Transgenic indica rice resistant to sap-sucking insects.

Agrobacterium-mediated genetic transformation has been optimized in indica rice susceptible to sap-sucking insects, viz., brown planthopper (BPH) and green leafhopper (GLH). Snowdrop lectin gene (gna) from Galanthus nivalis, driven by phloem-specific rice-sucrose-synthase promoter, along with herbicide resistance gene (bar) driven by CaMV 35S promoter, was employed for genetic transformation. Embryogenic calli--after co-cultivation with Agrobacterium strain LBA4404 harbouring Ti plasmid pSB111-bar-gna--were selected on the medium containing phosphinothricin. PCR and Southern blot analyses confirmed the stable integration of both the genes into genomes of transgenic (T0) rice plants. Northern and Western blot analyses revealed the expression of gna in the transgenic plants. In the T1 and T2 generations, the gna and bar transgenes showed co-segregation at a ratio of 3 : 1. Plant progenies expressing gna, in T1 and T2, exhibited substantial resistance against BPH and GLH pests. This is the first report dealing with transgenic indica rice exhibiting high resistance to both insects.

Crystal structure of a novel mid-gut procarboxypeptidase from the cotton pest Helicoverpa armigera.

The cotton bollworm Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is one of the most serious insect pests in Australia, India and China. The larva causes substantial economical losses to legume, fibre, cereal oilseed and vegetable crops. This pest has proven to be difficult to control by conventional means, mainly due to the development of pesticide resistance. We present here the 2.5 A crystal structure from the novel procarboxypeptidase (PCPAHa) found in the gut extracts from H. armigera larvae, the first one reported for an insect. This metalloprotease is synthesized as a zymogen of 46.6 kDa which, upon in vitro activation with Lys-C endoproteinase, yields a pro-segment of 91 residues and an active carboxypeptidase moiety of 318 residues. Both regions show a three-dimensional structure quite similar to the corresponding structures in mammalian digestive carboxypeptidases, the most relevant structural differences being located in the loops between conserved secondary structure elements, including the primary activation site. This activation site contains the motif (Ala)(5)Lys at the C terminus of the helix connecting the pro- and the carboxypeptidase domains. A remarkable feature of PCPAHa is the occurrence of the same (Ala)(6)Lys near the C terminus of the active enzyme. The presence of Ser255 in PCPAHa instead of Ile and Asp found in the pancreatic A and B forms, respectively, enlarges the S1' specificity pocket and influences the substrate preferences of the enzyme. The C-terminal tail of the leech carboxypeptidase inhibitor has been modelled into the PCPAHa active site to explore the substrate preferences and the enzymatic mechanism of this enzyme.

Midgut carboxypeptidase from Helicoverpa armigera (Lepidoptera: Noctuidae) larvae: enzyme characterisation, cDNA cloning and expression.

Using synthetic substrates we have characterised carboxypeptidase activity in gut extracts from Helicoverpa armigera larvae. Carboxypeptidase A activity predominates, with only low levels of carboxypeptidase B activity present. Maximum carboxypeptidase A activity occurs over a broad pH range and is inhibited by phenanthroline and potato carboxypeptidase inhibitor. A cDNA clone encoding carboxypeptidase (the first such sequence from a lepidopteran insect) was isolated from a larval gut library. The sequence predicts a secreted polypeptide of Mr 46.6 k with homology to metallocarboxypeptidases from mammalian and invertebrate species. The presence of a serine residue at the active site suggests carboxypeptidase A activity. To further characterise the gene product, the complete cDNA sequence was expressed in insect cells using the baculovirus system. Culture supernatant from these cells contained carboxypeptidase A activity, with no activity against a carboxypeptidase B substrate; the carboxypeptidase B activity in gut extracts must thus be due to a separate enzyme. In agreement with this conclusion, the expressed carboxypeptidase cDNA is a member of a small multigene family. Chronic ingestion of soybean Kunitz trypsin inhibitor by H. armigera larvae results in increased accumulation of carboxypeptidase mRNA in the midgut cells, and an increase in carboxypeptidase A activity detected in gut extract.

