Eicosanoid Signaling in Insects: from Discovery to Plant Protection

Prostaglandins (PGs) and related eicosanoids are signal moieties derived from arachidonic acid and two other C20 polyunsaturated fatty acids. They were discovered in the 1930s in the context of mammalian reproductive physiology; PGs were associated with the prostate gland, hence their name, and they stimulate uterine smooth muscle contraction. Determining PG chemical structures in the early 1960s and demonstrating that they mediate many human pathophysiological events in the 1970s stimulated intensive research over the following decades in universities, governments and the private sector. Interest in the biological significance of PGs in insects arose in the 1970s and 1980s, which opened a new research frontier. PGs act in reproduction, releasing egg-laying behaviors in some species and signaling egg-maturation events in the Drosophila and silk moth models. They act in insect immunity, mediating and coordinating cellular and humoral responses to wounds, infection and invasion. PGs act in ion transport physiology in insect Malpighian tubules and recta. These compounds also mediate physiological trade-offs between insect immunity and reproduction. Finally, they are central players in the molecular ecology of interactions between blood-feeding insects and their vertebrate hosts. Some PG functions are critical at specific, crucial moments in insect lives, moments we consider ‘emergencies,’ such as the immediate reactions to infection. Certain microbial species have keyed into insect PG signaling and they evolved mechanisms to disable insect immune reactions to infection by inhibiting key enzymes in PG biosynthesis. We provide proof-of-principle that RNA interference treatments designed to silence genes in PG signaling disrupts insect immunity. In this review we describe the history, chemistry and biology of PGs. We use this background to argue that because PGs and other eicosanoids act in emergency situations, they are visible targets for development and deployment of novel insect pest management technologies.     

Eicosanoids act in nodulation reactions to bacterial infections in newly emerged adult honey bees, Apis mellifera, but not in older foragers

Nodulation is the first, and qualitatively predominant, cellular defense reaction to bacterial infections in insects. We tested the hypothesis that eicosanoids also mediate nodulation reactions to bacterial challenge in adults of a social insect, the honey bee, Apis mellifera. Treating newly-emerged experimental bees with the eicosanoid biosynthesis inhibitor, dexamethasone, impaired nodulation reactions to bacterial infections, and the influence of dexamethasone was reversed by treating infected insects with arachidonic acid, an eicosanoid precursor. Several other eicosanoid biosynthesis inhibitors, including the cyclooxygenase inhibitor, indomethacin, and the dual cyclooxygenase/lipoxygenase inhibitor, phenidone, also impaired the ability of experimental honeybees to form nodules in reaction to bacterial challenge. The influence of phenidone on nodulation was expressed in a dose-dependent manner. However, in experiments with older honey bees foragers, similar bacterial challenge did not evoke nodulation reactions. We infer from our results that while eicosanoids mediate cellular immune responses to bacterial infections in newly emerged honey bees, and more broadly, in most insect species, nodulation reactions to bacterial challenge probably do not occur in all phases of insect life cycles.     

Developmental modulation of immunity: changes within the feeding period of the fifth larval stage in the defence reactions of Manduca sexta to infection by Photorhabdus

In insect pathogen interactions, host developmental stage is among several factors that influence the induction of immune responses. Here, we show that the effectiveness of immune reactions to a pathogen can vary markedly within a single larval stage. Pre-wandering fifth-stage (day 5) larvae of the model lepidopteran insect Manduca sexta succumb faster to infection by the insect pathogenic bacterium Photorhabdus luminescens than newly ecdysed fifth-stage (day 0) caterpillars. The decrease in insect survival of the older larvae is associated with a reduction in both humoral and cellular defence reactions compared to less developed larvae. We present evidence that older fifth-stage larvae are less able to over-transcribe microbial pattern recognition protein and antibacterial effector genes in the fat body and hemocytes. Additionally, older larvae show reduced levels of phenoloxidase (PO) activity in the cell-free hemolymph plasma as well as a dramatic decrease in the number of circulating hemocytes, reduced ability to phagocytose bacteria and fewer melanotic nodules in the infected tissues. The decline in overall immune function of older fifth-stage larvae is reflected by higher bacterial growth in the hemolymph and increased colonization of Photorhabdus on the basal surface of the insect gut. We suggest that developmentally programmed variation in immune competence may have important implications for studies of ecological immunity.     

