Juan D. López

Bacillus thuringiensis-toxin resistance management: Stable isotope assessment of alternate host use by Helicoverpa zea

Data have been lacking on the proportion of Helicovera zea larvae that develop on noncotton host plants that can serve as a refuge from selection pressure for adaptation to transgenic cotton varieties that produce a toxin from the bacterium Bacillus thuringiensis. We found that individual H. zea moths that develop as larvae on cotton and other plants with C3 physiology have a different ratio of 13C to 12C than moths that develop on plants with C4 physiology, such as corn. We used this finding in determining the minimum percentage of moths that developed on noncotton hosts in two cotton-growing areas. Our results indicate that local corn can serve as a refuge for H. zea in midsummer. Our results contrast dramatically with the prevailing hypothesis that the large majority of late-season moths are produced from larvae feeding on cotton, soybean, and other C3 plants. Typically, <50% of moths captured in August through October have isotope ratios indicative of larval feeding on C3 plants. In one October sample, 100% of the moths originated from C4 hosts even though C4 crops were harvested at least 1 mo earlier, and no common wild C4 hosts were available. These findings support other research indicating that many late-season H. zea moths captured in Louisiana and Texas are migrants whose larvae developed on corn in more northern locations. Our isotope data on moths collected in Texas early in the season indicate that the majority of overwintering H. zea do not originate from cotton-feeding larvae and may be migrants from Mexico. Non-Bt corn in Mexico and the U.S. corn belt appears to serve as an important refuge for H. zea.

Enhanced in vivo activity of peptidase-resistant analogs of the insect kinin neuropeptide family

The diuretic/myotropic insect kinin neuropeptides, which share the common C-terminal pentapeptide core FX(1)X(2)WG-NH(2), reveal primary (X(2)-W) and secondary (N-terminal to F) sites of susceptibility to peptidases bound to corn earworm (H. zea) Malpighian tubule tissue. Analogs designed to enhance resistance to tissue-bound peptidases, and pure insect neprilysin and ACE, demonstrate markedly enhanced in vivo activity in a weight gain inhibition assay in H. zea, and strong in vivo diuretic activity in the housefly (M. domestica). The peptidase-resistant insect kinin analog pQK(pQ)FF[Aib]WG-NH(2) demonstrates a longer internal residence time in the housefly than the native muscakinin (MK), and despite a difference of over 4 orders of magnitude in an in vitro Malpighian tubule fluid secretion assay, is equipotent with MK in an in vivo housefly diuretic assay. Aminohexanoic acid (Ahx) is shown to function as a surrogate for N-terminal Lys, while at the same time providing enhanced resistance to aminopeptidase attack. Peptidaese-resistant insect kinin analogs demonstrate enhanced inhibition of weight gain in larvae of the agriculturally destructive corn earworm moth. Potent peptidase resistant analogs of the insect kinins, coupled with an increased understanding of related regulatory factors, offer promise in the development of new, environmentally friendly pest insect control measures.

Identification and Comparison of Fall Armyworm (Lepidoptera: Noctuidae) Host Strains in Brazil, Texas, and Florida

Abstract Fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is a major economic pest throughout the Western Hemisphere. Studies of populations in the southern United States and the Caribbean demonstrated the existence of two morphologically identical but genetically distinct host strains. These races can be distinguished by using polymorphisms in the mitochondrial cytochrome oxidase I gene that define two distinct maternal lineages that correlate with strain-specific behaviors in Florida populations. Although there is evidence of different biotypes in Brazil, it has not been demonstrated that these biotypes are equivalent to the U.S. strains. Sampling from Brazil indicates that its fall armyworm populations consist of the two strain-specific haplotypes found in Florida and also display the expected biases in plant host distribution. The same genetic markers also were present in samples from Texas, a major source of migrating fall armyworm in North America. These results indicate that the biology and behaviors of Brazilian fall armyworm populations are similar to those found in North America.

