Plastid Transformation of Micro-Tom Tomato with a Hemipteran Double-Stranded RNA Results in RNA Interference in Multiple Insect Species

Plant-mediated RNA interference (RNAi) holds great promise for insect pest control, as plants can be transformed to produce double-stranded RNA (dsRNA) to selectively down-regulate insect genes essential for survival. For optimum potency, dsRNA can be produced in plant plastids, enabling the accumulation of unprocessed dsRNAs. However, the relative effectiveness of this strategy in inducing an RNAi response in insects using different feeding mechanisms is understudied. To investigate this, we first tested an in vitro-synthesized 189 bp dsRNA matching a highly conserved region of the v-ATPaseA gene from cotton mealybug (Phenacoccus solenopsis) on three insect species from two different orders that use leaf-chewing, lacerate-and-flush, or sap-sucking mechanisms to feed, and showed that the dsRNA significantly down-regulated the target gene. We then developed transplastomic Micro-tom tomato plants to produce the dsRNA in plant plastids and showed that the dsRNA is produced in leaf, flower, green fruit, red fruit, and roots, with the highest dsRNA levels found in the leaf. The plastid-produced dsRNA induced a significant gene down-regulation in insects using leaf-chewing and lacerate-and-flush feeding mechanisms, while sap-sucking insects were unaffected. Our results suggest that plastid-produced dsRNA can be used to control leaf-chewing and lacerate-and-flush feeding insects, but may not be useful for sap-sucking insects.     

A simple and rapid method for DNA isolation from xylophagous insects

Published methods to isolate DNA from insects are not always effective in xylophagous insects because they have high concentrations of phenolics and other secondary plant compounds in their digestive tracts. A simple, reliable and labor-effective cetyltrimethylammonium bromide-polyvinylpyrrolidone (CTAB-PVP) method for isolation of high quality DNA from xylophagous insects is described. This method was successfully applied to PCR and restriction analysis, indicating removal of common inhibitors. DNA isolated by the CTAB-PVP method could be used in most molecular analyses.     

Reduction in diet breadth results in sequestration of plant chemicals and increases efficacy of chemical defense in a generalist grasshopper

The lubber grasshopper,Romalea guttata, is a generalist feeding on a broad diet of many herbaceous plant species and has a metathoracic defensive secretion normally containing phenolics and quinones synthesized by the insect. When insects were reared on a restricted diet of wild onion, they sequestered sulfur volatiles from the plant into their defensive secretions. These compounds were not detected by gas chromatography-mass spectroscopy in secretions of insects on an artificial diet or a natural, generalist diet of 26 plants that included wild onion as a component, nor were they present in secretions from field-collected insects. Defensive secretions of insects reared on wild onion were significantly more deterrent, by as much as an order of magnitude, to two species of ant predators than secretions from insects on either of the other two diets, despite a reduction in the concentration of autogenous defensive chemicals in secretions of insects on the onion diet. Sequestration of plant chemicals that increased defensive efficacy occurred when diet breadth was reduced. We suggest that this occurs because under conditions of specialization, plant secondary metabolites are more likely to be ingested and bioaccumulated in sufficient concentrations to have biological activity against predators. What we define as casual bioaccumulation of bioactive plant chemicals following dietary specialization may lead to evolution of sequestered defense syndromes in insects, and this process may not necessarily require specific adaptation to or coevolution with a toxic host plant.     

Identification and bioactivity of alarm pheromone in the western flower thrips,Frankliniella occidentalis

Analysis by gas chromatography (GC) and GC-mass spectroscopy disclosed that droplets of anal fluid produced by second-instar western flower thrips,Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), contain a two-component alarm pheromone, comprised of decyl acetate and dodecyl acetate, in a molar ratio of approximately 1.51. Both nymphs and adults responded to the pheromone by walking away from the source. The synthetic pheromone was active at a concentration of 1.0 ng, and the proportions of insects responding to the pheromone, but not the distances moved, increased with increasing dose. Each component was active alone, although at low doses, the response to decyl acetate was less than to either dodecyl acetate or the blend. The pheromone also induced some second instars to drop from leaves and reduced oviposition by adult females in both two-choice and nochoice experiments. Because the response of western flower thrips to the alarm pheromone is relatively weak, the potential for its use in pest management is limited, unless it is used in conjunction with other control measures.     

