Selection for high levels of resistance to double-stranded RNA (dsRNA) in Colorado potato beetle (Leptinotarsa decemlineata Say) using non-transgenic foliar delivery.

Insecticidal double-stranded RNAs (dsRNAs) silence expression of vital genes by activating the RNA interference (RNAi) mechanism in insect cells. Despite high commercial interest in insecticidal dsRNA, information on resistance to dsRNA is scarce, particularly for dsRNA products with non-transgenic delivery (ex. foliar/topical application) nearing regulatory review. We report the development of the CEAS 300 population of Colorado potato beetle (Leptinotarsa decemlineata Say) (Coleoptera: Chrysomelidae) with > 11,100-fold resistance to a dsRNA targeting the V-ATPase subunit A gene after nine episodes of selection using non-transgenic delivery by foliar coating. Resistance was associated with lack of target gene down-regulation in CEAS 300 larvae and cross-resistance to another dsRNA target (COPI ß; Coatomer subunit beta). In contrast, CEAS 300 larvae showed very low (~ 4-fold) reduced susceptibility to the Cry3Aa insecticidal protein from Bacillus thuringiensis. Resistance to dsRNA in CEAS 300 is transmitted as an autosomal recessive trait and is polygenic. These data represent the first documented case of resistance in an insect pest with high pesticide resistance potential using dsRNA delivered through non-transgenic techniques. Information on the genetics of resistance and availability of dsRNA-resistant L. decemlineata guide the design of resistance management tools and allow research to identify resistance alleles and estimate resistance risks.

Insecticidal activity of Vanillosmopsis arborea essential oil and of its major constituent α-bisabolol against Callosobruchus maculatus (Coleoptera: Chrysomelidae).

Vigna unguiculata, one of the most important legumes, mainly in underdeveloped countries, is susceptible to post-harvest losses in storage by Callosobruchus maculatus (Fabricius, 1775) (Coleoptera: Chrysomelidae). The work evaluated the toxicity, inhibition of oviposition, instantaneous rate of population growth (ri) and the development of fumigated C. maculatus with the essential oil of Vanillosmopsis arborea and its major constituent, α-bisabolol. The experimental units consisted of 0.8 L flasks treated with concentrations of 1.2-11.2 µL L-1of air of the essential oil of V. arborea or its major constituent applied to disks of filter paper. α-Bisabolol was quantified as 409.33 mL L-1 of the essential oil. The development rate of C. maculatus was evaluated by daily adult counts. Oviposition was evaluated at lethal concentrations (LC50, LC25, LC10 and LC1). The LC50 and LC95 of the essential oil of V. arborea and α-bisabolol were 5.23 and 12.97 µL L-1 of air and 2.47 and 8.82 µL L-1 of air, respectively. At some concentrations, the α-bisabolol was more toxic to males than to females of the insect. Increased concentrations of the essential oil reduced the ri, rate of development, oviposition, and number of eggs of C. maculatus and therefore have potential for pest control.

Transgenic potato lines expressing hairpin RNAi construct of molting-associated EcR gene exhibit enhanced resistance against Colorado potato beetle (Leptinotarsa decemlineata, Say).

Most of the commercialized insect resistant transgenic crops express cry gene(s) isolated from Bacillus thuringiensis; however, intensive cultivation of Bt crops over almost two decades has been questioned regarding its sustainability and durability in pest management. The present study focused on silencing of highly specific molting-associated Ecdysone receptor (EcR) gene of Colorado potato beetle (CPB) using RNA interference (RNAi) approach. The partial cDNA of EcR gene of CPB was amplified using specific primers in sense and anti-sense orientations, and cloned in pRNAi-GG vector flanked by an intronic sequence (pdk). Leaf and internodal explants of Agria and Lady Olympia potato cultivars were infected with Agrobacterium strain LBA4404 harboring constructs under the control of CaMV 35S promoter. Standard molecular analysis of primary transformants showed proper integration of T-DNA in plant genome. The transgenic plants of both cultivars were evaluated for their efficacy against first, second and third instar CPB larvae. The leaf biotoxicity assays revealed 15-80% of CPB mortality. A significantly lower fold-change (0.87-4.14×) in larval weight was observed in insects fed on transgenic plants compared to the ones fed on control plants (1.87-6.53×). Furthermore, CPB larvae fed on transgenic plants exhibited reduced EcR transcripts, indicating the functionality of dsRNA EcR in silencing EcR gene expression. This study is an excellent example of the integration of an alternative, effective and reliable method to cope with potato insect pests that incur significant losses to potato production in the world.

