Field-collected Insects Research Articles

Detecting nutritional state and food source use in field‐collected insects that synthesize honeydew oligosaccharides

1. During the adult stage many arthropod species, including aphid predators and parasitoids, depend on nectar and honeydew as a source of carbohydrates. Despite the importance of sugar feeding for these organisms, we know little about their energy and nutrient provision under field conditions. 2. Here we assessed the nutritional state of adult parasitoids, hoverflies and lacewings in a Swiss winter wheat (WW) and a spring wheat (SW) field and studied the contribution of honeydew to the diet of these aphidophagous insects. The total sugar level and the glucose-fructose ratio were used as indicators for nutritional state and sugar feeding. 3. Over 76% of the collected individuals from each of the three insect groups in both fields had recently consumed carbohydrates. The average nutritional state was significantly higher in the SW field for Chrysoperla carnea and Aphidius spp. 4. Honeydew consumption by insects is commonly investigated by analyzing target insects for the presence of honeydew 'signature' sugars, such as melezitose and erlose. However, our laboratory studies show that adults of the three insect orders investigated synthesize these 'honeydew-specific' sugars after sucrose feeding. 5. As the erlose-melezitose ratio of sucrose-fed Aphidius spp. and the hoverfly Episyrphus balteatus differed clearly from the honeydew sugar profiles of wheat infesting aphids, this ratio could be used as an alternative indicator of honeydew feeding. However, this method could not be used for the lacewing C. carnea. 6. Our data show that 55% (WW) and 59% (SW) of field-collected Aphidius spp. which showed evidence of sugar feeding could be classified as having consumed honeydew within the 24–48 h before collection. Evidence of honeydew feeding by hoverflies, on the other hand, was found to be much more variable, ranging from 0% in the WW field to 44% in the SW field. 7. This study shows that the detection of honeydew consumption in field-collected insects based on honeydew oligosaccharides can be feasible even when insects synthesize these oligosaccharides themselves.

Isolation of Paecilomyces lilacinus (Thom) Samson (Ascomycota: Hypocreales) from the Chagas disease vector, Triatoma infestans Klug (Hemiptera: Reduviidae) in an endemic area in Argentina

A survey for entomopathogenic fungi of the Chagas disease vector Triatoma infestans was conducted in two provinces of Argentina from March-December 2003. Field-collected insects that died in the laboratory were individually maintained in moist chamber and incubated at 22 degrees C. Triatominae adults infected with the fungus Paecilomyces lilacinus were found at El Quebracho (27 degrees 34'S-64 degrees 31'W), Santiago del Estero province, Argentina, in December 2003. Paecilomyces lilacinus was cultured and isolated from infected insects in SDAY, PYG and MEA media. Pathogenicity tests were conducted and positive results were recorded. The median survival time (MST) of T. infestans exposed to a P. lilacinus conidial suspension was 12.8 days, and 100% mortality occurred at 30 days post-treatment. This is the first record of natural infection caused by P. lilacinus in T. infestans in the world.

Host range of an NPV and a GV isolated from the common cutworm, Agrotis segetum: pathogenicity within the cutworm complex

The term cutworm covers a range of species with a similar life history that can be very damaging pests on a wide range of crops. Attacks by cutworms are often made up of more than one species; thus, the most cost effective microbial control agent needs to be pathogenic for multiple species within this complex. In this study we investigate the host range of Agrotis segetum NPV and A. segetum GV for other cutworm species and closely related Noctuinae. Eight species, A. segetum, Agrotis ipsilon, Agrotis exclamationis, Agrotis puta, Noctua comes, Peridroma saucia, Xestia sexstrigata, and Xestia xanthographa, were clearly susceptible to AgseNPV, which was confirmed by DNA analysis. Aglais urticae, Diarsia rubi, Noctua pronuba, and Xestia c-nigrum were not susceptible to AgseNPV at the doses used. Noctua fimbriata, Noctua janthina, and Ochroplura plecta gave ambivalent results: larvae died of NPV infection when they were challenged with AgseNPV, but these individuals only produced weak positives in a squash blot analysis and there was insufficient DNA for confirmation by restriction endonuclease profiling. These ambivalent results could suggest either a weak infection by AgseNPV or partial homology between their own virus and AgseNPV. The untreated control insects of several species died of NPV infection, which indicates that these field-collected insects were probably carrying a vertically transmitted NPV. Fewer species were tested with AgseGV and only N. pronuba and N. comes were susceptible. N. fimbriata and Helicoverpa armigera were not susceptible to AgseGV.