Pyrethrins Protect Pyrethrum Leaves Against Attack by Western Flower Thrips, <Emphasis Type="Italic">Frankliniella occidentalis</Emphasis>

Pyrethrins are active ingredients extracted from pyrethrum flowers (Tanacetum cinerariifolium), and are the most widely used botanical insecticide. However, several thrips species are commonly found on pyrethrum flowers in the field, and are the dominant insects found inside the flowers. Up to 80 % of western flower thrips (WFT, Frankliniella occidentalis) adults died within 3 days of initiating feeding on leaves of pyrethrum, leading us to evaluate the role of pyrethrins in the defense of pyrethrum leaves against WFT. The effects of pyrethrins on WFT survival, feeding behavior, and reproduction were measured both in vitro and in planta (infiltrated leaves). The lethal concentration value (LC50) for pyrethrins against WFT adults was 12.9 mg/ml, and pyrethrins at 0.1 % (w/v) and 1 % (w/v) had significantly negative effects on feeding, embryo development, and oviposition. About 20-70 % of WFT were killed within 2 days when they were fed chrysanthemum leaves containing 0.01-1 % pyrethrins. Chrysanthemum leaves containing 0.1 % or 1 % pyrethrins were significantly deterrent to WFT. In a no-choice assay, the reproduction of WFT was reduced significantly when the insects were fed leaves containing 0.1 % pyrethrins, and no eggs were found in leaves containing 1 % pyrethrins. Our results suggest that the natural concentrations of pyrethrins in the leaves may be responsible for the observed high mortality of WFT on pyrethrum.     

新品种丰花玫瑰鲜花细胞液提取率及化学成分分析初报

玫瑰是一种药食两用的芳香植物,丰花玫瑰是生产型玫瑰新品种,一年能多次开花,用其鲜花提取的细胞液,含有0.2%的玫瑰精油,具有天然的玫瑰花香,应用于化妆品和食品中,具有美容和保健作用。所用原料不同,细胞液的提取率不同;同一年不同花期的鲜花不仅细胞液提取率不同,其所含的化学成分亦不同。     

Physical Processes and Real-Time Chemical Measurement of the Insect Olfactory Environment

Odor-mediated insect navigation in airborne chemical plumes is vital to many ecological interactions, including mate finding, flower nectaring, and host locating (where disease transmission or herbivory may begin). After emission, volatile chemicals become rapidly mixed and diluted through physical processes that create a dynamic olfactory environment. This review examines those physical processes and some of the analytical technologies available to characterize those behavior-inducing chemical signals at temporal scales equivalent to the olfactory processing in insects. In particular, we focus on two areas of research that together may further our understanding of olfactory signal dynamics and its processing and perception by insects. First, measurement of physical atmospheric processes in the field can provide insight into the spatiotemporal dynamics of the odor signal available to insects. Field measurements in turn permit aspects of the physical environment to be simulated in the laboratory, thereby allowing careful investigation into the links between odor signal dynamics and insect behavior. Second, emerging analytical technologies with high recording frequencies and field-friendly inlet systems may offer new opportunities to characterize natural odors at spatiotemporal scales relevant to insect perception and behavior. Characterization of the chemical signal environment allows the determination of when and where olfactory-mediated behaviors may control ecological interactions. Finally, we argue that coupling of these two research areas will foster increased understanding of the physicochemical environment and enable researchers to determine how olfactory environments shape insect behaviors and sensory systems.     