The occurrence and formation of monoterpenes in herbivore-damaged poplar roots.

Volatiles are often released upon herbivory as plant defense compounds. While the formation of volatiles above-ground has been intensively studied, little is known about herbivore-induced root volatiles. Here, we show that cockchafer larvae-damaged roots of Populus trichocarpa and P. nigra release a mixture of monoterpenes, including (-)-α-pinene, (-)-camphene, (-)-ß-pinene, p-cymene, and 1,8-cineole. Three terpene synthases, PtTPS16 and PtTPS21 from P. trichocarpa and PnTPS4 from P. nigra, could be identified and characterized in vitro. PnTPS4 was found to produce 1,8-cineole as sole product. PtTPS16 and PtTPS21, although highly similar to each other, showed different product specificities and produced γ-terpinene and a mixture of (-)-camphene, (-)-α-pinene, (-)-ß-pinene, and (-)-limonene, respectively. Four active site residues were found to determine the different product specificities of the two enzymes. The expression profiles of PtTPS16, PtTPS21, and PnTPS4 in undamaged and herbivore-damaged poplar roots generally matched the emission pattern of monoterpenes, indicating that monoterpene emission in roots is mainly determined at the gene transcript level. Bioassays with Phytophtora cactorum (Oomycetes) revealed inhibitory effects of vapor-phase 1,8-cineole and (-)-ß-pinene on the growth of this important plant pathogen. Thus herbivore-induced volatile monoterpenes may have a role in defense against pathogens that cause secondary infections after root wounding.

Pest control. Full crop protection from an insect pest by expression of long double-stranded RNAs in plastids.

Double-stranded RNAs (dsRNAs) targeted against essential genes can trigger a lethal RNA interference (RNAi) response in insect pests. The application of this concept in plant protection is hampered by the presence of an endogenous plant RNAi pathway that processes dsRNAs into short interfering RNAs. We found that long dsRNAs can be stably produced in chloroplasts, a cellular compartment that appears to lack an RNAi machinery. When expressed from the chloroplast genome, dsRNAs accumulated to as much as 0.4% of the total cellular RNA. Transplastomic potato plants producing dsRNAs targeted against the ß-actin gene of the Colorado potato beetle, a notorious agricultural pest, were protected from herbivory and were lethal to its larvae. Thus, chloroplast expression of long dsRNAs can provide crop protection without chemical pesticides.

Herbivory responsive C2H2 zinc finger transcription factor protein StZFP2 from potato.

While C2H2 zinc finger transcription factors (TF) are often regulated by abiotic stress, their role during insect infestation has been overlooked. This study demonstrates that the transcripts of the zinc finger transcription factors StZFP1 and StZFP2 are induced in potato (Solanum tuberosum L.) upon infestation by either the generalist tobacco hornworm (THW, Manduca sexta L.) or the specialist Colorado potato beetle (CPB, Leptinotarsa decemlineata Say). StZFP1 has been previously characterized as conferring salt tolerance to transgenic tobacco and its transcript is induced by Phytophthora infestans and several abiotic stresses. StZFP2 has not been characterized previously, but contains the hallmarks of a C2H2 zinc finger TF, with two conserved zinc finger domains and DLN motif, which encodes a transcriptional repressor domain. Expression studies demonstrate that StZFP2 transcript is also induced by tobacco hornworm and Colorado potato beetle. These observations expand the role of the C2H2 transcription factor in potato to include the response to chewing insect pests.

