Spotted Lanternfly (Hemiptera: Fulgoridae) Nymphal Dispersion Patterns and Their Influence on Field Experiments.

The spotted lanternfly, Lycorma delicatula (Hemiptera: Fulgoridae) (White, 1845), is an invasive pest in the Mid-Atlantic region of the United States. Understanding this pest's dispersion patterns is fundamental for development of management and surveillance programs. To address this knowledge gap, we quantified spotted lanternfly nymph dispersion patterns by instar for rural and urban/suburban habitats, and we compared the number of sample units required for sticky traps and in situ visual counts to estimate population densities at several precisions. In addition, we assessed the ability of two experimental designs (completely random and randomized complete block) to detect management practices' impacts in the field. All instars typically followed an aggregated dispersion pattern. Sample size and time requirements for checking and replacing sticky traps and for conducting in situ counts were similar, but in situ counts do not require purchasing traps, installation time, or delays before treatment, and do not remove insects. Although the cost for using in situ counts is likely less than for sticky traps, early instar spotted lanternfly nymph populations are harder to visually detect than later instars because of their small size, which may negate any cost advantage when treatments are applied early. In general, using a randomized complete block design resulted in higher statistical power than a completely random design, allowing detection of proportional population reductions of 10-20% less with equal replication. Studies aiming to evaluate treatments that reduce spotted lanternfly numbers by less than 60% will require researchers to evaluate the feasibility of using the required large sample sizes.

Induction and transmission of tolerance to the synthetic pesticide emamectin benzoate in field and laboratory populations of diamondback moth.

Field surveys of pest insect pest populations in agroecosystems reveal low but significant levels of tolerance to synthetic and biological pesticides but fail to uncover resistance alleles in test crosses. To study the potential of inducible mechanisms to generate tolerance to synthetic pesticides, we performed baseline susceptibility studies in field and laboratory populations of diamondback moth, Plutella xylostella (L.), to commercial formulations of emamectin benzoate. Pesticide exposure in the field caused elevated levels of tolerance, which decreased in field-collected populations after maintaining insects with pesticide-free diet in the laboratory. Because no significant resistance alleles were identified in back-crossed individuals, the observed increase in tolerance was probably not based on preexisting recessive resistance mechanisms in the population. Instead, the genetic analysis after five and 12 generations is compatible with a transient up-regulation of an immune and metabolic status in tolerant insects that can be transmitted to offspring by a maternal effect. Although the epigenetic effects contributed to incremental increases in tolerance in the first five generations, other resistance mechanisms that are transmitted genetically predominate after 12 generations of increased exposure to the pesticide.

Economic, ecological, food safety, and social consequences of the deployment of bt transgenic plants.

Transgenic plants expressing insecticidal proteins from the bacterium, Bacillus thuringiensis (Bt), are revolutionizing agriculture. Bt, which had limited use as a foliar insecticide, has become a major insecticide because genes that produce Bt toxins have been engineered into major crops grown on 11.4 million ha worldwide in 2000. Based on the data collected to date, generally these crops have shown positive economic benefits to growers and reduced the use of other insecticides. The potential ecological and human health consequences of Bt plants, including effects on nontarget organisms, food safety, and the development of resistant insect populations, are being compared for Bt plants and alternative insect management strategies. Scientists do not have full knowledge of the risks and benefits of any insect management strategies. Bt plants were deployed with the expectation that the risks would be lower than current or alternative technologies and that the benefits would be greater. Based on the data to date, these expectations seem valid.

Greenhouse tests on resistance management of Bt transgenic plants using refuge strategies.

Experimental evaluation of the effectiveness of resistance management tactics is vital to help provide guidelines for the deployment of transgenic insecticidal crops. Transgenic broccoli expressing a Cry1Ac gene of Bacillus thuringiensis (Bt) and the diamondback moth, Plutella xylostella (L.), were used in greenhouse tests to evaluate the influence of size and placement of nontransgenic refuge plants on changes in resistance allele frequency and pest population growth. In the first test with an initial Cry1Ac-resistance (R) allele frequency of 0.007, P. xylostella were introduced into cages with the following treatments: 0, 3.3, 10, 20, and 100% refuge plants. Results after four generations showed that resistance could be delayed by increasing the proportion of refuge plants in the cage. Population growth was also influenced by refuge size with the highest populations occurring in treatments that had either no refuge plants or all refuge plants. In the second test, we evaluated the effect of refuge placement by comparing 20% separate and 20% mixed refuges. P. xylostella with an initial frequency of resistant alleles at 0.0125 were introduced into cages and allowed to cycle; later generations were evaluated for resistance and population growth. Separating the refuge had a pronounced effect on delaying resistance and slowing establishment of resistant larvae on Bt plants. Combining information from both trials, we found a strong negative correlation between the number of larvae on Bt plants and the mortality of the population in leaf dip bioassays. Results from larval movement studies showed that separate refuges delayed resistance better than mixed refuges because they conserved relatively more susceptible alleles than R alleles and did not increase the effective dominance of resistance.

