Characterization of Limonoids Isolated from the Fruits of Melia toosendan and Their Antifeedant Activity against Pieris rapae.

The fruits of Melia toosendan Sieb. et Zucc. (Meliaceae) are a source of bioactive limonoids that can be used as effective pesticides. In this study, two novel limonoids, 6-acetylsendanal and 6-ketocinamodiol, were isolated together with fourteen known compounds, namely four protolimonoids, six trichilin-class limonoids, and four C-seco limonoids. The structures of the new compounds were determined by extensive spectroscopic analyses (HR-ESI-MS, UV, IR, 1D and 2D NMR). The bioassay results revealed that eleven of the extracted limonoids exhibited interesting antifeedant activities against the larvae of Pieris rapae with AFC50 values in the range of 0.11-1.79 mm. Particularly, mesendanin H, with an AFC50 value of 0.11 mm, exhibited a higher activity than the positive control toosendanin. Information on new bioactive limonoids may provide further insight into M. toosendan as a source of bioactive components.

Potential impact of genetically modified Lepidoptera-resistant Brassica napus in biodiversity hotspots: Sicily as a theoretical model.

The general increase of the cultivation and trade of Bt transgenic plants resistant to Lepidoptera pests raises concerns regarding the conservation of animal and plant biodiversity. Demand for biofuels has increased the cultivation and importation of oilseed rape (Brassica napus L.), including transgenic lines. In environmental risk assessments (ERAs) for its potential future cultivation as well as for food and feed uses, the impact on wild Brassicaeae relatives and on non-target Lepidoptera should be assessed. Here we consider the potential exposure of butterflies as results of possible cultivation or naturalization of spilled seed in Sicily (Italy). Diurnal Lepidoptera, which are pollinators, can be exposed directly to the insecticidal proteins as larvae (mainly of Pieridae) through the host and through the pollen that can deposit on other host plants. Adults can be exposed via pollen and nectar. The flight periods of butterflies were recorded, and they were found to overlap for about 90% of the flowering period of B. napus for the majority of the species. In addition, B. napus has a high potential to hybridise with endemic taxa belonging to the B. oleracea group. This could lead to an exposure of non-target Lepidoptera if introgression of the Bt gene into a wild population happens. A rank of the risk for butterflies and wild relatives of oilseed rape is given. We conclude that, in environmental risk assessments, attention should be paid to plant-insect interaction especially in a biodiversity hotspot such as Sicily.

Evaluation of the potential exposure of butterflies to genetically modified maize pollen in protected areas in Italy.

Environmental impacts of genetically modified crops are mandatorily assessed during their premarket phase. One of the areas of concern is the possible impact on nontarget organisms. Crops expressing Cry toxins might affect Lepidoptera larvae living outside cultivated fields, through pollen deposition on wild plants, which constitute their food source. While pollen toxicity varies among different events, possible exposure of nontarget species depends on the agro-environmental conditions. This study was conducted in two protected areas in Italy, characterized by different climatic conditions, where many Lepidoptera species thrive in proximity to maize cultivations. To estimate the possible exposure in absence of the actual stressor (e.g., Cry1-expressing maize plants), we conducted a two-year field survey of butterflies and weeds. Indicator species were selected-Aglais (Inachis) io in the Northern site and Vanessa cardui in the Southern site-and their phenology was investigated. Pollen dispersal from maize fields was measured by collection in Petri dishes. Duration and frequency of exposure was defined by the overlap between pollen emission and presence of larvae on host plants. Different risk scenarios are expected in the two regions: highest exposure is foreseen for A. io in the Northern site, while minimal exposure is estimated for V. cardui in the Southern site. In the latter case, locally grown maize cultivars flower in mid-summer in coincidence with an aestivation period for several butterfly species due to hot and dry conditions. Moreover, host plants of V. cardui are at the end of their life cycle thus limiting food availability.

Inhibitory effects of an extract from non-host plants on physiological characteristics of two major cabbage pests.