Comparative peptidomics of four related hemipteran species: Pyrokinins, myosuppressin, corazonin, adipokinetic hormone, sNPF, and periviscerokinins

We performed the first comprehensive peptidomic analysis of neurohormones from hemipteran insects by analyzing the neuropeptides of two major neurohemal organs, namely the corpora cardiaca and abdominal perisympathetic organs. For the experiments we selected four related species of polyphagous stinkbugs (Pentatomidae), three of which are known to attack several important food crops. Peptide sequences were identified by MALDI-TOF mass spectrometry; tandem fragmentation of myosuppressin, sNPF, CAPA-periviscerokinins and pyrokinins revealed novel sequences not known from other insects so far. Most Leu/Ile and Glu/Lys ambiguities could be solved by either specific side-chain fragmentations or on-plate acetylation experiments. The identification of the specific sequences provides a solid basis for forthcoming pharmacological tests to study the neuroendocrine system of these pest insects. However, it should be mentioned in this context that the sequences of the peptides from different stinkbugs are likely not representative of Hemiptera in general. The forthcoming release of the genome from the reduviid Rhodnius prolixus will provide sufficient data to clear this point.

A Polymerase Chain Reaction Screen of Field Populations of Heliothis virescens for a Retrotransposon Insertion Conferring Resistance to Bacillus thuringiensis Toxin

Abstract The evolution of pest resistance to transgenic crop plants producing insecticidal toxins from Bacillus thuringiensis (Bt) Berliner poses a continuing threat to their sustainable use in agriculture. One component of the U.S.-wide resistance management plan for Bt cotton, Gossypium hirsutum L., involves monitoring the frequency of resistance alleles in field populations. However, existing methods are expensive and may not detect recessive resistance alleles until their frequencies are too high for countermeasures to be effective; therefore, more sensitive methods are needed. The first Bt resistance-causing mutation described at the molecular level was a retrotransposon insertion into the gene encoding a 12-cadherin-domain protein expressed in the midgut of larval Heliothis virescens (F.). We report the first large-scale screen for this mutation using a polymerase chain reaction (PCR)-based approach on >7,000 field-collected individuals. The specific insertion was not detected in any of these samples, nor was it detected in three progeny-tested, field-caught males thought to carry a Bt resistance gene. Unlike the targets of many chemical insecticides where a limited number of resistance-causing mutations compatible with viability can occur; a very large number of such mutations seem possible for the 12-cadherin-domain gene. However, even if these mutations are viable in the laboratory, they may not threaten the effectiveness of transgenic crops because of a high fitness cost in the field. The challenge remains to detect the subset of possible resistance-conferring alleles that are still rare but are viable in the field and increasing due to selection by Bt cotton. This situation will complicate PCR-based Bt resistance monitoring strategies.

Susceptibility of isofamilies of Spodoptera frugiperda (Lepidoptera: Noctuidae) to Cry1Ac and Cry1Fa proteins of Bacillus thuringiensis.

Abstract. The fall armyworm, Spodoptera frugiperda (J. E. Smith), is one of the most important insect pests on the American continent. Its control has relied primarily on multiple applications of insecticides that can amount to 1,000 g of active ingredient per hectare on some of approximately 30 crops the insect damages. The use of genetically engineered crops that express Bacillus thuringiensis (Bt) Berliner toxins, Bt-corn, Zee meys L.; and Bt-cotton, Gossypium hirsutum L.; are other ways to control this insect. However, fall armyworm is one of the Lepidoptera species least susceptible to Bt proteins, and a case of high tolerance to Bt-corn has already being reported. We found the susceptibility to Cry1Ac and Cry1Fa proteins of Bt in 133 isofamilies from five regions of three countries was similar to the susceptibility of two Bt-susceptible laboratory colonies to these proteins. Four isofamilies from Puerto Rico were very tolerant to Cry1Fa and not so tolerant to Cry1Ac. Two of the four isofamilies were backcrossed with a Bt-susceptible laboratory colony and their progeny was as susceptible to both Bt proteins as was the Bt-susceptible colony, indicating that resistance to Bt is a recessive trait.

Using Haplotypes to Monitor the Migration of Fall Armyworm (Lepidoptera: Noctuidae) Corn-Strain Populations from Texas and Florida

Abstract Fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), infestations in most of North America north of Mexico arise from annual migrations of populations that overwinter in southern Texas and Florida. A comparison of the cytochrome oxidase I haplotype profiles within the fall armyworm corn-strain, the subgroup that preferentially infests corn (Zea mays L.) and sorghum (Sorghum vulgare Pers.), identified significant differences in the proportions of certain haplotypes between the Texas and Florida populations. These proportional differences were preserved as the populations migrated, providing a molecular metric by which the source of a migrant population could be identified. The migratory pattern derived from this method for several southeastern states was shown to be consistent with predictions based on analysis of historical agricultural and fall armyworm infestation data. These results demonstrate the utility of haplotype proportions to monitor fall armyworm migration, and they also introduce a potential method to predict the severity of cotton crop infestations in the short term.