Characterization and authentication of insect cell lines using RAPD markers

Random amplified polymorphic DNA (RAPD) analysis was used to characterize 11 insect cell lines, including six from lepidoptera (five species), one from diptera and four from coleoptera (one species: Leptinotarsa decemlineata). Whatever the order and even when comparing two closely related species from the same genus ( Spodoptera), the DNA fingerprints are very different from one species or from one primer to the other. On the other hand, two independently isolated cell lines from the lepidopteran Phthorimaea operculella produce nearly identical profiles with only minor differences. Finally, a statistical analysis based on Nei’s similarity coefficient was performed on the fingerprints of four independent cell lines from the Colorado potato beetle, L. decemlineata. Each possesses a common recognizable pattern also found in field-collected insects, while also showing a series of polymorphic markers which allow one to distinguish each cell line from the three others. RAPD fingerprinting, together with the use of appropriate statistics, thus constitutes a highly specific method both for the authentication of the species from which a cell line was developed and for the individual characterization of each cell line from a given species.

Resistance to the Cry1Ac δ-Endotoxin of Bacillus thuringiensis in the Cotton Bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

Three laboratory strains of Helicoverpa armigera (Hübner) were established by mating of field-collected insects with an existing insecticide-susceptible laboratory strain. These strains were cultured on artificial diet containing the Cry1Ac protoxin of Bacillus thuringiensis using three different protocols. When no response to selection was detected after 7–11 generations of selection, the three strains were combined by controlled mating to preserve genetic diversity. The composite strain (BX) was selected on the basis of growth rate on artificial diet containing Cry1Ac crystals. Resistance to Cry1Ac was first detected after 16 generations of continuous selection. The resistance ratio (RR) peaked approximately 300-fold at generation 21, after which it declined to oscillate between 57- and 111-fold. First-instar H. armigera from generation 25 (RR = 63) were able to complete their larval development on transgenic cotton expressing Cry1Ac and produce fertile adults. There appeared to be a fitness cost associated with resistance on cotton and on artificial diet. The BX strain was not resistant to the commercial Bt spray formulations DiPel and XenTari, which contain multiple insecticidal crystal proteins, but was resistant to the MVP formulation, which only contains Cry1Ac. The strain was also resistant to Cry1Ab but not to Cry2Aa or Cry2Ab. Toxin binding assays showed that the resistant insects lacked the high affinity binding site that was detected in early generations of the strain. Genetic analysis confirmed that resistance in the BX strain of H. armigera is incompletely recessive.

Resistance to the Cry1Ac delta-endotoxin of Bacillus thuringiensis in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae).

Three laboratory strains of Helicoverpa armigera (Hübner) were established by mating of field-collected insects with an existing insecticide-susceptible laboratory strain. These strains were cultured on artificial diet containing the Cry1Ac protoxin of Bacillus thuringiensis using three different protocols. When no response to selection was detected after 7-11 generations of selection, the three strains were combined by controlled mating to preserve genetic diversity. The composite strain (BX) was selected on the basis of growth rate on artificial diet containing Cry1Ac crystals. Resistance to Cry1Ac was first detected after 16 generations of continuous selection. The resistance ratio (RR) peaked approximately 300-fold at generation 21, after which it declined to oscillate between 57- and 111-fold. First-instar H. armigera from generation 25 (RR = 63) were able to complete their larval development on transgenic cotton expressing Cry1Ac and produce fertile adults. There appeared to be a fitness cost associated with resistance on cotton and on artificial diet. The BX strain was not resistant to the commercial Bt spray formulations DiPel and XenTari, which contain multiple insecticidal crystal proteins, but was resistant to the MVP formulation, which only contains Cry1Ac. The strain was also resistant to Cry1Ab but not to Cry2Aa or Cry2Ab. Toxin binding assays showed that the resistant insects lacked the high affinity binding site that was detected in early generations of the strain. Genetic analysis confirmed that resistance in the BX strain of H. armigera is incompletely recessive.