不同产地望春玉兰花蕾红外光谱及挥发性成分比较研究

采用红外光谱法、气相色谱-质谱联机分析法及文献对比法对不同产地的望春玉兰花蕾和从其中用水蒸气蒸馏提取出的挥发性成分进行了研究。结果表明,不同产地的望春玉兰花蕾化学成分有所不同,红外谱图显示,高频区的3 399 cm-1左右的强吸收波数,中频区2 000~1 200 cm-1,1 657和1 612 cm-1,指纹区1 200~700cm-1,1 037、932.86 cm-1和854.25、617 cm-1可作为区别不同产地辛夷的标准峰。其中1 657、1 612、1 037 cm-1的肩峰是有效成分含量的标志,可作为评价其质量的标准;气质联用分析和文献比较显示,正宗产地甘肃和河南产辛夷的挥发性成分中,相对质量分数较大的化合物主要是桉油精、α-松油醇、桧烯、松油烯-4-醇、桉醇类等,可用桉油精、松油醇两种主要药用成分的质量分数作为望春玉兰花蕾这一药物的质量评价标准;其他产地的桧烯、月桂烯的质量分数较大,可用桧烯、月桂烯、金合欢醇质量分数作为望春玉兰花蕾用作香料时的质量标准;GC分离时可用SE-54作分离柱。     

Variation in methylglucosinolate and insect damage toCleome serrulata (Capparaceae) along a natural soil moisture gradient

We tested the hypothesis that plant loss to insects, and thus the relative fitness of an annual, was inversely related to spatial variation in the concentration of its characteristic secondary compound, methylglucosinolate, a mustard oil precursor. We found that methylyglucosinolate concentrations decreased significantly and linearly from the dry to the wet end along short soil moisture gradients in dry shortgrass prairie. Both leaf damage and capsule predation increased from the dry to the wet end. Thus, the glucosinolate appears to function defensively. Plant growth and flower production were favored at the wet end of the gradient; yet plants in the wet portion of the gradient were also more vulnerable to significant insect damage. The net result was that seed production by individual plants after predation was actually greater in the drier, harsher half of the gradient. The outcome strongly suggests that environmentally related variation in secondary compound concentration along environmental gradients can mediate and influence host-plant population abundance and distribution.     

Collection of pheromone from atmosphere surrounding boll weevils,Anthonomus grandis

An effluvial method was developed to collect the pheromone, grandlure from actively calling male boll weevils,Anthonomus grandis Boheman. The adsorbant, Porapak Q (ethylvinylbenzene-divinylbenzene), was utilized to trap and concentrate the pheromone. Captured pheromone was desorbed from columns packed with Porapak Q by elution withn-pentane and quantified by capillary column gas-liquid chromatography. In recovery studies with known amounts of synthetic grandlure, we found that the amount of each pheromone component collected was a function of collection duration, elution volume, and initial concentration. This effluvial method was capable of recovering as much as 94.9% of a known quantity (80 μg) of grandlure. The chromatograms were free of extraneous peaks. In studies of insect-produced pheromone, the effluvial method was used to collect pheromone from the air space surrounding male boll weevils as they fed on flower buds from CAMD-E cotton. The quantity and quality of boll-weevil-produced pheromone was determined for days 6, 8, 10, 11, 12, 13, and 14 of boll weevil adulthood. The maximum quantity of natural pheromone was produced on day 13 (4.2 μg/weevil) with a pheromone component ratio of 2.41∶2.29∶0.95∶1 for components I, II, III, and IV, respectively. The effluvial method described in this report is an efficient method to collect and quantify boll weevil pheromone from the atmosphere surrounding actively calling insects. Other applications of this method are suggested.     

蜡梅花头香的富集及其化学成分研究

用顶空和不同吸附剂吸附法收集蜡梅花的头香,并用GC-MS法对其进行了化学成分研究。对比了不同吸附剂在相同条件下对蜡梅花头香的吸附量与吸附成分。结果表明,不同吸附剂吸附的头香量和化学成分不同。大孔树脂的吸附量是0.22 g,且不具有蜡梅花香味,是自身分解物;阳离子交换树脂、凹凸棒石、活性炭、分子筛吸附量分别是0.52、0.42、0.26、0.29 g,检测出的化学成分种类分别是27、24、12、10种,且与蜡梅花的香味相似;后4种吸附剂所吸附的头香总成分约39种。凹凸棒石和阳离子交换树脂是蜡梅花头香成分的适宜吸附剂。     

