Quantitative trait locus mapping and functional genomics of an organophosphate resistance trait in the western corn rootworm, Diabrotica virgifera virgifera.

The western corn rootworm, Diabrotica virgifera virgifera, is an insect pest of corn and population suppression with chemical insecticides is an important management tool. Traits conferring organophosphate insecticide resistance have increased in frequency amongst D. v. virgifera populations, resulting in the reduced efficacy in many corn-growing regions of the USA. We used comparative functional genomic and quantitative trait locus (QTL) mapping approaches to investigate the genetic basis of D. v. virgifera resistance to the organophosphate methyl-parathion. RNA from adult methyl-parathion resistant and susceptible adults was hybridized to 8331 microarray probes. The results predicted that 11 transcripts were significantly up-regulated in resistant phenotypes, with the most significant (fold increases ≥ 2.43) being an α-esterase-like transcript. Differential expression was validated only for the α-esterase (ST020027A20C03), with 11- to 13-fold greater expression in methyl-parathion resistant adults (P < 0.05). Progeny with a segregating methyl-parathion resistance trait were obtained from a reciprocal backcross design. QTL analyses of high-throughput single nucleotide polymorphism genotype data predicted involvement of a single genome interval. These data suggest that a specific carboyxesterase may function in field-evolved corn rootworm resistance to organophosphates, even though direct linkage between the QTL and this locus could not be established.

Comparison of major biocontrol strains of non-aflatoxigenic Aspergillus flavus for the reduction of aflatoxins and cyclopiazonic acid in maize.

Biological control of toxigenic Aspergillus flavus in maize through competitive displacement by non-aflatoxigenic strains was evaluated in a series of field studies. Four sets of experiments were conducted between 2007 and 2009 to assess the competitiveness of non-aflatoxigenic strains when challenged against toxigenic strains using a pin-bar inoculation technique. In three sets of experiments the non-aflatoxigenic strain K49 effectively displaced toxigenic strains at various concentrations or combinations. The fourth study compared the relative competitiveness of three non-aflatoxigenic strains (K49, NRRL 21882 from Afla-Guard®, and AF36) when challenged on maize against two aflatoxin- and cyclopiazonic acid (CPA)-producing strains (K54 and F3W4). These studies indicate that K49 and NRRL 21882 are superior to AF36 in reducing total aflatoxin contamination. Neither K49 nor NRRL 21882 produce CPA and when challenged with K54 and F3W4, CPA and aflatoxins were reduced by 84-97% and 83-98%, respectively. In contrast, AF36 reduced aflatoxins by 20% with F3W4 and 93% with K54 and showed no reduction in CPA with F3W4 and only a 62% reduction in CPA with K54. Because AF36 produces CPA, high levels of CPA accumulate when maize is inoculated with AF36 alone or in combination with F3W4 or K54. These results indicate that K49 may be equally effective as NRRL 21882 in reducing both aflatoxins and CPA in maize.

Utilization of early soybeans for food and reproduction by the tarnished plant bug (Hemiptera: Miridae) in the delta of Mississippi.

Commercially produced maturity group (MG) IV soybeans, Glycine max L., were sampled during bloom for tarnished plant bugs, Lygus lineolaris (Palisot de Beauvois), during May and June 1999 (3 fields) and 2001 (18 fields). The adults and nymphs were found primarily in single population peaks in both years, indicating a single new generation was produced during each year. The peak mean numbers of nymphs were 0.61 and 0.84 per drop cloth sample in 1999 and 2001, respectively. Adults peaked at 3.96 (1999) and 3.76 (2001) per sweep net sample (25 sweeps). Tests using laboratory-reared and field-collected tarnished plant bugs resulted in very poor survival of nymphs on 16 different soybean varieties (MG III, one; IV, four; V, nine; VI, two). A large cage (0.06 ha) field test found that the number of nymphs produced on eight soybean varieties after mated adults were released into the cages was lower than could be expected on a suitable host. These results indicated that soybean was a marginal host for tarnished plant bugs. However, the numbers of adults and nymphs found in the commercially produced fields sampled in the study may have been high enough to cause feeding damage to the flowering soybeans. The nature of the damage and its possible economic importance were not determined. Reproduction of tarnished plant bugs in the commercially produced early soybean fields showed that the early soybeans provided tarnished plant bugs with a very abundant host at a time when only wild hosts were previously available.