Detection and diversity assessment of Xylella fastidiosa in field-collected plant and insect samples by using 16S rRNA and gyrB sequences.

The causal agent of diseases in many economically important plants is attributed to the xylem-limited bacterium Xylella fastidiosa. The detection of this plant pathogen has been hampered due to its difficult isolation and slow growth on plates. Nearly complete nucleotide sequences of the 16S rRNA gene and partial sequences of the gyrB gene were determined for 18 strains of X. fastidiosa isolated from different plant hosts. A phylogenetic analysis, based on gyrB, grouped strains in three clusters; grape-isolated strains formed one cluster, citrus-coffee strains formed another cluster, and a third cluster resulted from all other strains. Primer pairs designed for the 16S rRNA and gyrB genes were extensively searched in databases to verify their in silico specificity. Primer pairs were certified with 30 target and 36 nontarget pure cultures of microorganisms, confirming 100% specificity. A multiplex PCR protocol was developed and its sensitivity tested. Sequencing of PCR products confirmed the validity of the multiplex PCR. Xylella fastidiosa was detected in field-collected plants, disease vector insects, and nonsymptomatic but infected plants. Specific detection of X. fastidiosa may facilitate the understanding of its ecological significance and prevention of spread of the disease.

Transmission of Xylella fastidiosa to grapevines by Homalodisca coagulata (Hemiptera: Cicadellidae).

Pierce's disease (PD) of grapevines is caused by a xylem-limited bacterium Xylella fastidiosa (Wells, Raju, Hung, Weisburg, Mandelco-Paul, and Brenner) that is transmitted to plants by xylem sap-feeding insects. The introduction of the sharpshooter leafhopper Homalodisca coagulata (Say) into California has initiated new PD epidemics in southern California. In laboratory experiments, the major characteristics of H. coagulata's transmission of X. fastidiosa to grapevines were the same as reported for other vectors: short or absent latent period; nymphs transmitted but lost infectivity after molting and regained infectivity after feeding on infected plants; and infectivity persisted in adults. Adult H. coagulata acquired and inoculated X. fastidiosa in <1 h of access time on a plant. Inoculation rates increased with access time, but acquisition efficiency (20% per individual) did not increase significantly beyond 6-h access. Estimated inoculation efficiency per individual per day was 19.6, 17.9, and 10.3% for experiments where plant access was 1, 2, and 4 d, respectively. Freshly molted adults and nymphs acquired and transmitted X. fastidiosa more efficiently than did older, field-collected insects. H. coagulata transmitted X. fastidiosa to 2-yr-old woody tissues of grapevines as efficiently as to green shoots. H. coagulata transmitted X. fastidiosa 3.5 mo after acquisition, demonstrating persistence of infectivity in adults. About half (14/29) of the H. coagulata from which we failed to culture X. fostidiosa from homogenized heads (with a detection threshold of 265 CFU/head) transmitted the pathogen to grape, and 17 of 24 from which we cultured X. fastidiosa transmitted.

Frequency and Seasonal Distribution of Pear Psylla Infected with the Pear Decline Phytoplasma in California Pear Orchards

ABSTRACT Pear decline (PD) is an important disease of Pyrus communis fruiting cultivars in Europe, Asia, and the Americas. PD is caused by a phloem-limited phytoplasma that, in California, is transmitted from diseased to healthy trees by pear psylla, Cacopsylla pyricola. The percentage of phytoplasma-infected pear psylla has never been assessed in the United States in field-collected insects. Pear psylla were collected monthly from PD-infected trees from three orchards in northern California. Individual psylla were tested for the presence of PD phytoplasma, using both a quantitative DNA hybridization and PD phytoplasma-specific polymerase chain reaction (PCR) assays. The percentage of infected psylla ranged from 0 to 45% depending on the orchard, the month and year collected, and the method of detection. The PD phytoplasma was detected in both the winterform and summerform pear psylla. Significantly more infected psylla were detected with PCR than with DNA hybridization analysis in two of the three orchards. The number of PD phytoplasma per pear psylla was estimated to range from 1 x 10(6) to 8.2 x 10(7). The percentage of PD-infected pear psylla found in the three northern California pear orchards suggests that both winterform and summerform pear psylla could be important in the transmission of PD.