The PhytO3 Tech Crop Protection Technology for Microorganism and Insect Control using Ozone,UV, and Dipole-Electrical Air Jet Spray Technologies – Technical Basis and Possible Chemistries Involved

Plants can't walk away when they are attacked by pathogenic microorganisms and insects, or if they are exposed to any form of stress. They do not have a central nervous system that allows them to defend themselves or to ask for help! In the early part of the 20th century, it was discovered that growing plants can be stimulated to respond to stresses by developing a Systemic Acquired Resistance (SAR) to microorganisms and insects. During the last 50 years, and especially during the past decade, significant advances in this agronomical technology have been made. SARs have been proven to result from the application of many types of chemical formulations. Plants respond by generating their own chemicals internally that subsequently prevent attacks by microorganisms and insects. Recently it has been shown that sequential treatment of growing plants with (1) an aqueous spray of high voltage, pulsed negatively charged water, followed immediately with (2) a spray of ozonated water containing 8 mg/L of ozone generated from oxygen, and that followed immediately by (3) high energy UV-C radiation, also causes plants to develop SARs to microorganisms and insects, but without the use of chemicals. The primary advantages of this new ozone-UV-based technology are: (a) there are no harmful effects on the plants, (b) no toxic chemical residues remain on the plants, (c) the technology can be used in rainy weather, when crop protection is most necessary, (d) the technology is environmentally friendly (no chemical residues), and (e) the technology is cheaper for crop growers compared to current chemical approaches.     

Evidence for Behavioral Attractiveness of Methoxylated Aromatics in a Dynastid Scarab Beetle-Pollinated Araceae

Many plants attract their pollinators with floral scents, and these olfactory signals are especially important at night, when visual signals become inefficient. Dynastid scarab beetles are a speciose group of night-active pollinators, and several plants pollinated by these insects have methoxylated aromatic compounds in their scents. However, there is a large gap in our knowledge regarding the compounds responsible for beetle attraction. We used chemical analytical analyses to determine temporal patterns of scent emission and the composition of scent released from inflorescences of Philodendron selloum. The attractiveness of the main components in the scent to the dynastid scarab beetle Erioscelis emarginata, the exclusive pollinator of this plant, was assessed in field biotests. The amount of scent increased rapidly in the evening, and large amounts of scent were released during the activity time of the beetle pollinators. Inflorescences emitted a high number of compounds of different biosynthetic origin, among them both uncommon and also widespread flower scents. Methoxylated aromatic compounds dominated the scent, and 4-methoxystyrene, the most abundant compound, attracted E. emarginata beetles. Other compounds, such as (Z)-jasmone and possibly also the methoxylated aromatic compound 3,4-dimethoxystyrene increased the attractiveness of 4-methoxystyrene. Methoxylated aromatics, which are known from other dynastid pollinated plants as well, are important signals in many scarab beetles in a different context (e.g., pheromones), thus suggesting that these plants exploit pre-existing preferences of the beetles for attracting this group of insects as pollinators.     

Neutral sterol metabolism in insects

Since they are unable to biosynthesize sterols, many phytophagous and omnivorous insects satisfy their cholesterol requirement by side chain dealkylation of the C-24 alkyl group of dietary C₂₈ and C₂₉ phytosterols. However, not all insects that can dealkylate the phytosterol side chain produce cholesterol. In addition, certain insects,e.g., some Hymenoptera, Hemiptera, and Diptera, are unable to dealkylate the sterol side chain. Although C₂₇ ecdysteroids (molting hormones), which are biosynthesized from cholesterol, are the major ecdysteroids in most insects, many of those species that are unable to dealkylate phytosterols utilize campesterol as a precursor for the C₂₈ ecdysteroid makisterone A. The considerable diversity of steroid utilization between certain insect species makes it difficult to generalize about insect steroid biochemistry. The ability to disrupt certain unique aspects of steroid utilization and metabolism in insects might be exploited for developing new insect control technology. Based on a paper presented at the Symposium on Plant and Fungal Sterols: Biosynthesis, Metabolism and Function, held at the AOCS Annual Meeting, Baltimore, MD, April 1990.     