How Drosophila combats microbial infection: a model to study innate immunity and host-pathogen interactions

During the past year, dramatic progress has been achieved in our understanding of Drosophila immune reactions. The completion of the Drosophila genome sequencing project, microarray analysis and the use of genetic screens have led to the identification of several new genes required to combat microbial infection, filling in some important gaps in the understanding of innate immunity. At the same time, this insect was used as a model for the study of host-pathogen interactions. The recent major advances on the mechanisms by which this insect defends itself against intrusion of pathogens are discussed in this review.     

Insect cellular reactions to the lipopolysaccharide component of the bacterium Serratia marcescens are mediated by eicosanoids

Nodulation, which begins with the formation of cellular microaggregates, is the predominant cellular defense reaction to bacterial infections in insects. We suggested that these reactions to bacterial infections are mediated by eicosanoids. The lipopolysaccharide (LPS) component of some bacterial cells stimulates defense reactions in mammals and insects. Here, we report on experiments designed to test the hypothesis that eicosanoids mediate microaggregation reactions to LPS. Injections of LPS (purified from the bacterium, Serratia marcescens) into larvae of the tenebrionid beetle, Zophobas atratus, stimulated microaggregation reactions in a dose-dependent manner. Treatments with eicosanoid-biosynthesis inhibitors immediately prior to LPS challenge sharply reduced the microaggregation responses. Separate treatments with specific inhibitors of phospholipase A(2), cyclooxygenase and lipoxygenase reduced microaggregation, supporting our view that microaggregate formation involves lipoxygenase and cyclooxygenase products. The inhibitory influence of dexamethasone was apparent within 30min after injection, and microaggregation was significantly reduced, relative to control insects, over the following 90min. The dexamethasone effects were reversed by treating LPS-injected insects with the eicosanoid precursor, arachidonic acid. These findings indicate that cellular defense reactions to a specific component of bacterial cells are mediated by eicosanoids, and open up new possibilities for dissecting detailed hemocytic actions in insect immune reactions to bacterial infections.     

Utilization of glucose and amino acids in insect cell cultures: Quantifying the metabolic flows within the primary pathways and medium development

The current understanding of insect cell metabolism is very limited. In order to gain some insight into the growth and metabolism of insect cells Spodoptera frugiperda (Sf9), a comprehensive characterization of culture conditions for cells grown in the IPL-41 medium was made by measuring the amino acid composition of the growth medium and the cell extract, the macromolecular composition of the cells (DNA, RNA, and protein), medium concentrations of various metabolites and sugars, and the evolved CO(2). Since in the IPL-41-based serum-free medium all of the amino acids except cysteine are in great excess of what is needed by the cells for energy and protein production, a medium formulation with an osmolarity similar to the IPL-41 but with a lower amino acid content than IPL-41 was also developed. The new medium also lacks maltose and sucrose (contains only glucose), supported cell growth to a high cell density of 8 x 10(6) cells/mL. The cellular and energetic yields indicated that a tight coupling between the biosynthetic and energetic reactions was attained for cells grown in the new medium. Moreover, it was found that the intermittent feeding of glucose may not be required as the cell yield and growth rate were comparable whether the same total amount of glucose was provided intermittently or was included initially in the medium. The eventual cessation of growth in the new medium is believed to be due to the amino acid limitation because concentrations of both glutamine and glutamate were very low at the end of the growth phase. Thus, further optimization, which may include higher initial glutamine in the medium or its intermittent feeding, could lead to a further increase in the cell density. Finally, a stoichiometrically based analysis of metabolic reactions confirmed the operation of the key pathways and was used to quantify the distribution of metabolites among primary metabolic reactions. The quantitative flow values were used to highlight some key aspects of insect cell metabolism. (c) 1993 John Wiley & Sons, Inc.     