Development, survival, body weight, longevity, and reproductive potential of Oemena hirta (Coleoptera: Cerambycidae) under different rearing conditions.

Oemona hirta (F.) is a New Zealand native longicorn beetle, whose larvae bore into the wood of branches and stems of living trees and vines, causing serious damage. To explore effective methods for maintaining laboratory colonies and biology of immature stages of this species we evaluated four laboratory rearing methods with both natural and artificial diets and compared biological parameters of laboratory colonies with those of field-collected insects. On an artificial diet, approximately 40% of neonate and 70% of autumn- and 11% of winter-collected larvae reached adulthood. Neonate larvae could not complete their development in cut poplar (Populus nigra variety italica Koehne) twigs; however, when twigs were standing in water >46% of neonate larvae survived to adulthood. Mean larval development time ranged from approximately150 to almost 300 d, depending on rearing methods. Mean pupal stage ranged from 15 to 19 d. Adult females were significantly heavier than males. Although adult females from field-collected twigs and reared on the artificial diet had similar mean body weight, which was significantly greater than that of other rearing colonies, the potential fecundity was the highest in the former (83.1 +/- 29.4 eggs) and the lowest in the latter (33.5 +/- 9.1 eggs). In all laboratory-reared and field-collected insects, the total number of eggs produced (eggs laid + eggs that remained in dead female body) by females was positively correlated with their body weight. When larvae fed on the artificial diet, there was no positive correlation between the number of eggs successfully laid and female body weight. However, when larvae lived on natural food, a positive correlation was found. In laboratory colonies, mean longevity of females (36 to 52 d) was slightly greater than that of males (30 to 50 d) without significant difference between sexes but in the adults from field-collected twigs, males (52 d) lived significantly longer than females (33 d). In terms of time, labor, and the number of resulting adults, collecting larvae in the field in autumn and then transferring them onto artificial diet is the most effective method for maintaining a laboratory colony.

Prevailing triple infection with Wolbachia in Callosobruchus chinensis (Coleoptera: Bruchidae).

Prevailing triple infection with three distinct Wolbachia strains was identified in Japanese populations of the adzuki bean beetle, Callosobruchus chinensis. When a polymerase chain reaction (PCR) assay was conducted using universal primers for ftsZ and wsp, Wolbachia was detected in all the individuals examined, 288 males and 334 females from nine Japanese populations. PCR-restriction fragment length polymorphism (RFLP) analysis of cloned wsp gene fragments from single insects revealed that three types of wsp sequences coexist in the insects. Molecular phylogenetic analysis of the wsp sequences unequivocally demonstrated that C. chinensis harbours three phylogenetically distinct Wolbachia, tentatively designated as wBruCon, wBruOri and wBruAus, respectively. Diagnostic PCR analysis using specific primers demonstrated that, of 175 males and 235 females from nine local populations, infection frequencies with wBruCon, wBruOri and wBruAus were 100%, 96.3% and 97.0%, respectively. As for the infection status of individuals, triple infection (93.7%) dominated over double infection (6.1%) and single infection (0.2%). The amounts of wBruCon, wBruOri and wBruAus in field-collected adult insects were analysed by using a quantitative PCR technique in terms of wsp gene copies per individual insect. Irrespective of original populations, wBruCon and wBruOri (107 -108 wsp copies/insect) were consistently greater in amount than wBruAus (106 -107 wsp copies/insect), suggesting that the population sizes of the three Wolbachia strains are controlled, although the mechanism is unknown. Mating experiments suggested that the three Wolbachia cause cytoplasmic incompatibility at different levels of intensity.