Insect defenses against phototoxic plant chemicals

In addition to avoidance strategies, insects may use one or more biochemical defenses against phototoxic plant constituents or the reactive forms of oxygen they generate. These biochemical defenses may include metabolism; excited state quenching; deactivation of singlet oxygen, superoxide, or free radicals; and destruction of reaction products. This article is a survey of the antioxidative enzymes and simpler molecules produced by insects and their possible roles in combating phototoxic chemicals.     

Floral synomone of a wild orchid,Bulbophyllum cheiri,lures Bactrocera fruit flies for pollination

The major fruit fly attractant component in the floral fragrance of Bulbophyllum cheiri (fruit fly orchid) is methyl eugenol (ME). In the lowland rain forest of Malaysia, the solitary and nonresupinate flowers of the fruit fly orchid attract only males of the ME-sensitive fruit fly species (Bactrocera carambolae, B. papayae, and B. umbrosa. During the morning, the fruit fly orchid flower is visited by many fruit flies, which can sometimes cover the whole flower. The number of visitors dwindles in the afternoon. Headspace analysis of the flower shows a high ME peak in the morning, a small one between 12:00 and 14:00 hr, and no detectable ME peak after 14:00 hr. The process of pollination in the wild is initiated by attraction of fruit flies to floral ME. The flower, with the aid of its specialized hinged see-saw lip (labellum), temporarily traps (<1 min) a fruit fly pollinator between its lip and column. Just prior to this, the fly is rewarded by the opportunity to feed on the floral attractant found on surfaces of petals, sepals, and lip. The pollinaria borne by two wild B. papayae males (caught on and near the fruit fly orchid flower) are identical in morphology and structure with those obtained from the flower. Many of the B. papayae males (17 of 22 analyzed) attracted to the fruit fly orchid already possessed both ME metabolites, trans-coniferyl alcohol and 2-allyl-4,5-dimethoxyphenol, in their rectal glands, indicating that they had previously consumed ME. In this orchid–fruit fly association, both organisms gain direct reproductive benefits: the orchid flower gets pollinated without having to offer nectar, while the fruit fly boosts its pheromone and defense system, as well as its sexual competitiveness by feeding on the ME produced by the flower.     

Foraging in the Dark – Chemically Mediated Host Plant Location by Belowground Insect Herbivores

Root-feeding insects are key components in many terrestrial ecosystems. Like shoot-feeding insect herbivores, they exploit a range of chemical cues to locate host plants. Respiratory emissions of carbon dioxide (CO(2)) from the roots is widely reported as the main attractant, however, there is conflicting evidence about its exact role. CO(2) may act as a 'search trigger' causing insects to search more intensively for more host specific signals, or the plant may 'mask' CO(2) emissions with other root volatiles thus avoiding detection. At least 74 other compounds elicit behavioral responses in root-feeding insects, with the majority (>80?%) causing attraction. Low molecular weight compounds (e.g., alcohols, esters, and aldehydes) underpin attraction, whereas hydrocarbons tend to have repellent properties. A range of compounds act as phagostimulants (e.g., sugars) once insects feed on roots, whereas secondary metabolites often deter feeding. In contrast, some secondary metabolites usually regarded as plant defenses (e.g., dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA)), can be exploited by some root-feeding insects for host location. Insects share several host location cues with plant parasitic nematodes (CO(2), DIMBOA, glutamic acid), but some compounds (e.g., cucurbitacin A) repel nematodes while acting as phagostimulants to insects. Moreover, insect and nematode herbivory can induce exudation of compounds that may be mutually beneficial, suggesting potentially significant interactions between the two groups of herbivores. While a range of plant-derived chemicals can affect the behavior of root-feeding insects, little attempt has been made to exploit these in pest management, though this may become a more viable option with diminishing control options.     