Expression of snowdrop lectin (GNA) in transgenic rice plants confers resistance to rice brown planthopper.

Snowdrop lectin (Galanthus nivalis agglutinin; GNA) has been shown previously to be toxic towards rice brown planthopper (Nilaparvata lugens; BPH) when administered in artificial diet. BPH feeds by phloem abstraction, and causes 'hopper burn', as well as being an important virus vector. To evaluate the potential of the gna gene to confer resistance towards BPH, transgenic rice (Oryza sativa L.) plants were produced, containing the gna gene in constructs where its expression was driven by a phloem-specific promoter (from the rice sucrose synthase RSs1 gene) and by a constitutive promoter (from the maize ubiquitin ubi1 gene). PCR and Southern analyses on DNA from these plants confirmed their transgenic status, and that the transgenes were transmitted to progeny after self-fertilization. Western blot analyses revealed expression of GNA at levels of up to 2.0% of total protein in some of the transgenic plants. GNA expression driven by the RSs1 promoter was tissue-specific, as shown by immunohistochemical localization of the protein in the non-lignified vascular tissue of transgenic plants. Insect bioassays and feeding studies showed that GNA expressed in the transgenic rice plants decreased survival and overall fecundity (production of offspring) of the insects, retarded insect development, and had a deterrent effect on BPH feeding. gna is the first transgene to exhibit insecticidal activity towards sap-sucking insects in an important cereal crop plant.

Selective and conventional house-spraying of DDT and bendiocarb against Anopheles pseudopunctipennis in southern Mexico.

Indoor feeding behaviors and mortalities of Anopheles pseudopunctipennis females were evaluated following contact with selective (bands covering mosquitoes' preferred resting areas) and full applications of DDT and bendiocarb on indoor sprayable surfaces. The DDT residues provoked strong avoidance behavior. To a lesser degree, mosquitoes were also repelled by bendiocarb-sprayed surfaces. Because of strong irritancy/repellency, unfed mosquitoes were driven outdoors in proportionally higher numbers. The resting time on selectively or fully DDT-sprayed huts was greatly reduced in comparison to bendiocarb-sprayed huts. Although unfed mosquitoes tended to rest on non-DDT-sprayed surfaces in the selectively treated hut, the man-biting rate was similar with both types of treatments. Unfed mosquitoes were repelled less from selectively bendiocarb-treated surfaces. Similar reductions in postfed resting times were observed on all surfaces suggesting that once fed, mosquitoes rested on sprayed surfaces for shorter intervals of time. Engorged mosquitoes had normal resting behavior (pre- and postspray) within the range of preferred resting heights in both DDT- and bendiocarb-sprayed huts, but the proportion of mosquitoes fed in the DDT-treated huts was lower. Selective spraying of walls was as effective as spraying the complete walls with both insecticides, but DDT was more effective in reducing mosquito-human contact. These studies show that by more effectively targeting vector behavior, a cost-effective alternative to traditional control techniques can be achieved.

Selective loss of cholinergic receptors following unilateral intracortical injection of volkensin.

Experimental lesions and quantitative autoradiography were used to investigate the cellular distribution of neurotransmitter receptors in rats. Lesions were produced by intracortical injections of either volkensin or ricin. However, only the former is retrogradely transported and volkensin treatment causes significant loss of contralateral cortical pyramidal neurones. Binding of [3H]pirenzepine (muscarinic M1 receptors) and [3H]nicotine was reduced in contralateral cortex in volkensin compared with ricin and/or control (uninjected) animals. However, binding of [3H]8-hydroxy-2-(n-dipropylamino)tetralin (5-HT1A receptors), [3H]ketanserin (5-HT2A receptors), and [3H]1,3-dipropylcyclopentylxanthine (adenosine A1 receptors) was unchanged. The most likely explanation for these results is that M1 and nicotinic receptors are present in large numbers on those pyramidal neurones that are lost. The results are discussed in terms of the biology of cortical pyramidal neurones, drugs for Alzheimer's disease, and novel ligands for improving human brain scanning techniques.

