Steven R. Skoda

Population Variation of the Fall Armyworm, Spodoptera frugiperda, in the Western Hemisphere

Abstract Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), the fall armyworm is the most economically important maize pest in the western hemisphere. This research focused on the genetic variability of the maize host strain because there is a lack of information in this area of S. frugiperda research. Amplified fragment length polymorphism (AFLP) was used to assess the genetic variability of S. frugiperda over a large geographic area. Twenty populations were collected from the maize, one population was collected from princess tree, one population was collected from lemon tree, and one population was collected from bermudagrass. The 23 populations were from Mexico, the continental United States, Puerto Rico, Brazil, and Argentina. The objective of this research was to evaluate whether the majority of genetic variability was within populations or between populations. The AFLP results showed that the majority of the genetic variability is within populations and not between populations, indicating minor gene flow and suggesting that S. frugiperda in the Western Hemisphere are an interbreeding population.

Genetic Variability of the European Corn Borer, Ostrinia nubilalis, Suggests Gene Flow Between Populations in the Midwestern United States

Abstract The European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), is a widely distributed and serious economic pest to corn production in the U.S. Genetic variability of O. nubilalis was studied in 18 sub-populations in the upper Midwestern United States using amplified fragment length polymorphism. The relatively low GST values indicate that more variation exists within populations than between populations. High gene flow (Nm) values were indicated across the entire O. nubilalis population; the lowest degree of gene flow was in the northern samples (Nm = 1.96) and the highest degree of gene flow was in the southern samples (Nm = 2.77). The differences observed in the respective regions (north vs. south) may be explained by the voltinism patterns (univoltine vs. multivoltine, respectively) of O. nubilalis: southern multivoltine populations have opportunities for multiple matings for the duration of the year, further mix alleles. AMOVA results also indicated that most of the genetic variation was within sub-populations (≈ 81% of total variation); less variation (≈ 13%) was detected among populations within each of the three regions as designated for this study. However, the most striking and unexpected result was the low percentage of variation between all groups (≈ 6%), further supporting implications of a high degree of gene flow. These results provide support for current requirements of refugia corn planting in Bt-corn management. These results also indicate that if resistance to Bt were to evolve in O. nubilalis, quick action would be necessary to deter the rapid spread of the gene for resistance.

Volatiles Emitted From Eight Wound-Isolated Bacteria Differentially Attract Gravid Screwworms (Diptera: Calliphoridae) to Oviposit

ABSTRACT Bovine blood inoculated with bacteria isolated from screwworm [Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae) ]-infested animal wounds was tested as an attractant for oviposition for gravid screwworms. Eight species of gram-negative coliform (Enterobacteriaceae) bacteria mixed with bovine blood singly or all species combined and incubated for various times produced volatiles that attracted gravid flies in a cage bioassay in varying numbers. In 15-min duration tests, volatiles from five species of bacteria (Klebsiella oxytoca, Proteus mirabilis, Proteus vulgaris, Providencia rettgeri, and Providencia stuartii) attracted more females than volatiles of the three species (Enterobacter cloacae, Enterobacter sakazakii, and Serratia liquefaciens). In 1-h duration oviposition tests, volatiles from the substrate using the same five species of bacteria attracted more females to oviposit than the other three species. Volatiles from 24-h incubation period elicited least attraction and oviposition whereas volatiles from the 48- and 72-h incubation period resulted in significantly more attraction and oviposition. Attraction and oviposition decreased significantly when the substrates were incubated for 96 h. Volatiles from substrate with all species of bacteria combined attracted a significantly higher percentage of flies to land and oviposit than those from substrates prepared with single species. It is possible that multiple active chemicals present in volatiles of the all-species substrate may act as synergists resulting in greater response than those observed with volatiles from single-species substrate. Before oviposition flies took a bloodmeal from the oviposition substrate. It is possible that the oviposition is moderated by two different factors in screwworm—first, by using a chemical cue to land on a potential oviposition site and second, by using a bloodmeal to stimulate oviposition.

Resistance Status of House Flies (Diptera: Muscidae) from Southeastern Nebraska Beef Cattle Feedlots to Selected Insecticides

The status of resistance to three insecticides (permethrin, stirofos, and methoxychlor), relative to a laboratory-susceptible colony, was evaluated in field populations of house flies, Musca domestica L., collected from two beef cattle feedlots in southeastern Nebraska. Topical application and residual exposure to treated glass surfaces were suitable methods for determining the resistance status of house flies to permethrin, stirofos, or methoxychlor. However, in most cases, residual exposure was more sensitive in resistance detection (i.e., higher resistance ratios). The field populations tested were moderately resistant to permethrin (RR = 4.9-fold and RR = 7.3-fold, for topical application and residual exposure, respectively) and extremely resistant to stirofos and methoxychlor (not accurately quantifiable because of low mortality at the highest possible concentrations or doses). Probable explanations for the resistance status of these house fly populations and implications for global feedlot fly management are discussed.