Thrombostasin: purification, molecular cloning and expression of a novel anti-thrombin protein from horn fly saliva

Thrombostasin (TS), a novel protein found in the saliva of Haematobia irritans (horn fly), was purified by high-performance liquid chromatography from the saliva of field-collected insects. This protein, which inhibits thrombin, accounts for anti-clotting activity in horn fly saliva [J. Med. Entomol. 37 (2000) 416] and is the first purified anti-hemostatic factor described from the Stomoxyinae, a large group of blood-feeding insects that are major pests of livestock world-wide. The purified TS had an apparent molecular weight of 16.7 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and revealed two isoelectric groups with isoelectric points (p Is) of approximately 4.6 and 4.8. Mass spectroscopy analysis, however, resulted in at least three major isoforms that range in mass from 9213 to 9274 Da. A 243-bp coding sequence was obtained from cDNA by using a degenerate primer deduced from the N-terminal sequence of the purified TS. The conceptual translation of the 243-bp sequence showed that the 81-amino-acid peptide, whose first 30 amino acids match those of the N-terminal sequence, had a predicted mass of 9213 Da with p I 4.14. A full-length TS cDNA was generated by rapid amplification of cDNA ends of the 5′ and sequential polymerase chain reaction (PCR) amplification. It contained a 5′-end 12-bp segment preceding the putative ATG start codon, followed by a 54-bp sequence corresponding to a secretory signal and an additional 228-bp coding sequence preceding residues revealed by N-terminal sequencing of purified TS. The fidelity of the PCR-generated TS sequence was confirmed in genomic DNA and by biological activity of recombinant TS produced in a baculovirus expression system. Database comparisons revealed no homology between TS and other known molecules. Because of the paucity of other anti-hemostatic factors in horn fly saliva, TS may play a critical role in maintenance of the ectoparasitic lifestyle of horn flies.

Purification and characterization of the western spruce budworm larval midgut proteinases and comparison of gut activities of laboratory-reared and field-collected insects

Three proteolytic enzymes, trypsin, chymotrypsin, and aminopeptidase-N (APN), were purified from laboratory-reared western spruce budworm, Choristoneura occidentalis [Freeman], larvae. Budworm trypsin exhibited a high degree of substrate specificity, was inactivated by DFP and TLCK, and was inhibited by trypsin inhibitors. The western spruce budworm chymotrypsin hydrolyzed SAAPFpNA and SAAPLpNA, but not SFpNA, SGGFpNA, SGGLpNA or BTpNA. The chymotrypsin was inactivated by DFP, and was inhibited by chymostatin and the chymotrypsin inhibitor, POT-1. Purified budworm chymotrypsin exhibited little BTEE esterolytic activity and was insensitive to inhibition with TPCK. The N-terminal sequence of budworm trypsin, chymotrypsin, and APN were obtained. Similar levels of trypsin and APN gut activities were found in laboratory-reared and field-collected larvae. However, in comparison to laboratory-reared insects, considerably less chymotrypsin activity, and a much higher level of gut carboxypeptidase activity were found in field-collected western spruce budworm larvae.

General Program for Determining Instar Distributions from Headcapsule Widths: Example Analysis of Mountain Pine Beetle (Coleoptera: Scolytide) Data

Determining instar distributions from field-collected insect samples is a common problem in insect ecology. We describe a generalized computer program (HCAP) that determines an optimum separation rule based on the distribution of headcapsule widths. The program is initiated through visual interaction with the user to determine starting points for separation rules. HCAP then determines the optimum instar classification rules; estimates of means and standard deviations of headcapsule widths for each instar; estimated number in each instar; and probabilities of misclassification. Application of the program is illustrated by the analysis of 3 data sets of >10,000 observations of headcapsule widths obtained from field collections of larvae of the mountain pine beetle, Dendroctonus ponderosae Hopkins. This analysis resulted in finding significant differences between larvae collected from different hosts (ponderosa pine, Pinus ponderosae Lawson, and lodgepole pine, Pinus contorta Douglas variety latifolia Engelmann) and ecological circumstances (outbreak phase and geographic location). These results indicate that caution must be taken when extrapolating published results to new data or from one ecological situation to another.

