Eileen A. Buss

Metatranscriptomics and Pyrosequencing Facilitate Discovery of Potential Viral Natural Enemies of the Invasive Caribbean Crazy Ant, Nylanderia pubens

Background Nylanderia pubens (Forel) is an invasive ant species that in recent years has developed into a serious nuisance problem in the Caribbean and United States. A rapidly expanding range, explosive localized population growth, and control difficulties have elevated this ant to pest status. Professional entomologists and the pest control industry in the United States are urgently trying to understand its biology and develop effective control methods. Currently, no known biological-based control agents are available for use in controlling N. pubens. Methodology and Principal Findings Metagenomics and pyrosequencing techniques were employed to examine the transcriptome of field-collected N. pubens colonies in an effort to identify virus infections with potential to serve as control agents against this pest ant. Pyrosequencing (454-platform) of a non-normalized N. pubens expression library generated 1,306,177 raw sequence reads comprising 450 Mbp. Assembly resulted in generation of 59,017 non-redundant sequences, including 27,348 contigs and 31,669 singlets. BLAST analysis of these non-redundant sequences identified 51 of potential viral origin. Additional analyses winnowed this list of potential viruses to three that appear to replicate in N. pubens. Conclusions Pyrosequencing the transcriptome of field-collected samples of N. pubens has identified at least three sequences that are likely of viral origin and, in which, N. pubens serves as host. In addition, the N. pubens transcriptome provides a genetic resource for the scientific community which is especially important at this early stage of developing a knowledgebase for this new pest.

FLIGHT ACTIVITY AND RELATIVE ABUNDANCE OF PHYTOPHAGOUS SCARABS (COLEOPTERA: SCARABAEOIDEA) FROM TWO LOCATIONS IN FLORIDA

Abstract The seasonal abundance of phytophagous scarabs in Gainesville and Fort Lauderdale, Florida, was documented with ultraviolet blacklight traps operated from April 2002 to November 2004. Over 44,000 adult scarabs were trapped and identified, including 30 species from 14 genera. Hybosorus illigeri Reiche was the most abundant species trapped (n = 12,306 or 27.9% of total trap catches). Phyllophaga was the most diverse genus with ten species collected. Tomarus cuniculus (F.) and Dyscinetus morator (F.) adults were trapped every month of the year. Anomala innuba (F.), Cyclocephala lurida (Bland), C. parallela Casey, H. illigeri, and Phyllophaga bruneri Chapin exhibited bimodal flight patterns. Adults of these five species combined represented 49.1, 56.5, and 64.6% of the collections in 2002, 2003, and 2004, respectively. Species that occurred in both locations tended to be active earlier in Fort Lauderdale than in Gainesville. The flight activity and species composition of potential scarab pests in Florida appears to be different from those in the midwestern and northern U.S., suggesting that turfgrass and ornamental plant managers need to adjust their management strategies accordingly.

Billbug (Coleoptera: Curculionidae) Species Composition, Abundance, Seasonal Activity, and Developmental Time in Florida

ABSTRACT Billbugs (Coleoptera: Curculionidae: Sphenophorus spp.) are common pests whose damage is often misdiagnosed on turfgrass in the United States. Consequently, Florida turfgrass managers have been struggling to satisfactorily control billbug outbreaks. Thus, we sought to determine the species complex, abundance, seasonality, and fecundity of key Sphenophorus spp. from field collections, and quantify duration of developmental and daily activity periods through greenhouse rearing tests. From January 2006 to December 2007, > 18,000 adults of 10 different Sphenophorus spp. were collected from four linear pitfall traps on each of two golf courses in north central and two courses in southern Florida. Sphenophorus venatus vestitus Chittenden was the most abundant species, making up >94% of all specimens collected from three of the four golf courses (80.9% of all specimens collected). Adults were active and mature eggs were present in female ovaries nearly every week of the year. Adults were nocturnal. S. v. vestitus development from egg to adult can occur in 8–9 wk on ‘Tifway’ bermudagrass [Cynodon dactylon (L.) Pers.] or ‘Empire’ zoysiagrass (Zoysia japonica Steud.), with up to six overlapping generations per year, depending on environmental conditions. Thus, any time of year could be suitable to target either adults or larvae, and a repeated application may be needed to manage subsequently emerged larvae or eclosed adults.