Differentially regulated inhibitor-sensitive and insensitive protease genes from the phytophagous insect pest, Helicoverpa armigera, are members of complex multigene families.

Ingestion of soybean Kunitz trypsin inhibitor (SKTI) by larvae of the phytophagous insect pest Helicoverpa armigera induced production of inhibitor-insensitive protease activity. The induced activity was not due to proteolytic enzymes of different mechanistic classes, but rather to variants of the existing enzymes. Characterization of cDNAs showed that sequences encoding proteins of the serine protease family were abundant in gut tissue of both control and SKTI-fed insects. The majority of serine protease family cDNAs encode enzymes closely homologous to trypsin and chymotrypsin; comparison of these sequences shows variation in amino acid residues within the region which would be in contact with a protein protease inhibitor. More diverged sequences which may not encode active proteases are also present. All the cDNAs examined were found to derive from multigene families; at least 28 different genes are present in the serine protease family. Chronic ingestion of SKTI results in some serine protease-encoding mRNA species increasing in level, whereas others decrease. Chymotrypsin-encoding mRNAs tend to increase in level as a result of SKTI ingestion, but no clear trend is shown by trypsin-encoding mRNAs. It is suggested that multiple, varying protease-encoding genes are an adaptive mechanism for reducing the deleterious effects of plant protease inhibitors.

Behaviour of Anopheles albimanus in relation to pyrethroid-treated bednets.

Responses of the malaria vector Anopheles albimanus to pyrethroid impregnated bednets made of cotton or nylon, compared with untreated nets, were investigated in houses occupied by two people inside and/or outside two bednets, in coastal Chiapas, México. The pyrethroid used was lambdacyhalothrin 30 mg a.i./m2. Bioassay mortality rates of An.albimanus exposed to treated nets for 3 or 15 min, rose from 40-55% to 90-100% for nylon nets 3-19 weeks post-treatment, but were consistently lower for treated cotton nets. An.albimanus females (collected unfed on human bait) were released in houses surrounded by curtains for trapping mosquitoes that exited from the house. Floor sheets were used in and around each experimental house for retrieving any mosquitoes knocked-down and/or killed. During post-treatment assessment for 17 weeks, An.albimanus blood-feeding success rates were 23-24% with untreated nets, 14-18% with treated cotton nets and 8-15% with treated nylon nets, significantly reduced when both human baits were inside the treated bednets, but not when one or more baits were outside the treated bednet(s) within the house. Proportions of mosquitoes leaving houses < 3 h post-release were 53-59% from houses with untreated bednets versus 65-78% with treated bednets. Except in one case (when both humans were outside treated cotton nets), these increased early exit rates were significant, whether or not the human baits were inside the treated bednets indoors. Mortality rates of An.albimanus females exiting overnight (22.00-06.00 hours) averaged 15-39% from houses with treated cotton and 16-46% with treated nylon nets, very significantly greater than the control mean rates of 6-8% mortality with untreated nets. Observations on wild-caught An.albimanus females marked with fluorescent powder and released indoors revealed that few mosquitoes (3-11%) actually contacted the bednets unless both human baits remained under them - when contact rates were 22% on treated nylon, 23% on treated cotton and 42% on untreated nets (P approximately 0.05). The mean resting time was significantly longer on untreated (14.4 min) than on treated nylon (5.8 min) or cotton (9.5 min) bednets, whereas mean resting times on other surfaces indoors were 16.5-19.8 min. Proportions exiting within 2h of release were significantly more from houses with treated houses (33-35%) than with untreated nets (8%). However, mortality rates of mosquitoes that landed on treated nets were very significantly greater (90-100%) than after landing on untreated nets (10%). Thus, despite some excito-repellency, lambdacyhalothrin-impregnated bednets (especially made of nylon) proved to be effective as an alternative to house-spraying against An.albimanus.