Acephate resistance in populations of the tarnished plant bug (Heteroptera: Miridae) from the Mississippi River Delta.

A monitoring program that used a glass-vial bioassay to detect acephate resistance in populations of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Heteroptera: Miridae), was carried out with weed-collected populations from 20 sites in the delta of Arkansas, Louisiana, and Mississippi. Additional results from field tests using recommended rates of formulated acephate in cotton showed that plant bug populations with resistance ratio (RR50) values > 3.0 for acephate (from the glass-vial bioassay) would be difficult to control in the field. Over a 4-yr-period from 2001 through 2004, only one population tested with the glass-vial bioassay was found with an RR50 value > 3.0 for acephate, but six populations having RR50 values > 3.0 were found in the delta in 2005. In fall 2005, an additional 10 populations from the hill region (the cotton growing areas outside the delta) were tested and four of these populations had RR50 values > 3.0. The number of populations with RR50 values > 3.0 increased to five of 10 and 18 of 20 in the hills and delta, respectively, in fall 2006. Laboratory tests using resistant populations found that resistance to acephate was not sex-linked and the alleles controlling the resistance were semidominant in nature. Because of the large increase in resistant populations and the nature of the resistance found in this study, along with control problems experienced by growers in 2006, entomologists in the mid-South strongly recommended that alternation of insecticide classes in field treatments for plant bug control be used by growers in 2007. This control strategy probably helped control plant bugs in the hills of MS where plant bug pressure was low in 2007, and only one population was found in the fall with an RR50 value > 3.0. Plant bug pressure was very high in many parts of the delta in 2007, and 15 of the 20 populations tested in the fall had RR50 values > 3.0. In one field test in cotton, a population with multiple resistance was tested and not effectively controlled in treatments using recommended rates of carbamate, organophosphate, and pyrethroid insecticides. Alternation of insecticide classes may not work very well when populations are present that are resistant to three of the four main classes of cotton insecticides. New insecticides in different classes are badly needed for control of tarnished plant bugs in cotton in the mid-South.

Mycotoxin occurrence and Aspergillus flavus soil propagules in a corn and cotton glyphosate-resistant cropping systems.

The effects of cotton-corn rotation and glyphosate use on levels of soil-borne Aspergillus flavus, aflatoxin and fumonisin contamination in corn and cotton seed were determined during 2002-2005 in Stoneville, Mississippi (USA). There were four rotation systems (continuous cotton, continuous corn, cotton-corn and corn-cotton) for both glyphosate-resistant (GR) and non-GR cultivars-herbicide system arranged in a randomized complete block design with four replications. Aspergillus flavus populations in surface (5-cm depth) soil, sampled before planting (March/April), mid-season June) and after harvest (September), ranged from 1.47 to 2.99 log (10) cfu g(-1) soil in the four rotation systems. Propagules of A. flavus were higher in the continuous corn system compared to the continuous cotton system on three sample dates, and cotton rotated with corn decreased A. flavus propagules in three of nine sample dates. Propagules of A. flavus were significantly greater in plots with GR cultivars compared to non-GR cultivars in three samples. In cotton seed, aflatoxin and fumonisin levels were similar (< or = 4 microg kg(-1) and non-detectable, respectively) regardless of rotation and glyphosate. In corn grain, aflatoxin was above the regulatory level (> or = 20 microg kg(-1)) only in GR cultivar in 2004 and 2005. Fumonisin was higher in non-GR cultivar (4 mg kg(-1)) regardless of rotation in 2004; however, in 2002, 2003 and 2005, aflatoxin and fumonisin levels were similar regardless of rotation and glyphosate. These results indicate the potential for increased aflatoxin and fumonisin levels (1 of 4 years) in corn; however, climatic conditions encountered during this study did not allow for mycotoxin production. In laboratory incubation studies, fairly high concentrations of glyphosate were required to inhibit A. flavus growth; however no short-term effect of soil treatment with glyphosate on A. flavus populations were observed. These data suggest that altered populations of A. flavus or higher aflatoxin concentrations in corn grain were due to indirect effects of the GR cropping system.

Conventional resistance of experimental maize lines to corn earworm (Lepidoptera: Noctuidae), fall armyworm (Lepidoptera: Noctuidae), southwestern corn borer (Lepidoptera: Crambidae), and sugarcane borer (Lepidoptera: Crambidae).