Integration of insect parasitic nematodes (Rhabditida Steinernematidae) with insecticides for control of pest mole crickets (Orthoptera: Gryllotalpidae: Scapteriscus spp.)

Abstract To develop a successful integrated pest management program for pest mole crickets (Orthoptera: Gryllotalpidae: Scapteriscus spp.), it is important to ascertain the compatibility of infective juveniles of insect parasitic nematodes and chemical insecticides. Aqueous solutions of five pesticides (acephate, bifenthrin, deltamethrin, fipronil, and imidacloprid) used in turfgrass to control mole crickets were tested for compatibility with Steinernema scapterisci Nguyen & Smart (Rhabditida: Steinernematidae) in the laboratory. Survival of S. scapterisci was >95% in solutions of acephate, bifenthrin, and imidacloprid. Infectivity of S. scapterisci in adult Scapteriscus vicinus Scudder was >60% in acephate and bifenthrin; however, infectivity was <40% in imidacloprid. The entomopathogenic nematode was compatible with most insecticides tested without significantly reduced survival or infectivity.

Thermal Requirements and Development of Herpetogramma phaeopteralis (Lepidoptera: Crambidae: Spilomelinae)

ABSTRACT The tropical sod webworm, Herpetogramma phaeopteralis Guenée is a major turfgrass pest in the southeastern United States. We evaluated larval development on five artificial diets and at six temperatures (15, 20, 25, 30, 32.5, 35 ± 1°C) on St. Augustinegrass (Stenotaphrum secundatum (Walter) Kuntze). Only larvae fed St. Augustinegrass and soy-wheat germ diets completed their lifecycles. None of the artificial diets tested (corn-based, soy-wheat germ, corn cob-wheat germ, corn cob-soy flour, or pinto bean) were suitable for rearing this species, because of high mortality and slower developmental time. Total developmental time (oviposition to adult) on S. secundatum significantly decreased from 47.8 d at 20°C to 21.1 d at 30°C, and then increased to 32.6 d at 32.5°C. Tropical sod webworm failed to complete larval development at 15 and 35°C. The relationship between temperature and developmental rate was described using linear (common and polynomial) and nonlinear models (Briere-1, Briere-2, and Lactin-2). The estimated lower temperature thresholds using a linear model for eggs, first, second, third, fourth, fifth, and sixth instars, prepupa, pupa, and total development were 10.1, 6.9, 12.3, 10.5, 15.3, 13.9, 9.1, 13.1, 12.0, and 13.1°C, and the thermal constant of these stages were 62.9, 66.2, 38.2, 40.3, 24.9, 32.3, 51.9,106.4,109.9, and 370.4 degree-days, respectively. The Briere-1 model provided the best fit with estimated lower, upper, and optimum thresholds for total development of 14.9, 34.3, and 29.4°C, respectively. The developmental requirements of H. phaeopteralis can be used to help predict the distribution and seasonal phenology of this pest.

Steinernema phyllophagae n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from Florida, USA.

A new species of entomopathogenic nematode, Steinernema phyllophagae n. sp., was isolated from a white grub (Phyllophaga sp.) in a live oak (Quercus virginiana) nursery. The new species is characterised by morphometrics of the infective Sjuvenile (IJ) with body length 1289 (1133-1395) μm, distance from anterior end to the excretory pore 99 (84-120) μm, tail 89 (77-109) μm, ratio a = 31 (27.6-35.5), H% = 57 (51-60) and E% = 110 (70-139). The pattern of the lateral field of IJ of the new species is 2, 5, 6, 8, 6, 2. The male of the first generation can be recognised by the length and shape of the spicule and gubernaculum, position of the excretory pore, D% = 75 (68-80), and GS% = 71 (61-77). The female can be recognised by the vulva having low epiptygma and by the presence of several papilla-like structures near the tail tip. Steinernema phyllophagae n. sp. differs further from other species of Steinernema by characteristics of ITS and D2D3 regions of rDNA.

