Steven M. Valles

Fire Ant Control with Entomopathogens in the USA

Fire ants are stinging invasive ants from South America that infest over 129.5 million hectares in the southern United States, where eradication is no longer considered possible. The biological control of fire ants, especially by pathogens, is viewed by some as the only sustainable tactic for suppression. Microscopic-based surveys conducted in South America during the 1970s and 1980s led to the discovery of fungi and microsporidia infecting fire ants. Three of these microorganisms have been studied extensively: Beauveria bassiana 447, Thelohania solenopsae, and Vairimorpha invictae. B. bassiana 447 causes fire ant mortality but infections do not spread to queens and intercolony transmission was not evident. T. solenopsae has been found in the US and has been shown to spread naturally and debilitate colonies. Colony decline has also been associated with V. invictae, which is currently being evaluated for host specificity and possible release in the US. Through the use of molecular techniques, viruses infecting fire ant in the US have been discovered and characterized. Solenopsis invicta virus-1 can be transmitted easily to uninfected colonies and colony death often results. This virus apparently causes persistent, asymptomatic infections that actively replicate when the host is stressed. Research on fire ant-specific microsporidia and viruses, as well as other fire ant entomopathogens, is summarized to illustrate the efforts that have been undertaken to understand the biology of these pathogens and to facilitate their utilization in biological control of fire ants.

Loss of microbial (pathogen) infections associated with recent invasions of the red imported fire ant Solenopsis invicta

Loss of natural enemies during colonization is a prominent hypothesis explaining enhanced performance of invasive species in introduced areas. Numerous studies have tested this enemy release hypothesis in a wide range of taxa but few studies have focused on invasive ants. We conducted extensive surveys for the presence of six microbes in recently established populations (California, Australia, New Zealand, Taiwan, and China) of the invasive fire ant Solenopsis invicta. These microbes include Wolbachia, two microsporidia (Kneallhaziasolenopsae and Vairimorpha invictae) and three RNA viruses (SINV-1, -2 and -3), all of which previously have been reported in native South American populations of S. invicta. These surveys showed that the total number of enemy species is lower in the recently invaded areas compared with both South American and US populations. Only two microbes were found in any of these recently invaded areas: SINV-1 was detected in all surveyed populations except Australia and New Zealand, and SINV-2 was detected in California and Taiwan only. These results support the general prediction that invasive species lose many of their natural enemies during invasion. Further, the conspicuous absence of some of these microbes in these areas may result from strong selection against founders due to fitness costs associated with harboring detrimental infections rather than the alternative hypothesis that they simply were absent among the original founders. While the successful invasion of S. invicta in these recently invaded areas may be explained partly by the absence of natural enemies, other factors likely have been important as well.

Effects of the Synergists Piperonyl Butoxide and S,S,S-Tributyl Phosphorotrithioate on Propoxur Pharmacokinetics in Blattella germanica (Blattodea: Blattellidae)

Abstract Effects of the synergists piperonyl butoxide (PBO) and S,S,S-tributyl phosphorotrithioate (DEF) on propoxur pharmacokinetics were examined in the German cockroach, Blattella germanica (L.). Treatment of adult male German cockroaches with the cytochrome P450 monooxygenase inhibitor, PBO, or the esterase inhibitor, DEF, increased propoxur toxicity by 2- and 6.8-fold, respectively, implicating hydrolysis as a major detoxification route of propoxur in the German cockroach. However, significant hydrolytic metabolism could not be demonstrated conclusively in vitro resulting in a conflict between in situ bioassay data and in vitro metabolic studies. In vitro propoxur metabolism with NADPH-fortified microsomes produced at least nine metabolites. Formation of metabolites was NADPH-dependent; no quantifiable metabolism was detected with cytosolic fractions. However, microsomal fractions lacking an NADPH source did produce a low, but detectable, quantity of metabolites (1.6 pmol). PBO inhibited NADPH-dependent propoxur metabolism in a dose-dependent fashion, implicating cytochrome P450 monooxygenases as the enzyme system responsible for the metabolism. Interestingly, DEF also inhibited the NADPH-dependent metabolism of propoxur, albeit to a lower extent. Treatment with PBO or DEF also caused a significant reduction in the cuticular penetration rate of propoxur. The data demonstrate that unanticipated effects are possible with synergists and that caution must be exercised when interpreting synergist results.