Bacillus thuringiensis Cry1Ac resistance frequency in tobacco budworm (Lepidoptera: Noctuidae).

ABSTRACT The tobacco budworm, Heliothis virescens (F.) (Lepidoptera Noctuidae), is one of the most important pests of cotton, Gossypium hirsutum L., that has become resistant to a wide range of synthetic insecticides. Cry1Ac-expressing cotton has proven its effectiveness against this insect since its introduction in North America in 1996. However, the constant exposure of tobacco budworm to this protein toxin may result in the development of resistance to it. To estimate the frequency of alleles that confer resistance to a 1.0 µg of Bacillus thuringiensis Cry1Ac diagnostic concentration in field-collected insects, the second generation (F2) of 1,001 single-pair families from seven geographical regions representing 2,202 alleles from natural populations was screened in 2006 and 2007 without finding major resistant alleles. Neonates of 56 single-pair families were able to develop to second instar on the diagnostic concentration in the initial screen, but only seven of these lines did so again in a second confirmatory screen. Minor resistance alleles to Cry1Ac may be quite common in natural populations of H. virescens. Our estimated resistance allele frequencies (0.0036 – 0.0263) were not significantly different from a previously published estimate from 1993. There is no evidence that H. virescens populations have become more resistant to Cry1Ac.

Neurohormones implicated in the control of Malpighian tubule secretion in plant sucking heteropterans: The stink bugs Acrosternum hilare and Nezara viridula

Plant sucking heteropteran bugs feed regularly on small amounts of K(+)-rich plant material, in contrast to their hematophagous relatives which imbibe large volumes of Na(+)-rich blood. It was anticipated that this would be reflected in the endocrine control of Malpighian tubule (MT) secretion. To explore this, neuroendocrine factors known to influence MT secretion were tested on MT of the pentatomid plant sucking stink bugs, Acrosternum hilare and Nezara viridula, and the results compared with previously published data from Rhodnius prolixus. Serotonin had no effect on N. viridula MT, although it stimulates secretion by R. prolixus MT >1000-fold, and initiates a rapid diuresis to remove excess salt and water from the blood meal. Kinins had no effect on stink bug MT, but secretion was increased by Zoone-DH, a CRF-like peptide, although the response was a modest 2-3-fold acceleration compared with 1000-fold in R. prolixus. Native CAPA peptides, which have diuretic activity in dipteran flies, had antidiuretic activity in MT of the stink bug (Acrhi/Nezvi-CAPA-1 and -2), as previously shown with Rhopr-CAPA-2 in R. prolixus. The antidiuretic activity of Rhopr-CAPA-2 has been linked with terminating the rapid diuresis, but results with stink bugs suggest it is a general feature of heteropteran MT.

The control of Malpighian tubule secretion in a predacious hemipteran insect, the spined soldier bug Podisus maculiventris (Heteroptera, Pentatomidae).

Spined soldier bugs, Podisus maculiventris, are heteropteran insects that feed voraciously on other insects, particular the soft bodied larval forms of Lepidoptera and Coleoptera. The response of P. maculiventris Malpighian tubules (MTs) to serotonin and known diuretic and antidiuretic peptides has been investigated, and is compared with that of MT from the hematophagous and phytophagous heteropteran bugs Rhodnius prolixus and Acrosternum hilare, respectively. A CRF-related peptide diuretic hormone (DH) from the termite Zootermopsis nevadensis (Zoone-DH) stimulated MT secretion, which was reversed by a member of the CAP(2b) family of peptides from A. hilare (Acrhi-CAP(2b)-2), an antidiuretic effect. Serotonin had no effect on secretion, neither did a representative calcitonin-like DH, kinin, tachykinin-related peptide, and an antidiuretic factor from the mealworm Tenebrio molitor (Tenmo-ADFb) in both P. maculiventris or A. hilare. Serotonin is a DH in R. prolixus, and its lack of effect on MT from P. maculiventris and A. hilare suggests this is an adaptation to hematophagy. On the other hand, the antidiuretic activity of members of the CAP(2b) family in all three bugs is consistent with this being a heteropteran feature rather than a specialism for hematophagy.