Prevalence and impact of the microsporidium Thelohania solenopsae (Microsporidia) on wild populations of red imported fire ants, Solenopsis invicta, in Louisiana.

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.

Threshold distances and depths of nucleopolyhedrovirus in soil for transport to cotton plants by wind and rain.

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.

Interaction of an entomopathogen with an insect social form: an epizootic of Thelohania solenopsae (Microsporidia) in a population of the red imported fire ant, Solenopsis invicta.

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).

Comparative effectiveness of light-microscopic techniques and PCR in detecting Thelohania solenopsae (Microsporidia) infections in red imported fire ants (Solenopsis invicta).

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.

Deletion of pe38 attenuates AcMNPV genome replication, budded virus production, and virulence in heliothis virescens.

The pe38 gene product of Autographa californica M nucleopolyhedrovirus (AcMNPV) has been shown to be involved in transcriptionally transactivating viral genes and augmenting viral DNA replication in transient assays. To assess the role of pe38 during infection, we generated a knockout virus, Delta pe38-E9/E9, in which the pe38 open reading frame was replaced with that of the green fluorescent protein. We compared mutant and wild-type (WT) viral replication in insect cell culture and virulence in Heliothis virescens larvae. Compared to WT, Delta pe38-E9/E9 budded virus (BV) production was delayed by at least 3 h, and BV yields were reduced over 99%. Similarly, Delta pe38-E9/E9 DNA synthesis levels were greatly reduced relative to those of WT, but onset of DNA replication was the same for both viruses. In bioassays, nearly sevenfold more Delta pe38-E9/E9 virus than WT virus was required to achieve an LD(50) when administered orally, but not hemocoelically. These results support the hypothesis that the kinetics of AcMNPV BV production greatly impact virulence in larvae infected orally (the natural route of infection) and that PE38 is an important, but not essential, factor in viral DNA synthesis and BV production.