SIMULTANEOUS DETECTION OF VAIRIMORPHA INVICTAE (MICROSPORIDIA: BURENELLIDAE) AND THELOHANIA SOLENOPSAE (MICROSPORIDIA: THELOHANIIDAE) IN FIRE ANTS BY PCR

A PCR-based method capable of detecting Thelohania solenopsae and/or Vairimorpha invictae infection in the red imported fire ant, Solenopsis invicta, was developed. Multiplex PCR allows simultaneous detection of both species of microsporidia in a single reaction.

Inter- and intra-instar food consumption in the German cockroach, Blattella germanica

Food consumption was measured in an insecticide‐susceptible (Orlando) and ‐resistant (Village Green) strain of German cockroach, Blattella germanica (Dictyoptera: Blattellidae), throughout each stadium. All instars exhibited a similar pattern of consumption over the course of the instar; consumption was low to moderate at the beginning of each instar, climbed steadily to a maximum near the middle, declined progressively from the mid‐instar maximum, and ceased or was minimal several days before the next molt. Consumption ceased or was minimal during the final two to four days of each instar. Cumulative consumption for the six instars was significantly greater in the Village Green strain as compared with the Orlando strain. Village Green nymphs consumed 79.6 ± 0.7 mg of rat chow while Orlando nymphs consumed 63.7 ± 3.4 mg. Total consumption by instar increased significantly with each successive instar in the Village Green strain. In addition, Village Green sixth instar females consumed significantly more food than sixth instar males. A similar trend was observed for the Orlando strain; consumption tended to increase in each successive instar. Relative consumption rate (food consumed/cockroach/instar divided by the mean weight of the instar) was highest in the first two instars and decreased in subsequent instars.

Kneallhazia (= Thelohania ) solenopsae Infection Rate of Pseudacteon curvatus Flies Determined by Multiplex PCR

ABSTRACT A multiplex PCR method was developed and used to determine the infection rate of Kneallhazia solenopsae in individual Pseudacteon curvatus flies in north-central Florida. Among P. curvatus flies infected with K. solenopsae, 2 amplicons were produced, one of 800 nucleotides from the P. curvatus 18S rRNA gene, and one of 318 nucleotides from the K. solenopsae 16S rRNA gene. Multiplex PCR of DNA extracted from P. curvatus flies was capable of detecting 117.5 ± 82.7 K. solenopsae spore equivalents. The mean K. solenopsae infection rate of P. curvatus from 4 sites in Gainesville and Williston, Florida, was 12.3 ± 5.0%. The K. solenopsae infection rate for P. curvatus was independent of the K. solenopsae infection rate observed among S. invicta nests from where the fly collections took place. Not all P. curvatus flies that developed in K. solenopsae-infected fire ants were positive for K. solenopsae upon eclosion. Among 50 P. curvatus flies known to develop in K. solenopsae-infected S. invicta workers, 12 (24%) were positive for K. solenopsae at eclosion.

Characterization of Solenopsis invicta (Hymenoptera: Formicidae) Populations in Virginia: Social Form Genotyping and Pathogen/Parasitoid Detection

ABSTRACT Red imported fire ant, Solenopsis invicta Buren, workers were sampled from 26 colonies in Virginia during the 2007–2008 time period. Polymerase chain reaction (PCR) assays were used to determine colony social form (monogyny or polygyny) by genotyping ants at the Gp9 locus. Twenty of the colonies (76.9%) were found to be polygyne. Multiplex PCR was also used to detect the presence of several organisms currently being used as biological control agents for fire ants in the U.S., including the microsporidian parasite Kneallhazia solenopsae and Pseudacteon spp. parasitioid decapitating phorid flies in the sampled colonies. Kneallhazia solenopsae was detected in 11 of 26 colonies (42%). In addition, Pseudacteon spp. flies were detected in 2 (7.7%) colonies. The sampled colonies were examined by reverse transcription PCR (RT-PCR) for the presence of Solenopsis invicta viruses -1 and -2. Results indicated that 5 colonies were infected with SINV-1 (19%) and none were infected with SINV-2. This study is the first to characterize the red imported fire ant infestation in Virginia and documents the presence of biological control agents in this area. RESUMEN Se realizó un muestreo de las obreras de la hormiga de fuego roja importada, Solenopsis invicta Buren, en 26 colonias en el estado de Virginia durante el período del 2007 al 2008. Se utilizaron ensayos de la reacción en cadena por la polimerasa (RCP) para determinar la forma social de la colonia (monoginia = una sola reina reproductive) o poliginia = reinas multiples) por medio del genotipo de las hormigas en el locus Gp-9. Se encontró que veinte de las colonias (76.9%) fueron poliginias. Se usó RCP múltiplex para detectar la presencia de varios organismos que estan usando actualmente como agentes de control biológico para las hormigas de fuego en los Estados Unidos, incluyendo el parasito microesporidiano Kneallhazia solenopsae, y la mosca Pseudacteon spp. (Diptera: Phoridae) que son parasitoides que decapitan las hormigas en las colonias muestreadas. Kneallhazia solenopsae fue detectado en 11 de las 26 colonias (42%). Además, se detectaron las moscas Pseudacteon spp. en dos (7.7%) de las colonias. Se examinaron las colonias muestreadas por la transcripción reversa de RCP (TR-RCP) para la presencia de los virus 1 y 2 de Solenopsis invicta. Los resultados indican que las 5 colonias fueron infectadas con SINV-1 (19%) y ninguna fue infectada con SINV-2. Este es el primer studio que caractiza la infestación de la hormiga de fuego roja importada en Virginia y documenta la presencia de agentes de control biológico en esta área.