Development and characterization of diamondback moth resistance to transgenic broccoli expressing high levels of Cry1C.

A field-collected colony of the diamondback moth, Plutella xylostella, had 31-fold resistance to Cry1C protoxin of Bacillus thuringiensis. After 24 generations of selection with Cry1C protoxin and transgenic broccoli expressing a Cry1C protein, the resistance that developed was high enough that neonates of the resistant strain could complete their entire life cycle on transgenic broccoli expressing high levels of Cry1C. After 26 generations of selection, the resistance ratios of this strain to Cry1C protoxin were 12,400- and 63,100-fold, respectively, for the neonates and second instars by a leaf dip assay. The resistance remained stable until generation 38 (G38) under continuous selection but decreased to 235-fold at G38 when selection ceased at G28. The Cry1C resistance in this strain was seen to be inherited as an autosomal and incompletely recessive factor or factors when evaluated using a leaf dip assay and recessive when evaluated using Cry1C transgenic broccoli. Saturable binding of (125)I-Cry1C was found with brush border membrane vesicles (BBMV) from both susceptible and Cry1C-resistant strains. Significant differences in Cry1C binding to BBMV from the two strains were detected. BBMV from the resistant strain had about sevenfold-lower affinity for Cry1C and threefold-higher binding site concentration than BBMV from the susceptible strain. The overall Cry1C binding affinity was just 2.5-fold higher for BBMV from the susceptible strain than it was for BBMV from the resistant strain. These results suggest that reduced binding is not the major mechanism of resistance to Cry1C.

Field tests on managing resistance to Bt-engineered plants.

Several important crops have been engineered to express toxins of Bacillus thuringiensis (Bt) for insect control. In 1999, US farmers planted nearly 8 million hectares (nearly 20 million acres) of transgenic Bt crops approved by the EPA. Bt-transgenic plants can greatly reduce the use of broader spectrum insecticides, but insect resistance may hinder this technology. Present resistance management strategies rely on a "refuge" composed of non-Bt plants to conserve susceptible alleles. We have used Bt-transgenic broccoli plants and the diamondback moth as a model system to examine resistance management strategies. The higher number of larvae on refuge plants in our field tests indicate that a "separate refuge" will be more effective at conserving susceptible larvae than a "mixed refuge" and would thereby reduce the number of homozygous resistant (RR) offspring. Our field tests also examined the strategy of spraying the refuge to prevent economic loss to the crop while maintaining susceptible alleles in the population. Results indicate that great care must be taken to ensure that refuges, particularly those sprayed with efficacious insecticides, produce adequate numbers of susceptible alleles. Each insect/Bt crop system may have unique management requirements because of the biology of the insect, but our studies validate the need for a refuge. As we learn more about how to refine our present resistance management strategies, it is important to also develop the next generation of technology and implementation strategies.

Distribution of parthenogenesis-inducing symbionts in ovaries and eggs of Aphytis (Hymentoptera: Aphelinidae).

In parasitic Hymenoptera, severe sex ratio distortions are sometimes associated with the presence of symbiotic microorganisms. Symbiont-induced uniparental reproduction is found in several species of the genus Aphytis and is suspected in others. These intracellular bacteria (called Wolbachia) reside within the egg cytoplasm. In order to follow their vertical transmission, the distribution pattern of the symbionts in ovaries and eggs of two uniparental lines (A. lingnanensis and A. yanonensis) was studied by electron, confocal, and two-photon microscopy. The results obtained suggest that Wolbachia move from nurse cells to developing oocytes. In freshly laid eggs, the symbionts are concentrated in the posterior pole, away from the micropyle, and in later stages of embryogenesis they are found surrounding nuclei throughout the embryo.