Eicosanoids mediate melanotic nodulation reactions to viral infection in larvae of the parasitic wasp, Pimpla turionellae

Nodulation is the quantitatively predominant insect cellular immune function activated in response to bacterial, fungal and some viral infections. We posed the hypothesis that parasitoid insects express melanotic nodulation reactions to viral challenge and that eicosanoids mediate these reactions. Treating fifth-instar larvae of the ichneumonid endoparasitoid Pimpla turionellae with Bovine Herpes Simplex Virus-1 (BHSV-1) induced nodulation reactions in a challenge dose-dependent manner. Experimental larvae treated with the cyclooxygenase inhibitor, indomethacin, the lipoxygenase inhibitor, esculetin, and the phospholipase A2 inhibitor, dexamethasone, resulted in severely impaired nodulation reactions to our standard BHSV-1 challenge dose. The immunoinhibitory influence of dexamethasone was reversed in larvae reared on culture medium amended with arachidonic acid, the fatty acid precursor of eicosanoid biosynthesis. Larvae that had been reared on media amended with indomethacin, esculetin, or dexamethasone were also compromised in their nodulation reactions to viral challenge. The influence of the orally administered pharmaceutical was expressed in a dose-dependent manner. Finally, wasp larvae reared in the presence of indomethacin and dexamethasone expressed significantly decreased levels of phenoloxidase activity in response to viral challenge. These findings draw attention to the idea that endoparasitoid insects express cellular immune reactions to viral challenge; they also support our hypothesis that eicosanoids mediate nodulation reactions to viral challenge in these highly specialized insects.     

Insect hemolymph clotting: evidence for interaction between the coagulation system and the prophenoloxidase activating cascade

Here we describe a novel approach to isolate proteins involved in insect hemolymph coagulation. In order to avoid problems in purifying clot proteins after they had been crosslinked, we performed an in vitro coagulation reaction with cell-free hemolymph from the lepidopteran Galleria mellonella and used the resulting complexes to produce a specific antiserum. The antiserum reacted with a subset of hemolymph proteins as well as with granular cells, but not with other hemocyte types of Galleria. Screening expression libraries identified some positive clones, which turned out to code for some previously characterized components of immune cascades, as well as some novel candidates for clotting factors. Known components include members of both the coagulation system and the prophenol-activating cascade, lending support to the idea that both systems work together during the formation of a hemolymph clot. Novel candidates for insect clotting factors include a mucin-like protein, a glutathione-S-transferase, and a distant member of the alpha-crystallin/small heat shock protein family. Using assays measuring the activity of transglutaminase, a key enzyme in clotting reactions in both vertebrates and invertebrates, we found a partial overlap between transglutaminase substrates and proteins recognized by the antiserum against the in vitro-induced clot.     

The bacterium Xenorhabdus nematophilus depresses nodulation reactions to infection by inhibiting eicosanoid biosynthesis in tobacco hornworms,Manduca sexta

The bacterium, Xenorhabdus nematophilus, is a virulent insect pathogen. We tested the hypothesis that this bacterium impairs insect cellular immune defense reactions by inhibiting biosynthesis of eicosanoids involved in mediating cellular defense reactions. Fifth instar tobacco hornworms, Manduca sexta, produced melanized nodules in reaction to challenge with living and heat-killed X. nematophilus. However, the nodulation reactions were much attenuated in insects challenged with living bacteria (approximately 20 nodules/larva for living bacteria vs. approximately 80 nodules/larva in insects challenged with heat-killed bacteria). The nodule-inhibiting action of living X. nematophilus was due to a factor that was present in the organic, but not aqueous, fraction of the bacterial cultural medium. The nodule-inhibiting factor in the organic fraction was labile to heat treatments. The immunodepressive influence of the factor in the organic fraction was reversed by treating challenged hornworms with arachidonic acid. The factor also depressed nodulation reactions to challenge with the plant pathogenic bacteria, Pseudomonas putida and Ralstonia solanacearum. These findings indicate that one or more factors from X. nematophilus depress nodulation reactions in tobacco hornworms by inhibiting eicosanoid biosynthesis.     