A Cellulose Fiber-Based Diet for Screwworm (Diptera: Calliphoridae) Larvae

Abstract A highly absorbent cellulose fiber from recycled paper was tested and compared with a polyacrylate gelling agent, Aquatain, normally used for bulking and solidifying larval rearing medium of screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae). The absorbent fiber, when mixed with water and dietary ingredients, produced a diet medium of homogeneous texture that supported larval growth and development comparable with the standard gelled diet. Larval and pupal weights from two concentrations of cellulose fiber-based diet were significantly higher than those obtained using gelled diet. The number of pupae per tray, percentage of adult emergence, oviposition, percentage of egg hatch, and adult longevity obtained from the insects reared in the cellulose fiber-based diet were comparable or slightly better than the biological parameters recorded from flies reared in the gelled diet. Moreover, results indicate that a lesser amount of the cellulose fiber-based diet than the normal amount of gelled diet per tray would support normal larval growth. Physical properties and texture of the new diet seem to allow the larvae to move and feed more freely than they do on the semisolid gelled diet, resulting in less wasted diet. The cellulose fiber is biodegradable and inexpensive, whereas the polyacrylate gel polymer is not biodegradable and is relatively expensive. Replacing gel with cellulose fiber in the screwworm larval diet for mass rearing should result in substantial cost savings in material and labor as well as eliminating concern of environmental pollution due to diet waste disposal.

Virulence of Mexican isolates of entomopathogenic fungi (Hypocreales: Clavicipitaceae) upon Rhipicephalus=Boophilus microplus (Acari: Ixodidae) larvae and the efficacy of conidia formulations to reduce larval tick density under field conditions.

The first objective was laboratory evaluation of the virulence of 53 Mexican isolates of fungi against larvae of Rhipicephalus (Boophilus) microplus. Thirty-three isolates of Metarhizium anisopliae var. anisopliae (Metschnickoff) Sorokin (Hypocreales: Clavicipitaceae) and 20 isolates of Isaria (Paecilomyces) fumosorosea (fumosoroseus) (Wize) (Eurotiales: Trichomaceae) were tested on 7-day-old larvae under laboratory conditions. Larvae were immersed in a suspension containing 10(8)conidia/mL and the CL(50) values were estimated. Then, field tests were conducted to determine the efficacy of formulations of the isolate with the highest virulence. M. anisopliae (Ma 14 isolate) was formulated with four carriers: Tween, Celite, wheat bran, and Citroline (mineral oil) and applied on pasture beds of Cynodon plectostachyus (L.), at a dose of 2 x 10(9)CFU/m(2). In the first trial, M. anisopliae was applied on plots naturally infested with larvae; in the second trial, tick populations in the experimental plots were eliminated and then re-infested with 20,000 7-day-old larvae. In the laboratory, all M. anisopliae isolates infected larvae with a mortality range between 2 and 100%; also, 13 of 20 I. fumosorosea isolates caused mortality rates between 7 and 94%. In the first field trial, 14 days post-application, conidial formulations in Celite and wheat bran caused 67.8 and 94.2% population reduction, respectively. In the second trial, the Tween formulation caused the highest larval reduction, reaching up to 61% (28 days post-application). Wheat bran formulation caused 58.3% larval reduction (21 days post-application) and was one of the most effective. The carriers and emulsifiers have a large impact on the effectiveness of conidial formulations.

Use of the entomopathogenic fungi Metarhizium anisopliae , Cordyceps bassiana and Isaria fumosorosea to control Diaphorina citri (Hemiptera: Psyllidae) in Persian lime under field conditions

The Asian citrus psyllid Diaphorina citri Kuwayama is a destructive insect pest in citriculture, because it is an efficient vector of the proteobacteria ‘Candidatus Liberibacter asiaticus’ (Las), ‘Ca. L. africanus’ (Laf) and ‘Ca. L. americanus’ (Lam). These bacteria cause the ‘huanglongbing’ disease or ‘greening’ or ‘yellow dragon’ disease. The disease kills the plant and reduces fruit production. This insect pest is susceptible to entomopathogenic fungi, and we report the use of different strains of Metarhizium anisopliae, Cordyceps bassiana and Isaria fumosorosea against the nymphs and adults of D. citri under field conditions. The fungi were applied four times using a concentration of 2 × 1013 conidia/ha with a time interval of 15 days between applications. The percentage of control of Cb 108, Ma 65, Ma 14 and Ifr 4 was 60, 50, 40 and 35% in nymphs, and 50, 50, 42 and 22% in adults, respectively. Metarhizium anisopliae, C. bassiana and I. fumosorosea applied on Persian lime groves are more effective in reducing higher density of nymphs than adults of D. citri.

