. Sporulation of M. anisopliae played a more important role than virulence in producing epizootics within small groups of termites, but this was not the case for B. bassiana. Isolates characterized by quick sporulation (day 2 after death) did not exhibit better transmission in termites than those with high total sporulation (day 11 after death) in either fungal species. An isolate of M. anisopliae ranking highly in all three categories (virulence, quick sporulation, and total sporulation) produced better epizootics than an isolate that was inferior in all three characteristics. High temperatures (35 degrees C) significantly reduced fungal germination rates, leading to significant reduction of epizootics. M. anisopliae was better than B. bassiana in producing epizootics at 27 degrees C. Thus, fungal characteristics other than virulence should be considered for the seasonal colonization approach to termite microbial control.

The sporulation of 22 total isolates of Metarhizium anisopliae and Beauveria bassiana was quantified on cadavers of the Formosan subterranean termite, Coptotermes formosanus. Conidial production increased significantly over 11 days post-death. Effects of isolates of M. anisopliae and B. bassiana on in vivo sporulation were significant. Although the overall effects of fungal species on in vivo sporulation were not significant, the interactions between fungal species and certain times post-death were significant, indicating different sporulation patterns between the two fungal species. B. bassiana isolates could be categorized into a group with high total sporulation (day 11) and low quick sporulation (on days 2 and 3), while M. anisopliae isolates fell into another group with high quick sporulation and low total sporulation. This could give M. anisopliae an advantage over B. bassiana in termite microbial control due to termite defensive social behaviors. Conidial production was significantly higher in vitro than in vivo. In vitro and in vivo sporulation differed by as much as 89x and 232x among the selected isolates of B. bassiana and M. anisopliae, respectively. Correlation between in vivo and in vitro conidial production was positive and significant. This may allow preliminary in vitro screening of a large number of isolates for high in vivo sporulation.

Four viruses were tested for vertical transmission in Trichoplusia ni: T. ni nucleopolyhedrovirus (TnSNPV), T. ni cypovirus (TnCPV), Autographa californica nucleopolyhedrovirus (AcMNPV), and AcMNPV engineered to express a scorpion toxin (AcMNPV.AaIT). Fifth instars were exposed to each virus, the survivors were reared and mated, and second-generation (F(1)) insects were examined for infection. TnSNPV was transmitted to offspring at a prevalence rate of 15.4%, TnCPV at 10.2%, and AcMNPV at 10.1%. Only one of 2484 F(1) insects was infected with AcMNPV.AaIT; this experiment was repeated, and none of 4774 insects was infected. Thus, vertical transmission is unlikely to contribute to AcMNPV.AaIT contacting non-target organisms after its field release. There was evidence that TnCPV and possibly TnSNPV were activated to overt infections by ingestion of a different virus. TnCPV, but not the NPVs, routinely infected 0.3-1.7% of non-treated insects, probably indicating that it is vertically transmitted at enzootic levels., (2002 Elsevier Science (USA))

Significantly more occlusion bodies (OB) of DuPont viral construct HzSNPV-LqhIT2, expressing a scorpion toxin, were transported by artificial rainfall to cotton plants from sandy soil (70:15:15 sand-silt-clay) than from silt (15:70:15) and significantly more from silt than from clay (15:15:70). The amounts transported by 5 versus 50 mm of precipitation were the same, and transport was zero when there was no precipitation. In treatments that included precipitation, the mean number of viable OB transported to entire, 25- to 35-cm-tall cotton plants ranged from 56 (clay soil, 5 mm of rain) to 226 (sandy soil, 50 mm of rain) OB/plant. In a second experiment, viral transport increased with increasing wind velocity (0, 16, and 31 km/h) and was greater in dry (-1.0 bar of matric potential) than in moist (-0.5 bar) soil. Wind transport was greater for virus in a clay soil than in silt or sand. Only 3.3 x 10(-7) (clay soil, 5 mm rain) to 1.3 x 10(-6) (sandy soil, 50 mm rain) of the OB in surrounding soil in experiment 1 or 1.1 x 10(-7) (-0.5 bar sandy soil, 16-km/h wind) to 1.3 x 10(-6) (-1.0 bar clay soil, 31-km/h wind) in experiment 2 were transported by rainfall or wind to cotton plants. This reduces the risk of environmental release of a recombinant nucleopolyhedrovirus (NPV), because only a very small proportion of recombinant virus in the soil reservoir is transported to vegetation, where it can be ingested by and replicate in new host insects.