Some like it hot: the influence and implications of climate change on coffee berry borer (Hypothenemus hampei) and coffee production in East Africa.

The negative effects of climate change are already evident for many of the 25 million coffee farmers across the tropics and the 90 billion dollar (US) coffee industry. The coffee berry borer (Hypothenemus hampei), the most important pest of coffee worldwide, has already benefited from the temperature rise in East Africa: increased damage to coffee crops and expansion in its distribution range have been reported. In order to anticipate threats and prioritize management actions for H. hampei we present here, maps on future distributions of H. hampei in coffee producing areas of East Africa. Using the CLIMEX model we relate present-day insect distributions to current climate and then project the fitted climatic envelopes under future scenarios A2A and B2B (for HADCM3 model). In both scenarios, the situation with H. hampei is forecasted to worsen in the current Coffea arabica producing areas of Ethiopia, the Ugandan part of the Lake Victoria and Mt. Elgon regions, Mt. Kenya and the Kenyan side of Mt. Elgon, and most of Rwanda and Burundi. The calculated hypothetical number of generations per year of H. hampei is predicted to increase in all C. arabica-producing areas from five to ten. These outcomes will have serious implications for C. arabica production and livelihoods in East Africa. We suggest that the best way to adapt to a rise of temperatures in coffee plantations could be via the introduction of shade trees in sun grown plantations. The aims of this study are to fill knowledge gaps existing in the coffee industry, and to draft an outline for the development of an adaptation strategy package for climate change on coffee production. An abstract in Spanish is provided as Abstract S1.

Organic agriculture promotes evenness and natural pest control.

Human activity can degrade ecosystem function by reducing species number (richness) and by skewing the relative abundance of species (evenness). Conservation efforts often focus on restoring or maintaining species number, reflecting the well-known impacts of richness on many ecological processes. In contrast, the ecological effects of disrupted evenness have received far less attention, and developing strategies for restoring evenness remains a conceptual challenge. In farmlands, agricultural pest-management practices often lead to altered food web structure and communities dominated by a few common species, which together contribute to pest outbreaks. Here we show that organic farming methods mitigate this ecological damage by promoting evenness among natural enemies. In field enclosures, very even communities of predator and pathogen biological control agents, typical of organic farms, exerted the strongest pest control and yielded the largest plants. In contrast, pest densities were high and plant biomass was low when enemy evenness was disrupted, as is typical under conventional management. Our results were independent of the numerically dominant predator or pathogen species, and so resulted from evenness itself. Moreover, evenness effects among natural enemy groups were independent and complementary. Our results strengthen the argument that rejuvenation of ecosystem function requires restoration of species evenness, rather than just richness. Organic farming potentially offers a means of returning functional evenness to ecosystems.

Biological activity of ethanolic extract fractions of Dracaena arborea against infestation of stored grains by two storage insect pests.

As part of on-going efforts to use eco-friendly alternatives to chemical pesticides, ethanolic extract of dried leaves of Dracaena arborea (Willd.) Link (Dragon tree; Dracaenaceae) dissolved in distilled water and partitioned between equal volumes of n-hexane, chloroform, ethyl acetate and butanol was assessed in the laboratory against infestation by Sitophillus zeamais Motsch. and Callosobruchus maculatus Walp. in stored maize and cowpea, respectively. One hundred grams each of maize grains and cowpea seeds were treated with 400 mg kg(-1) of each extract fraction to evaluate contact toxicity, damage assessment, effect on eggs and immature stages and progeny production in both insect species. Contact toxicity by topical application, toxicity upon filter paper application and repellency using area preference method were carried out on the two insect species. Results showed that the extract fraction caused significant (p < or = 0.05) mortality of both insect pests with a high residual contact activity against S. zeamais. Grain damage was significantly (p < or = 0.01) reduced, while progeny production and development of eggs within grains were inhibited. The extract fractions evoked a strong repellent action against S. zeamais but moderate action against C. maculatus. The full potentials of using extract fractions of D. arborea as grain protectant against infestation by insect pests is discussed.