AUGMENTATIVE APPLICATIONS OF STEINERNEMA SCAPTERISCI (NEMATODA: STEINERNEMATIDAE) FOR MOLE CRICKET (ORTHOPTERA: GRYLLOTALPIDAE) CONTROL ON GOLF COURSES

Abstract The insect parasitic nematode, Steinernema scapterisci Nguyen and Smart, is a non-chemical alternative to pest mole cricket control in the southern United States. These ambush nematodes can become established after one application and spread into untreated areas through host movement in the soil. However, the nematodes persistence from previous inoculative applications in 1988 and 1989 and the effectiveness of subsequent augmentative applications on intensively managed golf courses were unknown. In 2001, two linear pitfall traps were placed in the roughs of 10 holes on each of two golf courses (20 traps per course) near areas of adult mole cricket activity, and half of the plots with traps were treated with S. scapterisci. Ten to 15% of mole crickets trapped before the augmentative nematode applications were infected by S. scapterisci. After this application, the percentage of infected mole crickets was higher than the baseline for 8 mo at one golf course and 17 mo at the other. The percentage of mole crickets infected on treated plots equaled or exceeded pretreatment levels about 4-8 wk post-application. The percentage of infected mole crickets in untreated areas at both sites equaled the percent infection in treated areas after about 5 mo. Mole cricket trap catches and percent of infection declined in the second year, but continued to fluctuate with mole cricket population density, age, and environmental conditions. Augmentative applications of S. scapterisci for pest mole cricket control can enhance mole cricket mortality on golf courses.

A Synchronous Rearing Method for Blissus insularis (Hemiptera: Blissidae)

ABSTRACT The southern chinch bug, Blissus insularis Barber (Hemiptera: Blissidae), is the most destructive insect pest of St. Augustinegrass, Stenotaphrum secundatum (Walt.) Kuntze. Management of B. insulaiis has depended on frequent insecticide applications, which has resulted in populations becoming resistant to several insecticide classes. To facilitate developing a resistance management program for this pest, it is necessary to develop methods to rear insects of known age, generation, and pesticide exposure history. Synchronized rearing methods were developed after testing five different laboratory methods. The use of glass jars and a combined diet of fresh corn, Zea mays L., cob and St. Augustinegrass proved to be best for producing B. insuhris of known age and generation. Body size was consistent over nine generations of rearing. Production of a high proportion of brachypterous B. insuhris (the nondispersal adult form) also suggests that populations were not stressed during laboratory rearing. This work presents the first successful synchronized rearing method for B. insuhris.

Non-Enzymatic Hydrolysis of RNA in Workers of the Ant Nylanderia pubens

Abstract During preparation of total RNA from Nylanderia pubens (Forel) (Hymenoptera: Formicidae) workers for use in expression library construction, severe RNA degradation consistently occurred. This degradation was masked by spectrophotometric analysis but clearly evident by microfluidic-based assay. Although not specifically identified, the degrading entity was endogenous and localized to the abdomen (terminal abdominal segments) of adult ants. RNA degradation was not observed in preparations of larvae, non-melanized pupae, or eggs. Various RNase and protease inhibitors had no protective effect. However, the metal chelating agent ethylenediaminetetraacetic acid prevented RNA degradation and provides insight into the occurrence.

Susceptibility of Blissus insularis (Heteroptera: Hemiptera: Blissidae) Populations in Florida to Bifenthrin and Permethrin

ABSTRACT The southern chinch bug, Blissus insularis Barber, is a serious insect pest of St. Augustinegrass (Stenotaphrum secundatum [Walt.] Kuntze). Control for B. insularis is mainly achieved through insecticides. This pest has developed resistance to several insecticide classes because of near-constant exposure. The goals of this study were to sample select B. insularis populations in Florida to describe their susceptibility to bifenthrin, document new locations of bifenthrin resistance, and evaluate another pyrethroid, permethrin. Lethal concentration ratios (at the LC50) from B. insularis populations collected in 2006 and 2008 showed a 45-4,099-fold resistance to bifenthrin in Citrus, Escambia, Flagler, Hillsborough, Lake, Orange, Osceola, and Volusia counties. One population in Orange County demonstrated a 212-fold resistance to permethrin. There was a positive relationship between the number of insecticide applications made in 2006 and increasing insecticide resistance. This study documents the first case of insecticide resistance in the Florida Panhandle and the first report of B. insularis resistance to permethrin. Observations made during this study and possible causes for the development of insecticide resistance in B. insularis in Florida are discussed.