Effects of [3H]-BIDN, a novel bicyclic dinitrile radioligand for GABA-gated chloride channels of insects and vertebrates.

1. The radiolabelled bicyclic dinitrile, [3H]-3,3-bis-trifluoromethyl-bicyclo[2.2.1]heptane-2,2-dicarbonitrile ([3H]-BIDN), exhibited, specific binding of high affinity to membranes of the southern corn rootworm (Diabrotica undecimpunctata howardi) and other insects. A variety of gamma-aminobutyric acid (GABA) receptor convulsants, including the insecticides heptachlor (IC50, 35 +/- 3 nM) and dieldrin (IC50, 93 +/- 7 nM), displaced [3H]-BIDN from rootworm membranes. When tested at 100 microM, 1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]oct ane(EBOB), 4-t-butyl-2,6,7-trioxa-1-phosphabicy-clo[2.2.2]octane-1-thio ne (TBPS), 1-phenyl-4-t-butyl-2,6,7-trioxabicyclo[2.2.2]octane (TBOB) and picrotoxin failed to displace 50% of [3H]-BIDN binding to rootworm membranes indicating that the bicyclic dinitrile radioligand probes a site distinct from those identified by other convulsant radioligands. 2. Dissociation studies showed that dieldrin, ketoendrin, toxaphene, heptachlor epoxide and alpha and beta endosulphan displace bound [3H]-BIDN from rootworm membranes by a competitive mechanism. 3. Rat brain membranes were also shown to possess a population of saturable, specific [3H]-BIDN binding sites, though of lower affinity than in rootworm and with a different pharmacological profile. Of the insecticidal GABAergic convulsants that displaced [3H]-BIDN from rootworm, cockroach (Periplaneta americana) and rat brain membranes, many were more effective in rootworm. 4. Functional GABA-gated chloride channels of rootworm nervous system and of cockroach nerve and muscle were blocked by BIDN, whereas cockroach neuronal GABA(B) receptors were unaffected. 5. Expression in Xenopus oocytes of either rat brain mRNA, or cDNA-derived RNA encoding a GABA receptor subunit (Rdl) that is expressed widely in the nervous system of Drosophila melanogaster resulted in functional, homo-oligomeric GABA receptors that were blocked by BIDN. Thus, BIDN probes a novel site on GABA-gated Cl- channels to which a number of insecticidally-active molecules bind.

Toxicity of Bacillus thuringiensis Spore and Crystal Protein to Resistant Diamondback Moth (Plutella xylostella).

A colony of Plutella xylostella from crucifer fields in Florida was used in mortality bioassays with HD-1 spore, CryIA(a), CryIA(b), CryIA(c), CryIB, CryIC, CryID, CryIE, or CryIIA. The data revealed high levels of field-evolved resistance to HD-1 spore and all CryIA protoxins and no resistance to CryIB, CryIC, or CryID. CryIE and CryIIA were essentially not toxic. When HD-1 spore was combined 1:1 with protoxin and fed to susceptible larvae, spore synergized the activity of CryIA and CryIC 5- to 8-fold and 1.7-fold, respectively, and did not synergize the mortality of CryIIA. When fed to Florida larvae, spore failed to synergize the activity of all three CryIA protoxins, synergized the activity of CryIC 5.3-fold, and did not synergize the mortality for CryIIA. Binding studies with CryIA(b), CryIB, and CryIC were performed to determine possible mechanisms of resistance. The two techniques used were (i) binding of biotinylated toxin to tissue sections of larval midguts and (ii) binding of biotinylated toxin to brush border membrane vesicles prepared from whole larvae. Both showed dramatically reduced binding of CryIA(b) in resistant larvae compared with that in susceptible larvae but no differences in binding of CryIB or CryIC.

Blocking actions of BIDN, a bicyclic dinitrile convulsant compound, on wild-type and dieldrin-resistant GABA receptor homo-oligomers of Drosophila melanogaster expressed in Xenopus oocytes.

The receptor antagonist actions are described for a novel bicyclic dinitrile compound (BIDN, 3,3-bis-(trifluoromethyl)-bicyclo [2.2.1] heptane-2,2-dicarbonitrile) on a Drosophila melanogaster homo-oligomeric GABA receptor expressed in Xenopus oocytes. BIDN blocked the wild-type form of the receptor in a neither purely competitive, nor purely non-competitive manner, being dependent on the GABA concentration yet insurmountable, and block was independent of the membrane potential. BIDN was found to be less effective against a mutant (A(302) --> S) form of the receptor resistant to dieldrin and picrotoxinin. This cross resistance of dieldrin-resistant receptors to BIDN is of interest in the light of recent findings that BIDN binding to insect membranes is displaced competitively by dieldrin, but not by picrotoxinin.