Host plant choice experiments of Colorado potato beetle (Coleoptera: Chrysomelidae) in Virginia.

Field and laboratory-choice experiments were conducted to understand aspects of host plant orientation by the Colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), in Virginia. In laboratory bioassays, L. decemlineata oriented to volatiles emitted by potato, Solanum tuberosum L., foliage over both tomato, Lycopersicon esculentum L., and eggplant, Solanum melongena L., foliage, and eggplant over tomato foliage, all of which had been mechanically damaged. Field choice tests revealed more L. decemlineata adults, larvae, and egg masses on eggplant than on tomato. In other experiments, counts of live L. decemlineata on untreated paired plants and counts of dead beetles on imidacloprid-treated plants did not differ between potato and eggplant. L. decemlineata was significantly attracted to eggplant over both tomato and pepper. To determine whether feeding adults affected orientation to host plants, an imidacloprid-treated eggplant or potato plant was paired with an untreated eggplant or potato plant covered in a mesh bag containing two adult male beetles. Significantly more adults were attracted to eggplant with feeding male beetles paired with another eggplant than any other treatment combination. These results indicate that the presence of male L. decemlineata on plants affects host plant orientation and suggests that the male-produced aggregation pheromone may be involved.

Frutos de café "Conilon" brocados por Hypothenemus hampei (Ferrari) (Coleoptera: Scolytidae): qual a importância de sua queda no decorrer da fase de frutificação? / \"Conilon\" coffee berries bored by Hypothenemus hampei (Ferrari) (Coleoptera: Scolytidae): what matters if they drop down during the fruiting phase?

A queda dos frutos brocados por Hypothenemus hampei (Ferrari) pode ser o principal fator de perda durante a frutificação do café. Entretanto, apenas os frutos brocados que permanecem no solo antes de um novo período de frutificação têm sido reconhecidos como causadores de impacto no nível de broqueamento de frutos em formação. Neste trabalho, investigou-se, ao longo do período de frutificação, a presença, nas plantas e no solo, de frutos de Coffea canephora cv. Conilon brocados por H. hampei, em Ouro Preto d'Oeste, RO. As coletas foram realizadas, semanalmente, entre dezembro de 2000 e junho de 2001. Os dados obtidos foram submetidos à análise de regressão de sobrevivência, baseado no modelo de Weibull. Durante o período de frutificação do café, a queda dos frutos ocorre continuamente e a presença de frutos brocados por H. hampei chega a ser, em média, 4 a 20 vezes maior no solo (P < 2,3x10-18, n = 62747) do que nas plantas. Argumenta-se que a incorporação do "ambiente solo" na determinação de ações de manejo integrado possa apontar novas tecnologias para o controle da broca.

Falling of berries bored by Hypothenemus hampei (Ferrari) may be the major loosing factor during the fruiting period. However, only those bored berries which remain in the soil surface before a new yielding period have been recognized as responsible for the damage level Ho achieved by new developing berries. In this paper, we investigated in the plants and in the soil surface, the presence of Coffea canephora cv. Conilon berries bored by H. hampei during the yielding period in Ouro Preto d'Oeste, Rondônia, Brazil. We took samples, weekly, from December 2000 to June 2001. The data were submitted to the Surviving Regression Analysis, based on a censored Weibull model. During the yielding period, berries fall down continuously and, in average, the proportion of H. hampei bored berries was 4 to 20 times higher in the soil (P < 2,3x10-18, n = 62,747) than in the plants. Thus, we argue that adding the "soil environment" to the integrated management strategies could point to new technologies for the control of this insect.

Colorado potato beetle toxins revisited: evidence the beetle does not sequester host plant glycoalkaloids.