Persistence of recombinant and wild-type nucleopolyhedroviruses (NPV) was compared in field and laboratory microcosm experiments. Horizontal and vertical distribution of the viruses also was monitored in the field agricultural soil. Mixed populations of the bollworm, Helicoverpa zea, and tobacco budworm, Heliothis virescens, in cotton were sprayed five times during a growing season with wild-type H. zea NPV (HzSNPV.WT) or with a genetically modified H. zea NPV expressing an insect-specific neurotoxin (HzSNPV.LqhIT2). HzNPV.WT accumulated 2.3 times as many occlusion bodies (OB) as HzSNPV.LqhIT2 in soil by the end of the growing season in October 1997. Both NPVs were detected at all soil depths down to 26-35 cm. Both NPVs were randomly distributed among 0-2 cm soil samples throughout the plots according to analysis with Taylor's power law. By 4 August 1998, soil concentration of HzSNPV.WT was only 11-13 OB/g at depths from 0 to 14 cm, and the wild-type virus was not detected below 14 cm. HzSNPV.LqhIT2 was detected only in trace amounts at 0-2 cm at this time. Neither NPV was detected in bioassays of cotton leaves nor in insects sampled from the plots in 1998. Viral persistence also was monitored in laboratory soil microcosms. Three viruses-wild-type Autographa californica NPV (AcNPV.WT), A. californica NPV expressing a scorpion toxin (AcNPV.AaIT), and A. californica NPV expressing juvenile hormone esterase (AcNPV.JHE-S201G)-were introduced into soil microcosms by each of two methods, in water suspension or in host cadavers, for a total of six treatments plus controls. After 17 months, the number of viable OB remaining did not differ among the treatments. The results indicate that the only differences in soil populations of wild-type versus recombinant NPVs are due to the greater amounts of the wild-type viruses that accumulate, probably because they have a greater capacity to replicate in the host insect population.

The southern green stink bug, Nezara viridula (L.), and certain of its host plants were examined to determine the prevalence and biological characteristics of an intestinal trypanosomatid. Promastigotes with short (< or = 17.5 microm excluding flagellum) and long forms (> or = 25.0 microm) usually infected < 50% of the bugs before August and > 50% (maximum 95%) during August-October, but prevalence was not host-density dependent. The flagellate was detected in adults and in all nymphal instars, at all sampling sites where at least 10 bugs were captured, and in bugs from all host plants sampled (soybean, red clover, vetch). Of bugs with flagellates, 27% were heavily infected (> 20 flagellates per 160X microscope field). Weights of infected and uninfected adults did not differ. Live flagellates were detected in bug feces and in one stem of red clover. When bugs were fed soybean pods, tomatoes, or snap beans in the laboratory, only once were flagellates detected in plant tissue (snap beans). The flagellate was cultured in modified Medium 199. This flagellate is prevalent in N. viridula populations in Louisiana and apparently does not cause significant pathological effects in N. viridula or its host plants, including soybean.