Enzymic adaptations in leaf-feeding insects to host-plant allelochemicals

Herbivorous insects have the capacity to develop behavioral, physiological, and biochemical resistance mechanisms in response to chemical selection pressures. Among natural insect-plant associations, there are several cases of target-site insensitivity to and enhanced metabolism of plant allelochemicals. There are also known instances of physiological defenses such as extra rapid excretion or storage of toxic compounds. Multiple defenses seem to be prevalent in natural insect-plant interactions that involve toxic compounds, possibly reflecting the long time these interactions have had to evolve compared to insect-synthetic insecticide interactions. Synthetic insecticides were introduced about 45 years ago. Until recently they have been used as single-active-component preparations. As such, they have been and are very effective in producing insect populations with enhanced detoxification ability and target-site insensitivity. Most insecticide-resistant insect populations have one major defense mechanism. This feature makes the synthetic insecticides very useful tools for studies of insect defenses against toxic chemicals. Information gained from studies with insecticides can shed light on the capabilities of insects to adapt to toxicants in their environment. In assessing the validity of work with synthetic insecticides for natural systems, the fundamental differences between these substances and allelochemicals, and in their presentation to the insects, must, however, be considered. The prevalence of multiple defenses and reliance on modified physiological processes in natural interactions may reflect different properties of the natural chemicals in being generally highly biodegradable and often less acutely toxic than synthetic insecticides. In many cases, the plant allelochemicals are presented to the insects as mixtures. It is, however, to be expected that pest insects will evolve effective multiple defenses against synthetic insecticides. About 20% of all resistant populations have already developed multiple defenses, in most cases combinations of enhanced metabolism and target-site insensitivity. This implies that current crop protection practices need to be modified to ensure the continued usefulness of synthetic insecticides. To achieve this, it is important to study intensively not only insect-insecticide interactions but also the interactions operating in natural insect-plant associations.     

Contemporary frontiers in insect semiochemical research

Recent advances in analytical chemistry coupled with more definitive behavioral analyses have allowed more rigorous identification of many insect pheromones. This, in turn, has increased our understanding of the roles of pheromones in mediating insect behavior. Other semiochemicals that mediate insect behavior include those that enable parasitic and predatory insects to locate their hosts or prey. These may be produced by the host insects or by the plant on which they feed. Additionally, there are pheromones and plant-produced seminochemicals that regulate insect oviposition, a critical phase in the life cycle of insects. The challenge to chemists and biologists is to explore these areas to find new environmentally safe methods to control insect pests. One of the newer strategies used to investigate these semiochemicals is the study of the biochemistry of pheromone production and semiochemical perception in insects. These types of studies may reveal weak links in these systems that can be exploited to develop new, more effective and environmentally safe control methods.     

Induction of Plant Volatiles by Herbivores with Different Feeding Habits and the Effects of Induced Defenses on Host-Plant Selection by Thrips

Induced plant responses to attack by chewing insects have been intensively studied, but little is known about plant responses to nonchewing insects or to attack by multiple herbivores with different feeding habits. We examined volatile emissions by tobacco, Nicotiana tabacum, in response to feeding by the piercing–sucking insect western flower thrips (WFT), Frankliniella occidentalis, the chewing herbivore Heliothis virescens, and both herbivores simultaneously. In addition, we examined the effects of herbivore-induced plant defenses on host-plant selection by WFT. Plants responded to thrips feeding by consistently releasing five compounds. Simultaneous feeding by WFT and H. virescens elicited the same 11 compounds emitted in response to caterpillar feeding alone; however, two compounds, α-humulene and caryophyllene oxide, were produced in greater amounts in response to simultaneous herbivory. In choice tests, thrips consistently preferred uninduced plants over all other treatments and preferred plants damaged by caterpillars and those treated with caterpillar saliva over those treated with caterpillar regurgitant. The results are consistent with a previous finding that caterpillar regurgitant induces the release of significantly more volatile nicotine than plants damaged by caterpillars or plants treated with caterpillar saliva. A repellent effect of nicotine on WFT was confirmed by encircling unwounded plants with septa releasing volatile nicotine. Our results provide the first direct evidence that thrips feeding induces volatile responses and indicates that simultaneous herbivory by insects with different feeding habits can alter volatile emissions. In addition, the findings demonstrate that induced plant responses influence host-plant selection by WFT and suggest that the induction of volatile nicotine may play a role in this process.