Genetic Variation and Inheritance of Diapause Induction in Two Distinct Voltine Ecotypes of Ostrinia nubilalis (Lepidoptera: Crambidae)

ABSTRACT European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), displays a larval diapause in response to short photoperiods and is adapted to a variety of local conditions throughout North America. Hence, the effective photoperiod inducing larval diapause will differ among geographic ecotypes. This study considers the inheritance of photoperiodic larval diapause induction by hybridization and backcrossing two latitudinally distinct ecotypes of the European corn borer collected between 41° N, 96° W and 48° N, 96° W and under a range of photoperiods representative of their respective locations: from 14:10 to16:8 (L:D) h. The ecotype adapted to a bivoltine habitat (southeastern Nebraska) exhibited a shorter critical photoperiod (1480 h) than the ecotype (1533 h), originating from a univoltine habitat (northwestern Minnesota). Reciprocal F1 crosses exhibited intermediate values with indication of sex-linked inheritance. In addition, the male parent had significantly more influence on diapause incidence of subsequent progeny than the female. The F2 and backcross progeny further supported the supposition that diapause response is a sex-linked inheritance. The minimum number of genes estimates, and the response from backcross progeny, suggest that diapause response of European corn borer larva may be controlled by only a few loci. The overall results indicated that both ecotypes had adopted unique diapause responses, which ultimately lead to seasonal synchrony in their ecosystems.

Amplified fragment length polymorphism used for inter- and intraspecific differentiation of screwworms (Diptera: Calliphoridae).

Morphologically, early immature stages of the economically important pest called screwworms, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), and non-pest secondary screwworms, Cochliomyia macellaria (Fabricius) (Diptera: Calliphoridae), are nearly indistinguishable. Correct identification is crucial to the ongoing eradication and exclusion program protecting the United States, Mexico and Central America from reinvasion of screwworms persistent in South America and the Caribbean. Amplified fragment length polymorphism (AFLP) polymerase chain reaction was used to differentiate populations of C. hominivorax and to discriminate them from C. macellaria. Ten primer pairs screened for interspecific discrimination of C. hominivorax from C. macellaria showed 52 discrete bands, allowing the two species to be readily distinguished; divergent branches on resulting dendrograms showed 100% bootstrap support. C. macellaria populations grouped at the 92% level; C. hominivorax populations grouped at the 68% level. Of the 52 bands, seven were monomorphic for both species, 22 were specific to C. macellaria, ten were present only in C. hominivorax and the remaining 13 bands differentiated C. hominivorax populations. Separate studies using ten strains of C. hominivorax showed a higher level of genetic similarity within than between populations. Analyses using 72 bands (19 monomorphic bands, 53 bands grouped all ten strains at the 58% similarity level) resolved seven mutant strains from Mexico (85% similarity level); all ten strains were resolved at the 72% similarity level. Diagnostic bands were identified for species and strain identification. We conclude that AFLP can be a valuable tool for studies of interspecific and intraspecific genetic variation in screwworm populations.

Volatiles from Waste Larval Rearing Media Attract Gravid Screwworm Flies (Diptera: Calliphoridae) to Oviposit

ABSTRACT Gravid screwworm flies, Cochliomyia hominivorax (Coquerel), are attracted to the volatiles from waste larval rearing media to deposit eggs. Studies were conducted to identify volatile chemicals from the waste larval media and determine their effectiveness to attract gravid flies to oviposit. Volatiles were collected using solid-phase microextraction method, and five active chemicals, namely, dimethyl disulfide, dimethyl trisulfide, phenol, p-cresol, and indole, were identified using gas chromatography—mass spectrometry. In electroantennography studies, antennae of gravid screwworm flies, Cochliomyia macellaria (F.), responded positively to each of the identified compounds. A synthetic blend of these five compounds in the ratio of 335:200:57:1:12 was prepared and tested for its effectiveness to attract both C. hominivorax and C. macellaria using laboratory bioassay methods. Significantly more gravid C. macellaria were attracted to and landed on substrates treated with 10-fold diluted blends compared with those landed on substrates treated with ethanol only (as control). Only a few young females and young and old males were attracted to the substrates treated with the synthetic blend. The C. hominivorax females laid significantly more eggs on substrates treated with waste media, 10-fold diluted blend, and 100-fold diluted blend than on substrates with undiluted blend or ethanol. Similarly, C. macellaria deposited significantly more eggs on substrates treated with waste media, 10-fold diluted blend, and 100-fold diluted blend compared with substrates with undiluted blend or ethanol. C. macellaria females deposited significantly less amount of eggs than did C. hominivorax females. These results indicate that the synthetic blend of five compounds identified may serve as an oviposition attractant for C. hominivorax as well as for C. macellaria.