Low-volume application by mist-blower compared with conventional compression sprayer treatment of houses with residual pyrethroid to control the malaria vector Anopheles albimanus in Mexico.

Village-scale trials were carried out in southern Mexico to compare the efficacy of indoor-spraying of the pyrethroid insecticide lambda-cyhalothrin applied either as low-volume (LV) aqueous emulsion or as wettable-powder (WP) aqueous suspension for residual control of the principal coastal malaria vector Anopheles albimanus. Three indoor spray rounds were conducted at 3-month intervals using back-pack mist-blowers to apply lambda-cyhalothrin 12.5 mg a.i./m2 by LV, whereas the WP was applied by conventional compression sprayer at a mean rate of 26.5 mg a.i./m2. Both treatments caused mosquito mortality indoors and outdoors (collected inside house curtains) as a result of contact with treated surfaces before and after feeding, but had no significant impact on overall population density of An. albimanus resting indoors or assessed by human bait collections. Contact bioassays showed that WP and LV treatments with lambda-cyhalothrin were effective for 12-20 weeks (> 75% mortality) without causing excito-repellency. Compared to the WP treatment (8 houses/man/day), LV treatment (25 houses/man/day) was more than 3 times quicker per house, potentially saving 68% of labour costs. This is offset, however, by the much lower unit price of a compression sprayer (e.g. Hudson 'X-pert' at US$120) than a mist-blower (e.g. 'Super Jolly' at US$350), and higher running costs for LV applications. It was calculated, therefore, that LV becomes more economical than WP after 18.8 treatments/100 houses/10 men at equivalent rates of application, or after 7.6 spray rounds with half-rate LV applications.

Control of Anopheles albimanus mosquitos in southern Mexico by spraying their preferred indoor resting sites.

The traditional indoor spraying technique to control Anopheles albimanus mosquitos was compared with a selective method that targets their preferred resting sites in coastal villages of southern Mexico. We also determined whether mosquitos changed their preferred resting sites following insecticide applications. In the selective spraying approach, two 1-m wide horizontal swaths of bendiocarb insecticide were applied 0.75-1.75 m from the base of the walls and at 1-m upwards from the base of the inner roof at a target dose of 0.4 g/m2. A 2-year follow-up (1988-90), which included four insecticide treatments, showed that the selective spraying was as effective as full spraying for controlling A. albimanus. Also the mosquitos preferred resting sites that were not modified by the insecticide applications. Finally, the use of this techniques required 46% less time and cost 67% less than conventional spraying.

Intradomicillary pre- and postfeeding behavior of Anopheles pseudopunctipennis of southern Mexico: implications for malaria control.

The intradomicillary pre- and postfeed resting behavior of Anopheles pseudopunctipennis was studied in an experimental house in southern Mexico. During resting periods (both pre-/postfeed) mosquitos had greater contact (landings) with the inner roof than with the walls and other surfaces. A comparison of mean landing frequency and overall resting time (pre-/postfeed) showed that a greater periodic and prolonged contact occurred prefeed, probably as a result of disturbed activity associated with host movements. Pre-/postfeed resting patterns on walls were limited to a 0.6-0.5-m-wide band, nearly 1 m from the floor, and to a narrower band on the roof, 0.3-0.2 m wide, approximately 2.3 m from the floor, respectively. We calculated that with a band width of 0.8 m on the walls and another band 0.8 m wide on the roof, 87.2% of the mosquitoes had at least one contact with either the wall, the roof, or with both surfaces, along with an overall mean resting time (pre-/postfeed) of 8.1 min/landing. These findings suggest that a high potential for control can be achieved by spraying preferred wall and roof resting sites in this region where the intradomicillary application of residual insecticide is the primary malaria control measure.

Peri-/intradomicillary behavior in relation to host-seeking of Anopheles pseudopunctipennis in southern Mexico.