The Colorado potato beetle feeds only on glycoalkaloid-laden solanaceous plants, appears to be toxic to predators, and has aposematic coloration, suggesting the beetle may sequester alkaloids from its host plants. This study tested 4th instars and adults, as well as isolated hemolymph and excrement, to determine if the beetles sequester, metabolize, or excrete alkaloids ingested from their host plants. HPLC analysis showed: that neither the larvae nor the adults sequestered either solanine or chaconine from potato foliage; that any alkaloids in the beetles were at concentrations well below 1 ppm; and that alkaloids were found in the excrement of larvae at approximately the same concentrations as in foliage. Analysis of alkaloids in the remains of fed-upon leaflet halves plus excreta during 24 hr feeding by 4th instars, as compared to alkaloids in the uneaten halves of the leaflets, showed that equal amounts of alkaloids were excreted as were ingested. The aposematic coloration probably warns of a previously-identified toxic dipeptide instead of a plant-derived alkaloid, as the Colorado potato beetle appears to excrete, rather than sequester or metabolize, the alkaloids from its host plants.

A weevil pollinating the Canary Islands date palm: between parasitism and mutualism.

Palm pollination systems are highly diverse, including by wind and by several different groups of insects. Many palm species are associated with more or less specific pollinating weevils that are also floral herbivores. For many such palms, the importance of these "palm flower weevils" as pollinators has not been examined. Here we describe a new ex situ method of demonstrating insect pollination when pollinator exclusion is not possible. We show that Neoderelomus piriformis beetles carry pollen and deposit it on the stigma of Phoenix canariensis. Up until now, pollination systems in Phoenix have been unclear, despite the economic importance of the date palm P. dactylifera. We demonstrate here that small weevils that visit inflorescences and often inconspicuously hide there could be efficient pollinators.

Feeding response of Ips paraconfusus to phloem and phloem metabolites of Heterobasidion annosum-inoculated ponderosa pine, Pinus ponderosa.

In studies of feeding by the bark beetle, Ips paraconfusus, two pine stilbenes (pinosylvin and pinosylvin methyl ether), ferulic acid glucoside, and enantiomers of the four most common sugars present in ponderosa pine phloem (sucrose, glucose, fructose, and raffinose) did not stimulate or reduce male feeding when assayed on wet alpha-cellulose with or without stimulatory phloem extractives present. When allowed to feed on wet alpha-cellulose containing sequential extracts (hexane, methanol, and water) of ponderosa pine phloem, methanol and water extractives stimulated feeding, but hexane extractives did not. Males confined in wet alpha-cellulose containing aqueous or organic extracts of culture broths derived from phloem tissue and containing the root pathogen. Heterobasidion annosum, ingested less substrate than beetles confined to control preparations. In an assay using logs from uninoculated ponderosa pines, the mean lengths of phloem in the digestive tracts increased as time spent feeding increased. Males confined to the phloem of basal logs cut from ponderosa pines artificially inoculated with H. annosum ingested significantly less phloem than beetles in logs cut from trees that were (combined) mock-inoculated or uninoculated and did not contain the pathogen. However, individual pathogen-containing treatments were not significantly different from uninoculated controls. It was concluded that altered feeding rates are not a major factor which may explain why diseased ponderosa pines are colonized by I. paraconfusus.

Resistance of legume seeds to the bruchid, Callosobruchus maculatus: metabolites relationship.

A study was initiated to categorize the seeds of various wild and cultivar legume varieties on the basis of their relative resistance to the bruchid, C. maculatus, and to correlate the important primary and secondary metabolites (non-protein anti-metabolites) in these seeds to the developmental parameters of the bruchid. In general, the wild seed varities showed greater amount of resistance to the bruchid attack when compared to that of the cultivar varieties. All the cultivar varieties studied showed higher amounts of primary metabolites, namely, proteins, carbohydrates, lipids and free amino acids thus showing a positive correlation between the primary metabolites content and the infestation rate. The wild varieties, however, showed significantly lower amounts of these primary metabolites and consequently a lower level of infestation. The non-protein anti-metabolites such as total phenols, ortho- dihydroxy phenols and tannis were significantly lower in the cultivars. The wild varieties, in contrast, revealed higher amounts of these secondary metabolites showing a negative correlation between these secondary metabolites content and the infestation rate. The study revealed that these non-protein anti-metabolites are important in conferring resistance to the seeds.