Hyposensitisation to wasp venom in six hours.

Eleven patients with a history of anaphylaxis, positive reactions to skin tests, and specific IgE antibody to wasp venom underwent hyposensitisation in a six hour procedure. No general reactions occurred. Complement activation and proteinuria could not be shown. The patterns of specific IgE, IgG1, and IgG4 were as described in other procedures--namely, IgE increased sharply and then decreased; IgG1 and IgG4 increased steadily and then decreased--but increase and decrease came earlier. Challenge by a stinging insect at least four weeks after treatment proved complete protection. The skin reactivity two years later showed an unpredictable pattern.     

High basal defense gene expression determines sorghum resistance to the whorl‐feeding insect southwestern corn borer

Southwestern corn borer (SWCB, Diatraea grandiosella) and fall armyworm (FAW, Spodoptera frugiperda) are major pests of sorghum in the southern United States. Host plant resistance is a desirable means for reducing plant damage and yield losses from both insects. In this study, we evaluated 12 sorghum lines for whorl‐stage resistance to leaf‐feeding SWCB and FAW in greenhouse and laboratory bioassays. Differential plant responses were detected against the two insects. Among 12 lines tested, CM1821, Della and PI196583 were resistant to both insects, while BTx2752 was largely susceptible. Line R.09110 was resistant to SWCB, but susceptible to FAW, whereas Redbine‐60 was susceptible to SWCB, but not to FAW. In addition, we quantified various chemical components in the plants and determined their association with insect resistance. Tannin and chlorophyll in leaves did not show any significant correlation with resistance to either insects, but contents of soluble protein in general were negatively correlated with resistance to both insects. Endogenous soluble sugar and dhurrin were only positively correlated with resistance to SWCB, but not with FAW resistance. To gain some molecular insight into resistance mechanism of sorghum to SWCB, we performed qPCR reactions for key genes encoding enzymes involved in dhurrin and jasmonic acid (JA) biosynthesis on selected resistant or susceptible lines. Although these genes were rapidly and strongly induced by insect feeding in all lines, the observed resistance is likely explained by higher constitutive dhurrin contents in some resistant lines and higher basal JA biosynthesis in others. Our results suggest that sorghum utilizes multiple strategies to defend itself against SWCB.     

Eicosanoids mediate Galleria mellonella cellular immune response to viral infection

Nodulation is the predominant insect cellular immune response to bacterial and fungal infections and it can also be induced by some viral infections. Treating seventh instar larvae of greater wax moth Galleria mellonella with Bovine herpes simplex virus-1 (BHSV-1) induced nodulation reactions in a dose-dependent manner. Because eicosanoids mediate nodulation reactions to bacterial and fungal infection, we hypothesized that eicosanoids also mediate nodulation reactions to viral challenge. To test this idea, we injected G. mellonella larvae with indomethacin, a nonsteroidal anti-inflammatory drug immediately prior to intrahemocoelic injection of BHSV-1. Relative to vehicle-treated controls, indomethacin-treated larvae produced significantly reduced numbers of nodules following viral infection (down from approximately 190 nodules/larva to <50 nodules/larva). In addition to injection treatments, increasing dietary indomethacin dosages (from 0.01% to 1%) were associated with decreasing nodulation (by 10-fold) and phenoloxidase activity (by 3-fold) reactions to BHSV-1 injection. We infer from these findings that cyclooxygenase products, prostaglandins, mediate nodulation response to viral infection in G. mellonella.     