Peri-/intradomicillary resting and host-seeking behavior of Anopheles pseudopunctipennis was studied in an experimental house (surrounded by a curtain-net) that had not been sprayed with insecticide. Peak mosquito densities were recorded resting on vegetation 1 h earlier (1900-2000 h) than on the curtain-net, suggesting that a proportion of females prefer resting on adjacent vegetation prior to moving indoors. Between 2000 and 2100 h there was a marked decrease in numbers of mosquitoes resting on the exterior of the curtain-net. In separate experiments without the net, a single peak in numbers of mosquitoes resting on interior surfaces and/or biting human bait occurred between 2000 and 2100 h. Unfed mosquitoes had higher numbers of contacts with wall surfaces than with the roof. Moreover, a higher proportion of mosquitoes collected on human bait had > or = 1 dilatation(s), and higher parity rates than those resting on walls. Higher proportions of parous mosquitoes more frequently fed from 1800 to 2100 h, whereas higher proportions of nulliparous mosquitoes fed during morning hours. Nearly 25% of all mosquitoes exited 1 h following their release inside the house, whereas nearly 7% remained indoors for > 9 h. Overall results demonstrated that the behavior of An. pseudopunctipennis females depends on outdoor/indoor stimuli, being multivariable in nature. This includes contacts with a variety of biological (i.e., vegetation, human) and inert surfaces, which in part appears to be controlled by age structure.

[The focal control of malaria. Focal treatment using chemoprophylaxis and home insecticide spraying for the control of malaria in southern Mexico]. / Control focal del paludismo. Tratamiento focal usando quimioprofilaxis y rociado intradomiciliar con insecticida para el control del paludismo en el sur de México.

The efficacy of a focal control strategy for malaria was evaluated against a conventional scheme carried out in two groups of villages in the Soconusco, southern Chiapas, Mexico. Focal control consisted on the prophylactic administration of antimalarial drugs to people who had experienced malaria episodes two years previous to the study. Homes of these malaria patients were also sprayed indoors with DDT. The traditional strategy consisted on the treatment of all patients with antimalarial drugs as well as indoor spraying with DDT of all houses in the villages. Results from the focal control demonstrated similar efficacy as compared to conventional. However, in terms of cost, focal control was four fold more economical. Focal control had an additional advantage of incorporating community participation within the control operations.

Plasmodium vivax sporozoite rates from Anopheles albimanus in southern Chiapas, Mexico.

Anopheles albimanus mosquitoes were collected from August 1984 to November 1987 on intra- and peridomicile human bait in Rancheria El Gancho, Chiapas, Mexico. The mosquitoes were desiccated and stored in silicon chambers from 3 mo to 3 yr post-collection prior to being assayed using a direct enzyme-linked immunosorbent assay to detect Plasmodium vivax predominant-type sporozoite protein. Peridomicile-collected mosquitoes had a 10-fold higher sporozoite rate than those collected indoors, but only the latter correlate significantly with the seasonal human parasite index. Mosquito sporozoite burden was also significantly higher in the peridomicile-collected population.

Characterisation of potato (Solanum tuberosum L.) extensins: a novel extensin-like cDNA from dormant tubers.

A cDNA library from dormant potato tubers was screened for extensin (Ext)-like cDNAs (PTEL). The cDNAs recovered were partially sequenced and could be divided into two classes. One class, containing the repeating amino acid (aa) motifs, SPPPPSPKYVYK and SPPPPSPSPPPPYYYK, constitutes the potato counterpart of the tomato Ext-encoding gene previously isolated by Zhou et al. [Plant Mol. Biol. 20 (1992) 5-17]. A full-length cDNA from the second class was fully sequenced. The product of this cDNA has the repetitive sequence and proline-rich composition typical of Ext. Features worthy of note are the signal peptide sequence, which shares close homology with signal sequences of a number of Ext of diverse origin, and the presence of a novel, histidine-rich motif (SPPPHHHHPVYK) repeated within the sequence. Wounding of the potato tuber causes a marked increase in Ext-like mRNAs, although the species induced are not those present in dormant tissue.

Intradomiciliary behavior of Anopheles albimanus on the coastal plain of southern Mexico: implications for malaria control.