F2 Screen for Rare Resistance Alleles

The refuge plus high-dose resistance management strategy for transgenic crops producing Bacillus thuringiensis Berliner crystal protein toxins (Bt crops) assumes that resistance is rare and functionally recessive. We propose an F 2 screening procedure to estimate the frequency of rare resistance alleles in natural insect populations and acquire them for further testing. The procedure preserves genetic variation in isofemale lines and concentrates the resistance alleles in homozygous genotypes where they can be detected, whether they are recessive or dominant. Bayesian statistics for estimating allele frequency and credibility intervals, and the probability that the screen will not detect a resistance allele are derived. Compared with other methods for detecting and estimating resistance alleles, the F 2 screen will be efficient and accurate, extend the sensitivity of allele-frequency estimation for recessive traits by more than an order of magnitude compared with a discriminating-dose assay, and detect all resistance traits segregating in the initial sample of field-collected insects rather than a subset that responds most rapidly to laboratory selection.

Transmission ofSerratia marcescens(Enterobacteriaceae) in AdultHeliothis virescens(Lepidoptera: Noctuidae) Laboratory Colonies

The red strain ofSerratia marcescens(Bizio) has been isolated from healthy, diseased, or dead field-collected insects, but it is most frequently reported as a pathogen of insectary-reared insects. In this study, we determined the path of transmission ofS. marcescensin adultHeliothis virescens(F.) to the next generation and compared longevity of infected and uninfected adults. Our results show thatS. marcescenscan be present on or within eggs produced byS. marcescens-infected pairs. Highest external contamination and/or internal infection of surface sterilized eggs from moths fed an aqueous solution of 10% honey contaminated withS. marcescens(4 × 108bacterial cells/ml) was 45% and internal infection of eggs from the same group of moths was 19%. The highest percentage of adults producing eggs contaminated and/or infected withS. marcescenswas 70% and the highest percentage producing internally infected eggs was 17%. The highest percentage of total adult mortality at day 10, in bacterial treatments, was 90.1% and in the controls was 15%. Living infected moths are usually asymptomatic, but typically become red postmortem. We demonstrated thatS. marcescenscan be transmitted both vertically (genetic transmission; the passage of pathogen from one host generation to the next in or on egg) and horizontally (transmission of pathogen between insects at any age after birth) inH. virescens,and that the bacterium has detrimental effects on adult longevity. We point out in this paper thatS. marcescenshas been frequently reported from poor quality insectary-rearedH. virescens(poor vigor and high mortality). This insect which is commonly used for a host of beneficial endoparasitoids, production of viruses and protozoa, and testing various biological control agents, must be pathogen free (such asS. marcescens) to provide quality biological control agents or meaningful data.

Behavioral Response of Podisus maculiventris (Hemiptera: Pentatomidae) to its Synthetic Pheromone

A wind-tunnel bioassay was used to determine the effect of age and feeding history (starvation) on the response of Podisus maculiventris (Say) to its synthetic pheromone. Starved male and female adults showed positive anemotaxis toward the pheromone source; well-fed insects did not. This supports the hypothesis that P. maculiventris adults use the pheromone as a cue indicating presence of prey, in addition to a mating cue, although a physiological explanation for the lack of response by well-fed insects is also possible. In the presence of the pheromone, fed as well as starved insects increased activities such as extending antennae upwind; fluttering wings; and rubbing antennae, proboscis, forelegs, hindlegs, and abdomen. These activities may indicate stimulation of olfactory receptors on antennae and contact chemoreceptors elsewhere on the body. First- to third-generation offspring of field-collected P. maculiventris showed greater response to the synthetic pheromone compared with individuals from a 2-year-old laboratory colony, indicating the importance of using field-collected insects in behavioral studies. Fifth instars did not respond to the synthetic pheromone in the wind tunnel.

Baseline Susceptibility and Changes in Susceptibility to Bacillus thuringiensis subsp. kurstaki Under Selection Pressure in European Corn Borer (Lepidoptera: Pyralidae)