Host specificity of the microsporidian pathogen Vairimorpha invictae at five field sites with infected Solenopsis invicta fire ant colonies in Northern Argentina.

Abstract The microsporidian pathogen Vairimorpha invictae is being evaluated for release in the United States as a potential self-sustaining biological control agent for imported fire ants. We examined the host range of this pathogen at 5 sites in northern Argentina where Solenopsis invicta Buren fire ant colonies had high levels of infection (28-83%). At 3 sites near the city of Corrientes, we examined 509 non-Solenopsis ants from 61 collections, 12 genera, and 19 species with a polymerase chain reaction-based screening procedure. No V. invictae infections were detected in any of the samples. At 2 sites near San Javier in Santa Fe Province, 350 km to the south, we screened another 438 non-Solenopsis ants from 44 baits, 4 genera, and 4 species, again with no infections. At the Corrientes sites, we also examined 235 non-ant arthropods from 10 orders, 43 families, and more than 80 species. None were infected with V. invictae. The results of this study indicate that, in its native South American range, V. invictae is specific to Solenopsis fire ants.

TEMPERATURE EFFECTS ON k-CYHALOTHRIN TOXICITY IN INSECTICIDE-SUSCEPTIBLE AND RESISTANT GERMAN COCKROACHES (DICTYOPTERA: BLATTELLIDAE)

Pre- and post-treatment temperature effects on -cyhalothrin toxicity were determined in insecticide-susceptible and resistant German cockroach, Blattella germanica (L.), strains. Acclimation at 19, 26, or 31C for 10 days before insecticide treatment had no effect on l- cyhalothrin toxicity in either strain. No differences were observed in aldrin epoxidase and glutathione S-transferase activities when “Orlando” (susceptible) cockroaches were incubated for 10 days at 19, 26, and 31C. When temperature treatment followed insecticide application, a negative temperature coefficient of toxicity (greater toxicity at lower temperature) toward l- cyhalothrin was observed for the Orlando but not the kdr-type resistant “Village Green” cockroaches. Piperonyl butoxide synergized l- cyhalothrin in “Orlando” cockroaches 3 and 5-fold at 26 and 31C, respectively. Conversely, piperonyl butoxide pre-treatment did not cause a significant reduction in the LD 50 value in the “Village Green” strain regardless of temperature.

The Fire Ant (Hymenoptera: Formicidae) Pathogen, Vairimorpha invictae (Microsporidia: Burenellidae), Not Detected in Florida

Vairimorpha invictae Jouvenaz & Ellis (Micro sporidia: Burenellidae) is a microsporidian patho gen of Solenopsis fire ants in South America. Be cause Solenopsis invicta Buren fire ants infected with V. invictae were associated with higher mor tality, significantly slower colony growth, and re ductions in field populations in Argentina, there is considerable interest in utilizing this pathogen as a classical biological control agent against S. invicta in the U.S. (Briano & Williams 2002; Oi et al. 2005; Briano 2005). V. invictae was formally described by Jouvenaz & Ellis in 1986, however, it was most likely observed much earlier, being the undescribed, dimorphic microsporidium re ported from pathogen surveys conducted in Brazil in 1976 and 1979 (Jouvenaz et al., 1980; Briano & Williams 2002). Surveys for V. invictae in Ar gentina reported prevalence rates of 1, 2.3, and 10% after sampling 1836, 2528, and 2064 nests, respectively (Briano et al. 1995; 2006; Briano & Williams 2002). In the U.S., there have been surveys for vari ous fire ant pathogens since the 1970s. Limited surveys of red and black imported fire ants, S. in victa and Solenopsis richteri Forel, respectively, were conducted in northern Florida and in Mis