Drosophila GABA-gated chloride channel: modified [3H]EBOB binding site associated with Ala-->Ser or Gly mutants of Rdl subunit.

The non-competitive blocker site of the GABA-gated chloride ion channel in normal susceptible strains of Drosophila melanogaster and simulans binds 4-n-[3H]propyl-4'-ethynylbicycloorthobenzoate ([3H]EBOB) at specific sites with KdS of 1.6-1.9 nM and BmaxS of 171-181 fmol/mg protein. This specific binding of [3H]EBOB is strongly inhibited by: a large number and variety of insecticidal channel blockers at 20 nM (lindane, alpha-endosulfan, dieldrin, 12-ketoendrin, fipronil, and a representative bicycloorthobenzoate and dithiane) or 200 nM (picrotoxinin); the insecticidal channel activators avermectin and moxidectin at 20 nM; muscimol at 30 microM and GABA at 300 microM. Cyclodiene resistance in D. melanogaster has been attributed to a mutation resulting in an Ala302-->Ser replacement in the Rdl GABA receptor subunit and in D. simulans to an homologous Ala-->Ser or Gly replacement. These mutations are shown here to greatly reduce [3H]EBOB binding, i.e. lower affinity and apparent number of binding sites. The Ala-->Ser replacement with both melanogaster and simulans almost always reduces the potency in inhibiting [3H]EBOB binding of each of eight channel blockers and of muscimol and GABA. The Ala-->Gly replacement in D. simulans is generally less effective than the Ala-->Ser modification in reducing sensitivity to the channel blockers and to muscimol and GABA. The channel activators avermectin and moxidectin usually retain their inhibitory potency in the Rdl subunit mutants. Thus, it appears that replacement of Ala by Ser generally modifies the non-competitive blocker site and its coupling to the GABA-recognition site with less effect on the channel activator site. In contrast, the Ala-->Gly replacement has less impact in protecting the chloride channel from the action of insecticidal blockers. Each of the resistant strains has the same level of resistance to the lethal action of the five channel blockers examined but none to avermectins and muscimol.

Noninvolvement of the long terminal repeat of transposable element 17.6 in insecticide resistance in Drosophila.

Waters and colleagues recently suggested [Waters, L. C., Zelhof, A. C., Shaw, B. J. & Ch'ang, L.-Y. (1992) Proc. Natl. Acad. Sci. USA 89, 4855-4859] that an insertion of a long terminal repeat of transposable element 17.6 into the 3' untranslated region of a P450 gene leads to susceptibility to the insecticide DDT in Drosophila melanogaster. We tested this hypothesis by screening lines from around the world and found that the presence or absence of a long terminal repeat was uncorrelated with resistance in 31 strains of D. melanogaster and Drosophila simulans. Thus we must reject the hypothesis that the insertion of a long terminal repeat leads to DDT susceptibility in Drosophila.

Occurrence, genetics and management of insecticide resistance.

A lot has been learned about insecticide resistance in the past 40 years. The problem is more extensive and widespread than could have been predicted. In this review, Richard Roush discusses the genetics and management of resistance to insecticides (especially as related to arthropod vectors of human and animal diseases), with the objective of suggesting future directions.

The reconstruction and expression of a Bacillus thuringiensis cryIIIA gene in protoplasts and potato plants.

A Bacillus thuringiensis (B.t.) cryIIIA delta-endotoxin gene was designed for optimal expression in plants. The modified cry gene has the codon usage pattern of an average dicot gene and does not contain AT-rich nucleotide sequences typical of native B.t. cry genes. We assembled the 1.8 kb cryIIIA gene in nine blocks of three oligonucleotide pairs. For two DNA blocks, the polymerase chain reaction was used to enrich for correctly ligated pairs. We compared modified cryIIIA gene with native gene expression by electroporation of dicot (carrot) and monocot (corn) protoplasts. CryIIIA-specific RNA and protein was detected in carrot and corn protoplasts only after electroporation with the rebuilt gene. Transgenic potato lines were generated containing the redesigned cryIIIA gene under the transcriptional control of a chimeric CaMV 35S/mannopine synthetase (Mac) promoter. Out of 63 transgenic potato lines, 58 controlled first-instar Colorado potato beetle (CPB) larvae in bioassays. Egg masses which produced ca. 250,000 CPB larvae were placed on replicate clones of 56 transgenic potatoes. No CPB larvae developed past the second instar on any of these plants. Plants expressing high levels of delta-endotoxin were identified by their toxicity to more resistant third-instar larvae. We show there was good correlation between insect control and the levels of delta-endotoxin RNA and protein.