The postfeeding indoor resting behavior of Anopheles albimanus in experimental houses in southern México was investigated by using a mark-recapture procedure. The majority of mosquitoes rested inside houses after taking a blood meal indoors. There was a higher landing frequency on interior surfaces other than walls and roofs; however, mosquitoes rested for longer periods on these 2 surfaces. Successive landings on walls after short flights showed that mosquitoes gradually increased their mean landing height from 1.0 to 1.4 m. Similarly, mosquitoes resting at the base of inner roofs had a successive landing height range of about 0.5 m. Based on these observations and the potential for reduction of nearly 50% in the quantity of insecticide used and the time needed to apply it, village-scale studies involving the selective spraying of a 1-m-wide swath of insecticide on walls and on roofs are recommended in this area.

Indoor low-volume insecticide spray for the control of Anopheles albimanus in southern Mexico. Village-scale trials of bendiocarb, deltamethrin and cyfluthrin.

A comparative insecticide village-scale trial was carried out to determine the efficacy of low-volume (LV) indoor spray of bendiocarb, deltamethrin and cyfluthrin for the control of Anopheles albimanus in a coastal plain of southern Mexico. Low-volume spray was conducted with knapsack mist-blowers, giving an average discharge rate of 215 ml/min, which deposited droplets of 50-100 microns. Using this technique, 25 houses were treated/sprayman/day as compared with 8 houses/sprayman/day using conventional hand compression pumps. Indicators of LV mortality showed a residual activity of 10 wk as compared with 13 wk of activity for wettable powder (WP) (bendiocarb). When comparing expenditures, LV spray costs were 43% less when using bendiocarb, 83% less with deltamethrin and 74% less with cyfluthrin with respect to the conventional WP spray of the same insecticides. Low-volume spray time was reduced by one-third, with respect to WP. Malaria incidence was reduced by 53 and 56%, respectively, in the bendiocarb LV and deltamethrin LV treated villages as compared with the untreated village.

Anopheles albimanus (Diptera: Culicidae) host selection patterns in three ecological areas of the coastal plains of Chiapas, southern Mexico.

The host-feeding patterns of Anopheles albimanus Wiedemann were described and the effect of host availability on these patterns was assessed in three different ecological areas of coastal Chiapas, Mexico. Resting mosquitoes were collected indoors and outdoors during rainy seasons. A 20% sample of blood-fed mosquitoes was tested to determine the source of the blood meal using an ELISA. The unweighted human blood index (HBI) of An. albimanus in the three areas ranged from 0.11 to 0.21. This mosquito species fed more frequently on bovines than on any other host, but the forage ratio indicated that there was also a high preference for equines. Some females tended to rest or complete their gonotrophic cycle indoors after feeding on animals, but females also fed on man and rested outdoors. Host availability and ecological conditions appeared to be responsible for differences observed in the HBI among areas.

Sequence of a novel plant ras-related cDNA from Pisum sativum.

A clone isolated from a purple podded pea (Pisum sativum L.) cDNA library was shown to contain the complete coding sequence of a polypeptide with considerable homology to various members of the ras superfamily. The ras superfamily are a group of monomeric GTP-binding proteins of 21-25 kDa found in eukaryotic cells. Conserved sequences in the isolated clone include the GTP-binding site, GDP/GTP hydrolysis domain and C-terminal Cys residues involved in membrane attachment. Comparisons of the predicted amino acid sequence with those of other ras proteins show significantly higher homologies (ca. 70%) to two mammalian gene products, those of the BRL-ras oncogene, and the canine rab7 gene, than to any of the plant ras gene products so far identified (< 40% homology). The high percentage of amino acid identity suggests that this cDNA may be the product of a gene, designated Psa-rab, which is the plant counterpart of rab7. Rab/ypt proteins are a subfamily of the ras superfamily thought to be involved in intracellular transport from the endoplasmic reticulum to the Golgi apparatus and in vesicular transport. Northern blot hybridisation analysis of total RNA from green and purple podded pea revealed a mRNA species of approximately the same size as the isolated cDNAs.