Wild-type and recombinant nucleopolyhedroviruses (NPVs) were compared in their capability to be transported over limited distances by the predator Podisus maculiventris (Say) and scavengers Sarcophaga bullata (Parker) and Acheta domesticus (Linnaeus) in Trichoplusia ni (Hübner) larvae infesting collards in a greenhouse microcosm. Viruses tested were variants of Autographa californica (Speyer) NPV (AcNPV): wild-type virus (AcNPV.WT), AcNPV expressing a scorpion toxin (AcNPV.AaIT), and AcNPV expressing juvenile hormone esterase (AcJHE.SG). Podisus maculiventris transported AcNPV.WT and S. bullata transported AcNPV.WT and AcNPV.AaIT. Prevalence and transport of AcNPV.WT were greater than those of AcNPV.AaIT and AcJHE.SG, regardless of whether the nontarget organism carriers were present or absent. Podisus maculiventris and S. bullata transported recombinant and wild-type NPVs at a rate of up to 62.5 cm/day, and A. domesticus transported wild-type NPV at 125 cm/day. The infected host insects, T. ni, undoubtedly contributed to viral transport in the current research. In every experiment, both the wild-type and recombinant virus spread to some degree in the plots without predators or scavengers. The relative amounts of NPVs that accumulated in soil, as indicated by bioassay mortality percentages, generally exhibited spatial patterns similar to those of T. ni mortality due to NPV on the collards plants. Thus, the predator and scavengers in the current research demonstrated some capacity to transport wild-type as well as recombinant viruses at significant rates in a greenhouse microcosm.

Inability to consistently rear healthy Trichoplusia ni led to a study of its rearing diseases. Four diseases were designated after preliminary research which included electron microscopy: cytoplasmic polyhedrosis (due to cytoplasmic polyhedrosis virus, or CPV), nuclear polyhedrosis (due to nucleopolyhedrovirus, or NPV), "neonate death" syndrome (mortality in first or second instars), and "late-instar" syndrome (death in late instars accompanied by bacterial decomposition). Infectious agents were not detected by electron microscopy in insects with the latter two diseases. Prevalence of CPV and NPV, but not the neonate-death or late-instar diseases, in progeny was significantly associated with pairs of mating adults. In conjunction with egg-surface decontamination, this indicated that both viruses may be transmitted transovarially. Pupae killed by CPV had virions in the cytoplasm of infected cells, but polyhedra were empty, not occluding virions. None of the diseases had a consistent pattern of prevalence associated with the date on which eggs were laid after oviposition began. Prevalence rates of cytoplasmic polyhedrosis, nuclear polyhedrosis, and the late-instar disease were significantly greater at 95-100% relative humidity (RH) than at RH levels of 75% or below. These same three diseases killed significantly more insects in crowded rearing conditions (four or five larvae per cup with 10.2-cm(2) diet surface) than in uncrowded conditions (one to three larvae per cup). As a result of these experiments, healthy T. ni have been reared for 10 generations by use of a modified Pasteur method and rearing cups containing no more than two larvae., (Copyright 1999 Academic Press.)

A laboratory population of Anticarsia gemmatalis subjected to 80% mortality by the A. gemmatalis nucleopolyhedrovirus (AgNPV) developed a 5x resistance ratio (RR) within four generations. When exposure to AgNPV was discontinued, the resistant insects returned to their original level of susceptibility within three generations. Two additional cycles of resistance selection and reversion were repeated in the same insect population with similar results, except that the insects reached a 5x RR within three generations in the second cycle and two generations in the third. The insects returned to the original level of susceptibility within two generations in both the second and third resistance/reversion cycles. The resistant insects produced 61% as many viable neonates as susceptible insects. Resistant insects also had longer life spans, a lower rate of larval survival in rearing, and lower pupal weights than susceptible insects. These differences in life histories, all significant at P < 0.01, indicate that A. gemmatalis resistance to AgNPV might have associated costs in fitness. The addition of an optical brightener (OB) to the AgNPV suspension increased the susceptibility of resistant and susceptible insects by 24x and 58x, respectively, resulting in an increase of the RR from 4.7 to 11.2x. Thus, the LC50 of AgNPV with an optical brightener against resistant insects was less than the LC50 of AgNPV without brightener against susceptible insects. Copyright 1998 Academic Press.