A single-amino acid substitution in a gamma-aminobutyric acid subtype A receptor locus is associated with cyclodiene insecticide resistance in Drosophila populations.

Resistance to cyclodiene insecticides, documented in at least 277 species, is perhaps the most common kind of resistance to any pesticide. By using cyclodiene resistance to localize the responsible gene, a gamma-aminobutyric acid type A receptor/chloride ion-channel gene was previously cloned and sequenced from an insecticide-susceptible Drosophila melanogaster strain. We now describe the molecular genetics of the resistance allele. A single-base-pair mutation, causing a single-amino acid substitution (Ala-->Ser) within the second membrane-spanning region of the channel, was found to be the only consistent difference between resistant and susceptible strains of D. melanogaster. Some resistant strains of Drosophila simulans show the same mutation, whereas others show an alternative single-base-pair mutation in the same codon, resulting in the substitution of a different amino acid (glycine). These constitute single-box-pair mutations in insects that confer high levels of resistance to insecticides. The presence of the resistance mutations was then tested in a much larger set of strains by the PCR and subsequent digestion with a diagnostic restriction endonuclease. Both resistance-associated mutations cause the loss of a Hae II site. This site was invariably present in 122 susceptible strains but absent in 58 resistant lines of the two species sampled from five continents. PCR/restriction endonuclease treatment was also used to examine linkage of an EcoRI polymorphism in a neighboring intron in D. melanogaster, which was found associated with resistance in all but 3 of 48 strains examined. These PCR-based techniques are widely applicable to examination of the uniqueness of different resistance alleles in widespread populations, the identification of resistance mechanisms in different species, and the determination of resistance frequencies in monitoring.

Slowing the spread of anthelmintic resistant nematodes of sheep and goats in the United Kingdom.

Anthelmintic resistant nematodes pose an increasing threat to animal welfare and lamb production on lowland sheep farms in the United Kingdom. Based on published information on anthelmintic resistance in nematodes and insecticide resistance in arthropods, seven recommendations are made for reducing the development and spread of anthelmintic resistant nematodes. The two most important are probably to prevent the introduction of resistant worms through the purchase of stock and to reduce the reliance on frequent anthelmintic treatments by using epidemiological principles of nematode control.

Reduced neuronal sensitivity to dieldrin and picrotoxinin in a cyclodiene-resistant strain of Drosophila melanogaster (Meigen).

Toxicological and neurophysiological studies were performed to characterize the resistance mechanism in a cyclodiene-resistant strain of Drosophila melanogaster (Maryland strain). Dieldrin had an LC50 of 0.058 ppm against the larvae of susceptible D. melanogaster (Oregon-R wild type) when formulated in the rearing media. The LC50 of the resistant Maryland strain was 10.8 ppm, giving a resistance ratio (LC50-Maryland/LC50-susceptible) of 186-fold. Suction electrode recordings were made from peripheral nerves of the larval central nervous system to test whether reduced nerve sensitivity played any role in the observed resistance. In susceptible preparations (n = 5), inhibition of nerve firing by 1 mM gamma-aminobutyric acid (GABA) was effectively antagonized within 3-10 min by 10 microM dieldrin. In contrast, 30 min incubations with 10 microM dieldrin had no effect on preparations from cyclodiene-resistant individuals (n = 5). Similarly, 10 microM picrotoxinin blocked GABA-dependent inhibition in susceptible nerve preparations (n = 3). In recordings from resistant insects (n = 4), picrotoxinin displayed either weak antagonism of GABA or hyperexcitation indistinguishable from susceptible preparations. These results demonstrate that cyclodiene resistance in the Maryland strain of D. melanogaster 1) is expressed in immature stages, 2) is present at the level of the nerve, and 3) extends to picrotoxinin, albeit at a reduced level compared with dieldrin. The possible role of an altered GABA receptor in this resistance is discussed.