The ecology and evolution of host-plant resistance to insects

Genetic techniques have yielded new insights into plant-herbivore coevolution. Quantitative genetic tests of herbivory theory reveal that in some cases insect herbivores impose selection on resistance traits. Also, some resistance traits are costly while others appear not to be, and genetic models can explain these results. Genetic variation in plant resistance influences insect community structure by modifying interactions of herbivores with competitors and natural enemies. Therefore, models of multispecies coevolution are more realistic than pairwise coevolutionary models. Ecological genetics will facilitate further theoretical and empirical exploration of multispecies coevolution of plants and herbivores.     

A literature review of insect responses to fire, compared to other conservation managements of open habitat

This literature review concerns insect responses to fire, compared to other feasible and appropriate conservation managements of open habitats. Many insect groups decline markedly immediately after fire, with the magnitude of reduction related to the degree of exposure to the flames and mobility of the insect. Niche diversity is lower in recently burned habitat, and the rate of insect increase following fire also relates to the species' ability to gain access to the regrowing vegetation. Postburn flora can be quite attractive to some recolonizing insects, possibly to some degree a result of fire-caused insect mortality which provides plants with short-term release from insect herbivory. Insect declines may follow immediately after mowing, but usually of lesser degree and shorter duration than after a fire of comparable timing and size. Season and scale of cutting may affect how much and which species showed positive or negative responses. Cut areas offer the vegetational structure and composition preferred by some insects, but cutting – or cutting at certain scales, seasons, or frequencies – may also be unfavorable for some species. Heavy grazing results in niche and assemblage simplification. Nonetheless, some invertebrates prefer the short turfs and bare ground resulting from heavier grazing. Other species vary in whether they peak in abundance and diversity in intermediate, light, or no grazing. In comparisons of mowing/haying and grazing regimes of similar compatibility with maintenance of the same habitat types, responses of particular species and species groups varied as to whether they had a preference for one or the other. Characteristics associated with insect responses to fire related to the degree of exposure to lethal temperature and stress experienced in the post-fire environment, suitability of post-treatment vegetation as habitat, and ability to rebuild numbers in the site (from survivors and/or colonizers). These factors appear equally useful for explicating insect responses to other managements such as haying, mowing, and grazing. By contrast, the assumption that the most habitat-restricted species will be most adapted to ecological forces believed to be prevalent in that ecosystem appears less efficacious for predicting insect management preferences.     

Insect immune responses to nematode parasites

Host innate immunity plays a central role in detecting and eliminating microbial pathogenic infections in both vertebrate and invertebrate animals. Entomopathogenic or insect pathogenic nematodes are of particular importance for the control of insect pests and vectors of pathogens, while insect-borne nematodes cause serious diseases in humans. Recent work has begun to use the power of insect models to investigate host-nematode interactions and uncover host antiparasitic immune reactions. This review describes recent findings on innate immune evasion strategies of parasitic nematodes and host cellular and humoral responses to the infection. Such information can be used to model diseases caused by human parasitic nematodes and provide clues indicating directions for research into the interplay between vector insects and their invading tropical parasites.     

转新型双价抗虫基因杨树对光肩星天牛及美国白蛾的抗性鉴定

为明确双价基因chitinase-BmkIT对鞘翅目害虫光肩星天牛及鳞翅目害虫美国白蛾的抗虫效果, 分别以转该双价基因的杨树茎干和叶片为材料,采用室内茎干接虫和叶片接虫方法鉴定其对光肩星天牛Anoplophora glabripennis和美国白蛾Hyphantria cunea幼虫的抗性。结果表明：转基因株系132-2,144-1,123-1对美国白蛾幼虫的致死作用和生长发育影响显著高于取食未转化叶片的处理（P＜0.05）,且对其后代产生了影响,表现为幼虫不能化蛹或化蛹后不能羽化或羽化不正常;但供试的8个转基因株系对光肩星天牛幼虫无明显的致死和生长抑制作用,表现为幼虫死亡率和体重均与取食非转化茎干幼虫无显著差异（P＞0.05）。结果提示,双价基因chitinase-BmkIT可作为杨树鳞翅目害虫的抗虫基因源,并且对延缓害虫抗性的产生具有一定的意义。     