The diamondback moth (Plutella xylostella) and small white cabbage butterfly (Pieris rapae) are the two main serious pests of cruciferous crops (Brassicaceae) that have developed resistance to chemical control methods. In order to avoid such resistance and also the adverse effects of chemical pesticides on the environment, alternative methods have usually been suggested, including the use of plant enzyme inhibitors. Here, the inhibitory effects of proteinaceous inhibitors extracted from wheat, canola, sesame, bean and triticale were evaluated against the digestive α-amylases, larval growth, development and nutritional indecs of the diamondback moth and small white cabbage butterfly. Our results indicated that triticale and wheat extracts inhibited α-amylolytic activity in an alkaline pH, which is in accordance with the moth and butterfly gut α-amylase optimum pH. Dose-dependent inhibition of two crucifer pests by triticale and wheat was observed using spectrophotometry and gel electrophoresis. Implementation of specificity studies showed that wheat and triticale-proteinaceous extract were inactive against Chinese and purple cabbage amylase. Triticale and wheat were resistant against insects' gut proteases. Results of the feeding bioassay indicated that triticale-proteinaceous extract could cause a significant reduction in survival and larval body mass. The results of the nutritional indecs also showed larvae of both species that fed on a Triticale proteinaceous inhibitor-treated diet had the lowest values for the efficiency of conversion of ingested food and relative growth rate. Our observations suggested that triticale shows promise for use in the management of crucifer pests.

Soil-applied imidacloprid translocates to ornamental flowers and reduces survival of adult Coleomegilla maculata, Harmonia axyridis, and Hippodamia convergens lady beetles, and larval Danaus plexippus and Vanessa cardui butterflies.

Integrated Pest Management (IPM) is a decision making process used to manage pests that relies on many tactics, including cultural and biological control, which are practices that conserve beneficial insects and mites, and when needed, the use of conventional insecticides. However, systemic, soil-applied neonicotinoid insecticides are translocated to pollen and nectar of flowers, often for months, and may reduce survival of flower-feeding beneficial insects. Imidacloprid seed-treated crops (0.05 mg AI (active ingredient) /canola seed and 1.2 mg AI/corn seed) translocate less than 10 ppb to pollen and nectar. However, higher rates of soil-applied imidacloprid are used in nurseries and urban landscapes, such as 300 mg AI/10 L (3 gallon) pot and 69 g AI applied to the soil under a 61 (24 in) cm diam. tree. Translocation of imidacloprid from soil (300 mg AI) to flowers of Asclepias curassavica resulted in 6,030 ppb in 1X and 10,400 ppb in 2X treatments, which are similar to imidacloprid residues found in another plant species we studied. A second imidacloprid soil application 7 months later resulted in 21,000 ppb in 1X and 45,000 ppb in 2X treatments. Consequently, greenhouse/nursery use of imidacloprid applied to flowering plants can result in 793 to 1,368 times higher concentration compared to an imidacloprid seed treatment (7.6 ppb pollen in seed- treated canola), where most research has focused. These higher imidacloprid levels caused significant mortality in both 1X and 2X treatments in 3 lady beetle species, Coleomegilla maculata, Harmonia axyridis, and Hippodamia convergens, but not a fourth species, Coccinella septempunctata. Adult survival were not reduced for monarch, Danaus plexippus and painted lady, Vanessa cardui, butterflies, but larval survival was significantly reduced. The use of the neonicotinoid imidacloprid at greenhouse/nursery rates reduced survival of beneficial insects feeding on pollen and nectar and is incompatible with the principles of IPM.

Trans-generational parasite protection associated with paternal diet.

Multiple generations of hosts are often exposed to the same pathogens, favouring the evolution of trans-generational defences. Because females have more opportunities to transfer protective molecules to offspring, many studies have focused on maternally derived protection. However, males of many species can transfer compounds along with sperm, including chemicals that could provide protection. Here, we assess maternally and paternally derived protection in a monarch butterfly-protozoan parasite system where parasite resistance is heavily influenced by secondary plant chemicals, known as cardenolides, present in the larval diet of milkweed plants. We reared monarch butterflies on medicinal and non-medicinal milkweed species and then measured resistance of their offspring to infection. We also measured cardenolide content in adult monarchs reared on the two species, and in the eggs that they produced. We found that offspring were more resistant to infection when their fathers were reared on medicinal milkweed, while maternal diet had less of an effect. We also found that eggs contained the highest levels of cardenolides when both parents were reared on the medicinal species. Moreover, females reared on non-medicinal milkweed produced eggs with significantly higher levels of cardenolides if they mated with males reared on the medicinal milkweed species. However, we found an equivocal relationship between the cardenolides present in eggs and parasite resistance in the offspring. Our results demonstrate that males reared on medicinal plants can transfer protection to their offspring, but the exact mechanism remains unresolved. This suggests that paternal protection from parasitism might be important, particularly when there are environmental sources of parasite resistance and when males transfer spermatophores during mating.