Restriction endonuclease analysis was used to examine variation in DNA of 22 wild isolates of Spodoptera frugiperda nuclear polyhedrosis virus (SfNPV). Eleven of the 15 isolated from Louisiana were distinguishable based on restriction fragment profiles from the enzymes BamHI, HindIII, and EcoRI. There was significant genetic variation in SfNPV isolates within single agricultural fields. Nucleotide sequence divergence values, based on restriction fragment profiles, indicated that genetic variation among isolates foreign to Louisiana (Ohio, Ecuador, Mexico, Georgia, Colombia, and Venezuela) was greater than that among the Louisiana isolates. However, certain foreign isolates were similar to or identical with Louisiana isolates. Genetic variation of the viral DNA was not influenced by the insect's host plan species.

Baculoviruses have been researched extensively for insect control. Three of their features have been particularly attractive: their host specificity and consequential environmental safety, their virulence in host insects, and their capability for causing disease epizootics. There have been four approaches to their use for insect control: as microbial insecticides for short-term insect population suppression, through seasonal colonization or a recurring "booster shot" for control of more than one pest generation, through introduction-establishment where the viral species or strain was not indigenous, and through environmental manipulation to make the ecosystem more favorable for viral epizootics. Actual usage of baculoviruses in pest management has been disappointing, particularly with the microbial insecticide approach, primarily for three reasons: economics, slow speed of kill, and adverse effects of the environment on the viruses. The recombinant-DNA revolution has greatly increased the prospects for baculoviruses in insect pest management.

To determine the impact of α-tomatine at the third trophic level, the following model was developed:Nomuraea rileyi (Farlow) Samson, the secondary consumer, acting onHeliothis zea (Boddie), the primary consumer, fed an artificial diet modified with α-tomatine. In vitro, the allelochemical inhibited colony formation and growth of the fungus. The in vivo test revealed that larval growth and developmental time were affected by α-tomatine andN. rileyi. Detrimental effects on pupal development were observed in larvae fed diet containing α-tomatine and also treated withN. rileyi (LC90). The fungus was detected in the hemolymph and tissue of larvae treated with two lethal concentrations (LC50 and LC90) ofN. rileyi, including those fed α-tomatine. At the LC50, α-tomatine protected larvae againstN. rileyi and increased survivorship; at the LC90, it inhibited the development ofN. rileyi, thereby reducing production of conidia. Thus, the allelochemical α-tomatine retains its antifungal qualities beyond the second trophic level, inhibiting the development ofN. rileyi inH. zea.

Selection in the laboratory for Spodoptera frugiperda (Sf) resistant to nuclear polyhedrosis virus (NPV) affected the susceptibility of the insect to certain other mortality agents, including a chemical insecticide. Median lethal concentrations (LC50S) and associated statistics were compared for several mortality agents between colonies of NPV-resistant and -susceptible (control) insects. Compared to the susceptible insects, the NPV-resistant insects were cross-resistant to the S. frugiperda granulosis virus and to the Autographa californica NPV based on nonoverlap of 95% fiducial limits of the LC50S. The NPV-resistant insects were significantly more susceptible to methyl parathion than the control insects. The two colonies of S. frugiperda did not differ significantly in their response to Bacillus thuringiensis, Vairimorpha necatrix, or carbaryl. The cross-resistance experiments were based on per os exposure of the insects to the pathogens and insecticides; the susceptibility of the resistant and control insects did not differ significantly when the Sf NPV was injected into the hemocoel or when methyl parathion was applied topically.

Median lethal concentrations (LC) were determined for four nematode populations (two strains of Steinernema feltiae, a S. feltiae hybrid, and S. bibionis) against fifth-instar fall armyworm (Spodoptera frugiperda) larvae and for the most virulent of these nematodes against different instars and stages of the insect. Based on lack of overlap of 95% fiducial limits, there were significant differences in virulence among the four nematodes. The LC ranged from 7.6 to 33.3 nematodes/ 0.7 ml water, and slopes of the log dose-probit regression lines were similar except for the S. feltiae All strain. First-instar fall armyworms suffered virtually 100% mortality from the S. feltiae Mexican strain at 1.0 nematode/0.7 ml, and LC were 2.3 and 7.9 nematodes/0.7 ml in third-instar and fifth-instar larvae, respectively. Pupae had 7-20% mortality at doses ranging from 30 to 60 nematodes/0.7 ml.