Plant resistance is a useful component of integrated pest management for several insects that are economically damaging to maize, Zea mays L. In this study, 15 experimental lines of maize derived from a backcross breeding program were evaluated for resistance to corn earworm, Helicoverpa zea (Boddie); fall armyworm, Spodoptera frugiperda (J. E. Smith); southwestern corn borer, Diatraea grandiosella Dyar; and sugarcane borer, Diatraea saccharalis (F.). Experimental line 100-R-3 was resistant in the field to leaf feeding by fall armyworm and line 116-B-10 was resistant in the field to leaf feeding by fall armyworm and leaf and stalk feeding by southwestern corn borer. When corn earworm larvae were fed field harvested silks from experimental line 81-9-B in the laboratory, their pupal weights were significantly lower than the pupal weights of larvae that were fed silks from the resistant control, Zapalote Chico. Maysin levels lower than those commonly associated with corn earworm resistance were present in the resistant experimental line, 107-8-7, indicating a new basis confers resistance to corn earworm in this line. These resistant experimental lines will provide plant breeders with new sources of resistance to lepidopterous insects for the development of improved maize breeding populations.

Evaluation of conventional resistance to European corn borer (Lepidoptera: Crambidae) and western corn rootworm (Coleoptera: Chrysomelidae) in experimental maize lines developed from a backcross breeding program.

Plant resistance is a promising control method for the two most damaging insect pests of maize, Zea mays L.: the European corn borer, Ostrinia nubilalis (Hübner), and the western corn rootworm Diabrotica virgifera virgifera LeConte. Fifteen experimental lines of maize, derived from a backcross breeding program designed to introgress resistance to European corn borer from Peruvian maize into two U.S. Corn Belt adapted inbred lines, were evaluated for resistance to European corn borer and western corn rootwonrm. The experimental lines were in the second generation of backcrossing. All experimental lines were resistant to leaf blade feeding by European corn borer. These lines had low levels of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one, a chemical commonly associated with leaf blade feeding resistance, indicating that this was not the mechanism of resistance to leaf blade feeding in these lines. Eleven experimental lines were resistant to leaf sheath and collar feeding by European corn borer. Useful sources of European corn borer ovipositional nonpreference and root feeding resistance to western corn rootworm were not identified. Some of the lines evaluated in this study may provide useful sources of resistance to both leaf blade and leaf sheath and collar feeding by European corn borer.

Low sialic acid-bearing mouse thymocytes do not express helper T cell properties in vitro.

Immature, presumably cortical, mouse thymocytes were isolated by removing mature thymocytes by agglutination with the sialic acid-specific lectin, lobster agglutinin 1 (LAg1). These immature cells do not respond to the mitogenic effects of concanavalin A (Con A), even in the presence of interleukin 2. Moreover, they do not exhibit two properties of helper T cells; they do not secrete interleukin 2 when stimulated with Con A, nor do they provide T help for an in vitro immune response by spleen B cells to the T-dependent antigen, sheep erythrocytes. LAg1-negative thymocytes fail to provide T help even though Con A is added to the cultures, regardless of the number of LAg1-negative thymocytes added per culture, and even in the presence of exogenous macrophages. Unseparated thymocytes, LAg1-positive thymocytes and cortisone-resistant thymocytes all provide T cell help under these conditions. These experiments indicate that immature, presumably cortical mouse thymocytes, isolated by virtue of their low levels of surface sialic acid, are inherently unable to provide T cell help in vitro.

Studies on the structure and carbohydrate binding properties of lobster agglutinin 1 (LAg1), a sialic acid-binding lectin.

Lobster agglutinin 1 (LAg 1) was isolated from the hemolymph of the American lobster (Homarus americanus) by a sequential combination of ammonium sulfate precipitation, preparative starch block electrophoresis, gel filtration and affinity chromatography on Sepharose-Fetuin and Sepharose-Colominic acid columns. Two types of protomeric structures with molecular weights of 700 and 500 Kilodaltons respectively were isolated. These molecules are composed of noncovalently held subunits with a molecular weight of 70 Kilodaltons. Analysis of preparations by double immunodiffusion, polyacrylamide gel electrophoresis and isoelectrofocusing indicates that the LAg 1 obtained was a single molecular species. Hemagglutination inhibition experiments indicated that the best inhibitors were bovine mucin, glycophorin, fetuin and human IgM in that order. The desialylated forms of some of these proteins still bound lectin, although to a lesser degree than their intact sialylated counterparts. Affinity chromatography experiments indicated that LAg 1 binds to N-acetylneuraminic acid, N-acetylglucosamine and N-acetylgalactosamine. LAg 1 does not contain sialic acid nor neuraminidase activity: oligosaccharides associated with it appear to be either of the oligomannosyl or biantennary type. The sialic acid binding specificity of this lectin was used to separate immature mouse thymocytes (low sialic acid content) from mature thymocytes (high sialic acid content).

Immature thymocytes isolated using a sialic acid-specific lectin are unresponsive to concanavalin A.

Mouse thymocytes, which are approximately 90% immature cortical cells, are low in surface sialic acid when compared with more mature cortisone-resistant, presumably medullary, thymocytes and peripheral T lymphocytes. Thus, medullary thymocytes bind and are agglutinated by the N-acetylneuraminic acid-specific lectin, lobster agglutinin 1 (LAgl), whereas cortical thymocytes are not agglutinated by this lectin. It is demonstrated herein that mouse cortical thymocytes, purified using LAgl, do not respond to the mitogenic effects of concanavalin A (Con A). The lack of response to this lectin is not due to depletion of macrophages, since addition of macrophages does not restore the response. Populations of LAgl-negative thymocytes can be made to respond weakly to Con A by the addition of interleukin 2, but this response appears to be due to the presence of a few contaminating LAg1-binding thymocytes since it is abolished by treatment of the cells with rabbit anti-LAgl serum plus complement. Therefore highly purified cortical thymocytes not only cannot respond to Con A, but also they cannot be induced to respond by the addition of the T cell growth factor, interleukin 2. Thymocytes isolated by a single criterion, that is by virtue of their low amount of surface sialic acid, appear to be a truly immature population of thymocytes.

Stimulation of B cells, but not T cells or thymocytes, by a sialic acid-specific lectin.

Haemolymph of the American lobster, Homarus americanus, contains several lectins. One of these, lobster agglutinin 1 (LAg1) is specific for N-acetylneuraminic acid and agglutinates mouse and human erythrocytes. In addition, this lectin agglutinates peripheral T cells and cortisone-resistant thymocytes, but agglutinates whole thymocytes poorly. Because this material is being used to prepare purified populations of cortical thymocytes, and then to study their maturation, it was important to determine if it is mitogenic for thymocytes and T cells. Thus, the studies described here were conducted to find if LAg1 is a mitogen for mouse lymphocytes, and if so for what cell populations. The data show that purified LAg1, but not purified lobster agglutinin 2 (LAg2) is a mitogen for mouse spleen cells, and that LAg1 is a polyclonal activator. Furthermore, LAg1 is a B-cell mitogen since it stimulates nude spleen cells, nude spleen cells depleted of pre-T cells, and normal spleen cells which have been treated with rabbit anti-mouse brain antiserum and complement. Moreover, LAg1 does not stimulate division by thymocytes or T cells, that is, spleen cells which do not adhere to nylon wool columns. Mitogenic activity of LAg1 but not of LPS is inhibited by N-acetylmannosamine, demonstrating that the mitogenic effects of LAg1 are unlikely to be due to contaminants.

Sequential expression of fucosyltransferase and N-acetylneuraminyltransferase activities in human leukemic cells arrested at different stages of maturation.

Fucosyl transferase (Fuc-T) and N-acetylneuraminyl-transferase (NeuAc-T) activities were determined in lysates of human neoplastic hematopoietic cells arrested at different stages of maturation. Leukemic non-T/non-B lymphoblasts and myeloblasts arrested at a very early stage of maturation displayed high Fuc-T and low NeuAc-T activities. T-lymphoblasts, further developed along the T-lymphocyte lineage, showed increased NeuAc-T activity. In contrast, lymphoid cells further developed along the B-lymphocyte lineage revealed low Fuc-T, and either low or high levels of NeuAc-T activity. These results suggest that during the process of cell maturation the expression of Fuc-T precedes that of NeuAc-T, and that in T-lymphocytes the expression of NeuAc-T is concomitant with the emergence of the T-cell receptor for sheep erythrocytes.