Effect of milk thistle, Silybium marianum, extract on toxicity, development, nutrition, and enzyme activities of the small white butterfly, Pieris rapae.

The methanolic extract of milk thistle, Silybium marianum L. (Asterales: Asteraceae), was investigated for its effects on the mortality, growth, feeding indices, enzymatic activity, and levels of non-enzymatic molecules of the small white butterfly, Pieris rapae L. (Lepidoptera: Pieridae), a pest of cruciferous plants. Feeding indices including approximate digestibility (AD), efficiency of conversion of digested food (ECD), efficiency of conversion of ingested food (ECI), relative growth rate (RGR), and relative consumption rate (RCR) were measured. These indices were variously affected: the RGR, RCR, and AD decreased, but the ECD and ECI increased. The LC50 and LC25 values were estimated as 2.94% and 1.20%, respectively. At the lowest concentration of S. marianum extract (0.625%), the feeding deterrence index was 40.48%. The duration of the pupal stage and the rate of larval growth decreased. These changes may be due to alterations in metabolic activity, such as the increase in alkaline phosphatase activity, which is likely involved in detoxification. Additionally, the activities of alanine aminotransferase and aspartate aminotransferase, which are key components of amino acid catabolism, decreased. The amount of glucose (an energy source) and uric acid (the excreted end product) increased, while total protein (another energy source) and cholesterol decreased. These results indicate that this plant possesses potential secondary metabolites that may be useful for the future study of the control of insect pests.

Chemosensory basis of behavioural plasticity in response to deterrent plant chemicals in the larva of the Small Cabbage White butterfly Pieris rapae.

Behavioural and electrophysiological responsiveness to three chemically different secondary plant substances was studied in larvae of Pieris rapae L. (Lepidoptera: Pieridae). Three groups of caterpillars were studied that during their larval development were exposed to different rearing diets: an artificial diet or one of two host-plants, cabbage, Brassica oleracea, or nasturtium, Tropaeolum majus. In dual-choice leaf disc assays, caterpillars reared on cabbage were strongly deterred by the phenolic chlorogenic acid, the flavonol glycoside naringin and the alkaloid strychnine. However, behavioural plasticity was found in caterpillars reared on nasturtium or artificial diet in that these did not discriminate against chlorogenic acid. Caterpillars reared on the artificial diet were also significantly less sensitive to naringin and strychnine in the behavioural assay. Electrophysiological studies of the maxillary sensilla styloconica revealed that the deterrent neuron in the medial sensillum, but not in the lateral sensillum, of cabbage-reared caterpillars was more sensitive than the same neuron type of caterpillars reared on nasturtium or artificial diet. We conclude that (1) the diet-induced behavioural habituation to deterrents can at least partly be explained by chemosensory desensitisation of a generalist type of maxillary deterrent neuron; (2) behavioural cross-habituation to the three structurally diverse deterrent compounds can be traced back to cross-sensitivity for these compounds in the same gustatory neuron.

Use of buckwheat seed protease inhibitor gene for improvement of tobacco and potato plant resistance to biotic stress.

The possibility to use agrobacterial transformation of leaf discs to produce resistance to bacterial infections in tobacco and potato plants by introduction of a single gene encoding the serine proteinase inhibitor BWI-1a (ISP) from buckwheat seeds is shown. All studied PCR-positive transgenic plants exhibited antibacterial activity in biotests. It was shown that the presence of just a single gene of serine proteinase inhibitor provides sufficient protection at least against two bacterial phytopathogens, Pseudomonas syringae pv. tomato and Clavibacter michiganensis sbsp. michiganensis. The biotest including tobacco plant infection by the white wings butterfly in the green house has also demonstrated the existence of protective effect in transgenic tobacco plants. Significant genotypic variations in the protection efficiency were found between members of different genera of the same family (potato and tobacco) as well as between different lines of the same species. Northern blot analysis of four transgenic potato lines and three tobacco lines transformed by a vector plasmid containing the ISP gene of serine proteinases BWI-1a from buckwheat seeds has shown the presence of the expected size mRNA transcript.

Spatial distribution of Aglais urticae (L.) and its host plant Urtica dioica (L.) in an agricultural landscape: implications for Bt maize risk assessment and post-market monitoring.

