Constitutive Activation of the Midgut Response to Bacillus thuringiensis in Bt-Resistant Spodoptera exigua

Bacillus thuringiensis is the most effective microbial control agent for controlling numerous species from different insect orders. The main threat for the long term use of B. thuringiensis in pest control is the ability of insects to develop resistance. Thus, the identification of insect genes involved in conferring resistance is of paramount importance. A colony of Spodoptera exigua (Lepidoptera: Noctuidae) was selected for 15 years in the laboratory for resistance to Xentari™, a B. thuringiensis-based insecticide, reaching a final resistance level of greater than 1,000-fold. Around 600 midgut ESTs were analyzed by DNA-macroarray in order to find differences in midgut gene expression between susceptible and resistant insects. Among the differentially expressed genes, repat and arylphorin were identified and their increased expression was correlated with B. thuringiensis resistance. We also found overlap among genes that were constitutively over-expressed in resistant insects with genes that were up-regulated in susceptible insects after exposure to Xentari™, suggesting a permanent activation of the response to Xentari™ in resistant insects. Increased aminopeptidase activity in the lumen of resistant insects in the absence of exposure to Xentari™ corroborated the hypothesis of permanent activation of response genes. Increase in midgut proliferation has been proposed as a mechanism of response to pathogens in the adult from several insect species. Analysis of S. exigua larvae revealed that midgut proliferation was neither increased in resistant insects nor induced by exposure of susceptible larvae to Xentari™, suggesting that mechanisms other than midgut proliferation are involved in the response to B. thuringiensis by S. exigua larvae.     

Divergent immune priming responses across flour beetle life stages and populations

Growing evidence shows that low doses of pathogens may prime the immune response in many insects, conferring subsequent protection against infection in the same developmental stage (within‐life stage priming), across life stages (ontogenic priming), or to offspring (transgenerational priming). Recent work also suggests that immune priming is a costly response. Thus, depending on host and pathogen ecology and evolutionary history, tradeoffs with other fitness components may constrain the evolution of priming. However, the relative impacts of priming at different life stages and across natural populations remain unknown. We quantified immune priming responses of 10 natural populations of the red flour beetle Tribolium castaneum, primed and infected with the natural insect pathogen Bacillus thuringiensis. We found that priming responses were highly variable both across life stages and populations, ranging from no detectable response to a 13‐fold survival benefit. