Biological and molecular characterization of two Anticarsia gemmatalis multiple nucleopolyhedrovirus clones exhibiting contrasting virulence.

Baculovirus natural populations are known to be genetically heterogeneous and such genotypic diversity could have implications in the performance of biocontrol agents. The Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) has been widely used to control the velvetbean caterpillar, Anticarsia gemmatalis, in Brazil. In the present work, morphological and molecular analyses as well as the biological activity of AgMNPV genotypes derived from a Brazilian field isolate (AgMNPV-79) were carried out. The existence of genotypic variants in the population was confirmed by DNA restriction analysis. Although difference in virulence was observed among the variants, the most (Ag79-01) and the least (AgL-16) virulent clones do not show any morphological and cytopathological changes when compared to the most studied isolate (AgMNPV-2D). The complete genome analysis of the two viral clones showed the presence of single open reading frames (ORFs) of the pe-38 and he65 genes, which contrasts with the two split ORFs present in the genome of the AgMNPV-2D isolate. The viral clone AgL-16 has many variations in the ie-2 and pe-38 genes, which are transcription regulatory genes responsible for the regulation of viral early gene expression during insect cell infection. Furthermore, other genes showed alterations like the odv-e56, which have an essential role in the maturation and envelopment of the ODVs, and bro-a and bro-b genes which were fused to form a single ORF. For the Ag79-01, although the total number of single nucleotide variants (SNVs) was more prominent in the pe-38 gene, its genome showed very few modifications in comparison to the AgMNPV-2D genome.

Biological characteristics of Anticarsia gemmatalis (Lepidoptera: Noctuidae) for three consecutive generations under different temperatures: understanding the possible impact of global warming on a soybean pest.

Climate changes can affect the distribution and intensity of insect infestations through direct effects on their life cycles. Experiments were carried out during three consecutive generations to evaluate the effect of different temperatures (25°C, 28°C, 31°C, 34°C and 37±1°C) on biological traits of the velvetbean caterpillar Anticarsia gemmatalis Hübner, 1818 (Lepidoptera: Noctuidae). The insects were fed on artificial diet and reared in environmental chambers set at 14 h photophase. The developmental cycle slowed with the increase in the temperature, within the 25°C to 34°C range. Male and female longevities were reduced with an increase in temperature from 25°C to 28°C. Egg viability was highest at 25°C, and the sex ratio was not influenced by temperature, in the three generations. There was no interactive effect between development time and temperature on pupal weight. The results suggested that the increase in the temperature negatively impacted A. gemmatalis development inside the studied temperature range, indicating a possible future reduction of its occurrence on soybean crops, as a consequence of global warming, mainly considering its impact on tropical countries where this plant is cropped. A. gemmatalis was not able to adapt to higher temperatures in a three-generation interval for the studied temperature range. However, a gradual increase and a longer adaptation period may favor insect selection and consequently adaptation, and must be considered in future studies in this area. Moreover, it is important to consider that global warming might turn cold areas more suitable to A. gemmatalis outbreaks. Therefore, more than a future reduction of A. gemmatalis occurrence due to global warming, we might expect changes regarding its area of occurrence on a global perspective.

Construction of a recombinant Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV-2D) harbouring the beta-galactosidase gene.

We have constructed a transfer vector (pAgGal) containing the beta-galactosidase gene under control of the Escherichia coli gpt and AgMNPV polyhedrin (polh) promoters. The transfer vector was cotransfected with wild type Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) DNA into A. gemmatalis (UFL-AG-286) cells and a recombinant baculovirus (vAgGalA2) was isolated. The beta-galactosidase gene insertion was checked by polymerase chain reaction (PCR) using DNA from AgMNPV and vAgGalA2 and primers specific for regions upstream and downstream of the polh gene. Insect cells (UFL-AG-286) were infected with the recombinant vAgGalA2 and wild type AgMNPV viruses and the production of the heterologous protein analyzed by SDS-PAGE and Pulse-Chase. Beta-galactosidase was expressed at high levels late on infection as expected for a gene under the control of the polh promoter. The highly expressed beta-galactosidase protein was also shown to be biologically active by a beta-galactosidase assay.

Physical maps and virulence of Anticarsia gemmatalis nucleopolyhedrovirus genomic variants.

Seventeen plaque purified isolates of two viral preparations of Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV), were analyzed in terms of the genomic changes after digestion of their DNAs with HindIII and PstI restriction enzymes. The 1979 AgMNPV wild type preparation (AgMNPV-'79) resulted in six different variants and the 1985 viral commercial preparation (AgMNPV-'85), in eleven. The genomic variation of all the isolates was mapped showing that those from 1985 presented more heterogeneity with changes mapped in additional sites in comparison to the AgMNPV-'79 variants. Their virulence was compared by infecting two Lepidopteran cell lines, Spodoptera frugiperda (IPLB-SF-21AE) and Anticarsia gemmatalis (UFL-AG-286). The results indicated that there was some difference in virulence within the AgMNPV-'85 variants. This commercial preparation had been applied in soybean fields in Brazil over several years to control the velvetbean caterpillar defoliation.

Assessment of the application of baculoviruses for control of Lepidoptera.

Baculoviruses, among other insect viruses, are regarded as safe and selective bioinsecticides, restricted to invertebrates. They have been used worldwide against many insect pests, mainly Lepidoptera. Their application as microbial pesticides, however, has not met their potential to control pests in crops, forests, and pastures, with the exception of the nuclear polyhedrosis virus of the soybean caterpillar (Anticarsia gemmatalis), which is used on approximately 1 million ha annually in Brazil. Problems that have limited expansion of baculovirus use include narrow host range, slow killing speed, technical and economical difficulties for in vitro commercial production, timing of application based on frequent host population monitoring, variability of field efficacy due to climatic conditions, and farmers' attitudes toward pest control, which have been based on application of fast-killing chemical insecticides. Farmer education regarding use of biological insecticides and their characteristics is considered one of the major actions necessary for increased use of baculoviruses. Strategies to counteract some of the limitations of baculoviruses, especially their slow killing activity, have been investigated and are promising. These include the use of chemical or biological substances added to virus formulations and genetic engineering of the viruses themselves to express insect toxins or hormones. Such strategies can enhance viral activity and increase speed of kill as well as reduce larval feeding activity. The use of baculoviruses against Lepidoptera is reviewed, with the utilization of the nuclear polyhedrosis virus of A. gemmatalis in Brazil serving as a case-study.

Laboratory and field studies on the infection of stink bugs, nezara viridula, piezodorus guildinii, and euschistus heros (Hemiptera: pentatomidae) with metarhizium anisopliae and beauveria bassiana in brazil

Isolates of Metarhizium anisopliae (CNPSo-Ma12) and Beauveria bassiana (CNPSo-Bb56) were tested under field conditions as biological control agents of soybean stink bugs (Nezara viridula, Piezodorus guildinii, and Euschistus heros). Kaolin-based powder formulations of M. anisopliae or B. bassiana were applied to soybean plots at a rate of 1.5 x 10(13) conidia per ha. After treatment, field cages (0.25 m2) were placed in plots and stink bug adults were introduced into the cages. Mycosis for both P. guildinii and N. viridula was initially observed 7 days postapplication in the 1991 season and at 15 days postapplication in the 1992 season. For E. heros, mortality was detected on day 8 and on day 20 in 1991 and 1992, respectively. In 1991, infection levels of 48 and 41% were achieved at day 30 for P. guildinii and N. viridula, respectively, whereas the infection level in E. heros reached only 33%. In 1992, mortality caused by M. anisopliae on the three stink bug species was lower than that observed in 1991, with cumulative mycosis at day 30 being 15, 17, and 20% for P. guildinii, N. viridula, and E. heros, respectively. The 1991 season was humid and warm, whereas in the 1992 season there was an 8-day drought spell (<75% relative humidity (RH)) after application, suggesting that %RH was responsible for an earlier occurrence and a higher prevalence of both fungi on stink bug species in the 1991 trial. Despite the higher deposition of B. bassiana on the plants (17 colony forming units [CFU] per mm2 of leaflet) 1 day after application compared to M. anisopliae (5.1 CFU per mm2 of leaflet), B. bassiana was less efficient against stink bugs than M. anisopliae. Field results were confirmed through laboratory bioassays, in controlled conditions (T = 26 +/- 1.5 degreesC, 90% RH, and 8D:16L light regimen). The mean time to mortality by M. anisopliae was 4.3 +/- 0.2 days for P. guildinii, 4. 6 +/- 0.2 days for N. viridula, and 7.4 +/- 0.5 days for E. heros. E. heros was less susceptible and P. guildinii was the most susceptible species to M. anisopliae. Since these species usually occur as a complex in soybean in Brazil, these results represent important information toward the development of entomopathogenic fungi as microbial insecticides of these pests. Copyright 1998 Academic Press.

[Antineurotrophil cytoplasmic antibodies (ANCA) in the elderly]. / Anticuerpos anti citoplasma del neutrofilo (ANCA) en ancianos.

Aging in animals and human beings is frequently accompanied by a disarray of the immune response. In subjects 70 years of age or older the frequency of some autoantibodies (anti-nuclear, RF, antithyroid anti-parietal cell and others) without any associated pathology is clearly increased when compared against a younger population. ANCA (antineutrophil cytoplasmic antibodies) is considered to be a good marker for necrotizing systemic vasculitis. This auto-antibody is known to show two patterns when investigated by indirect immunofluorescence on alcohol fixed normal neutrophils: c-ANCA (cytoplasmatic fluorescence) and p-ANCA (perinuclear fluorescence), each one associated with different pathologies. The purpose of this work was to investigate how frequently was ANCA found in subjects 70 years of age or older without evidence of any associated pathology. ANCA was investigated in 447 ambulatory patients from our hospital and the only requirement to be included was age. The ANCA+ patients were divided into having or not clinical suspicion of vasculitis, according to the medical charts we reviewed. The same methodology was applied to investigate the frequency of antinuclear antibodies (ANA+) without clinical evidence of any associated pathology. This frequency was compared with that described in the literature as "ANA+ in healthy old subjects". In the 447 patients studied, 8 were positive for ANCA (1.8%), 3 for c-ANCA and 5 for p-ANCA. None of the 3 c-ANCA and only 1 of 5 p-ANCA belonged to patients without any associated pathology. A possible relationship between c-ANCA and cancer is discussed. As ANCA+ cases were very few no relationship with gender or age was investigated. The extremely low finding of ANCA+ in the absence of pathology, even considering old people's frequent immunological disarray suggests that in opposition to other autoantibodies, the presence of ANCA is always related to disease. The frequency of ANA+ without associated pathology was 2.9%, being this value on the lower limits of what is described in the literature. This can be due to different methodology or to the scope of the definition of "without associated pathology".