Baseline susceptibility of European corn borer larvae, Ostrinia nubilalis (Hubner), to a commercial formulation of Bacillus thuringiensis Berliner Subsp. kurstaki (Dipel ES), was determined for colonies from 3 different geographic locations. The KS-NE colony, collected near Manhattan in northeast Kansas, was more tolerant to Dipel than those collected from near St. John in south central Kansas (KS-SC colony) and colonies provided by the USDA Corn Insects Laboratory in Ames, IA (Iowa colony). Five colonies of European corn borers were exposed to laboratory selection using a meridic diet incorporating Dipel ES. Decreasing susceptibility of European corn borer to Dipel ES was observed in each colony selected. In the colony collected in south central Kansas (KS-SC), the LC50 and survival of larvae at specific concentrations increased more rapidly than it did in the other colonies. After only 3 selected generations, the LC50 of this colony increased 35.8-fold, and after 7 selected generations, it reached 72.9-fold. The LC50s of the 3 Iowa colonies increased 25.0- to 35.2-fold after 9-14 selected generations. The LC50 of the colony collected in northeast Kansas increased 16.2-fold after 4 selected generations and it reached 35-fold after 6 selected generations. This indicates that European corn borer populations can respond rapidly to intense selection pressure with a commercial formulation of Bacillus thuringiensis . The LC90s of the 5 colonies also increased significantly in a pattern similar to that observed in the LC50s, but the increases in LC90s were not as large as the increases for the LC50s. The bioassay data suggest that a dose of ≈2.4 ml Dipel per kilogram of diet could be used as a preliminary discriminating dose. Neonates from field-collected insects could be placed on diets incorporating this concentration of Dipel to monitor for changes and regional differences in susceptibility among field populations of European corn borer. Studies on the genetic basis, the mechanism of action, and the stability over time (in the absence of selection pressure) of this change in susceptibility remain to be carried out.

Response of xylem-feeding leafhopper to host plant species and plant quality

Carneocephala floridana, an oligophagous leafhopper that inhabits the salt marshes along the coasts of Florida, utilizesBorrichia frutescens andSalicornia virginica (both herbs) as primary summer hosts, but uses two grasses,Distichlis spicata andSpartina alterniflora, during the winter. We tested whether the seasonal patterns of abundance and apparent host-switching byCarneocephala are related to plant quality. In laboratory experiments, nymphs ofCarneocephala reared on nonfertilized control plants of the two herbs produced adults that were similar in size to field-collected insects. OnlyCarneocephala raised at the lowest densities onSpartina andDistichlis from the highest fertilizer treatments produced adults similar in body mass to those reared on nonfertilizedBorrichia andSalicornia. ForDistichlis, superior quality (high foliar nitrogen) plants were able to mitigate the negative effect of nymphal crowding on adult body mass. However, laboratory fertilization regimes produced an extremely high foliar nitrogen content in the two herbs and the organic acid concentration in the xylem fluid ofBorrichia, the only host species suitable for xylem fluid extraction, increased 2.5- to 3-fold. Total amino acid concentration in the xylem fluid of fertilizedBorrichia decreased compared to nonfertilized plants.Carneocephala demonstrated reduced feeding efficiencies on high nitrogenBorrichia. Our results suggest thatCarneocephala prefers, and performs better on, plants with high nitrogen content up to a threshold, beyond which high nitrogen levels result in reduced leafhopper feeding rates and assimilation efficiencies.

Distinguishing Fall Armyworm (Lepidoptera: Noctuidae) Strains Using a Diagnostic Mitochondrial DNA Marker

The fall armyworm, Spodoptera frugiperda (J. E. Smith), includes morphologically indistinguishable corn and rice strains. The two strains were surveyed for diagnostic restriction patterns in mitochondrial DNA (mtDNA) using 25 restriction endonucleases. Polymorphic mtDNA restriction patterns were identified for BstNI, HinfI and MspI. The MspI pattern was the most distinctive since the molecular size of each DNA fragment differed between the two strains. Analyses of laboratory and field-collected insects showed the MspI mtDNA pattern to be a diagnostic marker for corn and rice strain insects. Strain identification by the MspI mtDNA profile correlated exactly with nuclear DNA markers. Since no HaeIII sites are present in fall armyworm mtDNA, a double-digest of total fall armyworm DNA using HaeIII and MspI allowed the direct detection of mtDNA restriction fragments from total DNA on a stained agarose gel. In contrast to conventional techniques utilizing mtDNA markers, this rapid and simple procedure does not require the isolation of mtDNA, or avoids the use of DNA blots and labeled mtDNA.