Slaves of the environment: the movement of herbivorous insects in relation to their ecology and genotype

The majority of insect species do not show an innate behavioural migration, but rather populations expand into favourable new habitats or contract away from unfavourable ones by random changes of spatial scale. Over the past 50 years, the scientific fascination with dramatic long-distance and directed mass migratory events has overshadowed the more universal mode of population movement, involving much smaller stochastic displacement during the lifetime of the insects concerned. This may be limiting our understanding of insect population dynamics. In the following synthesis, we provide an overview of how herbivorous insect movement is governed by both abiotic and biotic factors, making these animals essentially ''slaves of their environment''. No displaced insect or insect population can leave a resource patch, migrate and flourish, leaving descendants, unless suitable habitat and/or resources are reached during movement. This must have constrained insects over geological time, bringing about species-specific adaptation in behaviour and movements in relation to their environment at a micro- and macrogeographical scale. With insects that undergo long-range spatial displacements, e.g. aphids and locusts, there is presumably a selection against movement unless overruled by factors, such as density-dependent triggering, which cause certain genotypes within the population to migrate. However, for most insect species, spatial changes of scale and range expansion are much slower and may occur over a much longer time-scale, and are not innate (nor directed). Ecologists may say that all animals and plants are figuratively speaking ''slaves of their environments'', in the sense that their distribution is defined by their ecology and genotype. But in the case of insects, a vast number must perish daily, either out at sea or over other hostile habitats, having failed to find suitable resources and/or a habitat on which to feed and reproduce. Since many are blown by the vagaries of the wind, their chances of success are serendipitous in the extreme, especially over large distances. Hence, the strategies adopted by mass migratory species (innate pre-programmed flight behaviour, large population sizes and/or fast reproduction), which improve the chances that some of these individuals will succeed. We also emphasize the dearth of knowledge in the various interactions of insect movement and their environment, and describe how molecular markers (protein and DNA) may be used to examine the details of spatial scale over which movement occurs in relation to insect ecology and genotype.     

寄主植物影响害虫药剂敏感性的研究进展

害虫取食不同寄主植物后,对杀虫剂的反应可归为3类：敏感性降低、增高和无明显变化。害虫对药剂的敏感性变化与不同植物中次生物质诱导激活/抑制昆虫体内相关解毒酶活性有关。这种诱导作用可受到植物营养、次生物质种类及其含量分布、害虫种类与发育阶段、以及环境温度等多种因素影响。经诱导的昆虫解毒酶对不同类型杀虫剂的代谢能力并不相同,进而导致对不同药剂的敏感性变化有明显差异。解毒酶系的诱导激活在害虫抗药性形成早期被认为有利于提高隐性抗性基因频率,从而可促进害虫抗药性的发展。最后,就寄主植物影响害虫对药剂敏感性在害虫治理中的应用作了探讨。     

Dissecting the immune response to the entomopathogen Photorhabdus

Bacterial pathogens either hide from or modulate the host's immune response to ensure their survival. Photorhabdus is a potent insect pathogenic bacterium that uses entomopathogenic nematodes as vectors in a system that represents a useful tool for probing the molecular basis of immunity. During the course of infection, Photorhabdus multiplies rapidly within the insect, producing a range of toxins that inhibit phagocytosis of the invading bacteria and eventually kill the insect host. Photorhabdus bacteria have recently been established as a tool for investigating immune recognition and defense mechanisms in model hosts such as Manduca and Drosophila. Such studies pave the way for investigations of gene interactions between pathogen virulence factors and host immune genes, which ultimately could lead to an understanding of how some Photorhabdus species have made the leap to becoming human pathogens.