Over the past decades, genes of Bacillus thuringiensis var. kurstaki (Berliner) (Bt) coding for protein toxins have been engineered into maize for protection against the European Corn Borer (Ostrinia nubilalis (Hbn.)). However, these transgenic plants may have an impact on non-target organisms. In particular, a potential hazard was identified for non-target lepidopteran larvae, if they consume Bt maize pollen on their host plants. Risk can be defined as a function of the effect of an event (hazard) and the likelihood of this event occurring. Although data on toxicity (hazard) are available from many lab and field studies, knowledge about the environmental exposure of European lepidopteran larvae is incomplete at the population level. Therefore we studied the distribution of small tortoiseshell caterpillars (Aglais urticae (L.)) and its host plant in an agricultural landscape in Germany, to estimate the potential population exposure to maize pollen. The results showed that larvae of the small tortoiseshell developed primarily on freshly sprouted nettle stands (Urtica dioica (L.)) in field margins, rather than adjacent to hedges and groves. However, the main distribution was at margins of cereal (non-maize) fields, where 70% of all larvae were found. This may be due the fact that cereals covered 54% of the survey area, while maize only covered 6.1%. On the other hand, maize fields seem so show higher food plant densities than cereal crops. The results must be interpreted carefully, as the data basis of the present study is very small, and the situation can vary between years due to crop rotation or other changes in agricultural practices. Therefore it is still questionable whether the small tortoiseshell is significantly exposed to maize pollen. For a conclusive risk assessment, more replications and surveys of larger areas in different intensively managed agricultural landscapes over several years are needed.

Genotype-specific response of a lycaenid herbivore to elevated carbon dioxide and phosphorus availability in calcareous grassland.

Effects of elevated CO2 and P availability on plant growth of the legume Lotus corniculatus and consequences for the butterfly larvae of Polyommatus icarus feeding on L. corniculatus were investigated in screen-aided CO2 control chambers under natural conditions on a calcareous grassland in the Swiss Jura mountains. Elevated CO2 conditions and P fertilisation increased the biomass production of L. corniculatus plants and affected the plant chemical composition. CO2 enrichment increased the C/N ratio and sugar concentration and decreased the N and P concentrations. C- and N-based allelochemicals (cyanoglycosides, total polyphenols and condensed tannins) were only marginally affected by CO2 enrichment. P fertilisation increased the specific leaf area and concentrations of water, N, sugar and P, while the C/N ratio and the concentration of total polyphenols decreased. Furthermore, P availability marginally enhanced the effect of elevated CO2 on the total dry mass and sugar concentration while the opposite occurred for the total polyphenol concentration. The changes in food-plant chemistry as a result of P fertilisation positively affected larval mass gain and accelerated the development time of P. icarus. Only a marginal negative effect on larval mass gain was found for CO2 enrichment. However, we found genotype-specific responses in the development time of P. icarus to elevated CO2 conditions. Larvae originating from different mothers developed better either under elevated CO2 or under ambient CO2 but some did not react to CO2 elevation. As far as we know this is the first finding of a genotype-specific response of an insect herbivore to elevated CO2 which suggests genetic shifts in insect life history traits in response to elevated CO2.

Autecology of the tailed jay butterfly Graphium agamemnon (Lepidoptera : Rhopalocera : Papilionidae).

The Tailed Jay Graphium agamemnon is one of the attractive papilionid butterflies that enliven the environment of Visakhapatnam. It occurs throughout the year. It lays eggs singly on young leaves of the mast tree Polyalthia longifolia var. pendula (Annonaceae). The eggs take 3-4 days to hatch. The larvae go through 5 instars over a period of 15-16 days. The pupal period is 13-14 days. The total period from egg to adult emergence spans over 33-36 days. Based on this short life cycle, and larval and pupal development success studied every month, this butterfly can be multivoltine with a minimum of 7-8 broods in a year. Both CI and GR decreased with the age of larva, their average figures being 3.78 and 0.43 respectively. AD values are high (average 92%) and decreased through successive instars. Both ECD and ECI followed a similar pattern with an increase from instar I up to II, then a decrease up to IV and again an increase in instar V and the highest value is with fifth instar. Adults frequently visited flowers (12-35 flowers in a min) spending 1.0 to 3.2 seconds on a flower. The nectar concentration ranged between 16 and 58%. Peak foraging activity mostly fell between 0900-1000 h. The proboscis received pollen in most of the floral species visited, thus satisfying one of the characteristics of butterfly pollination. Being a fast and strong flier it is treated as "high energy" pollinator promoting cross-pollination.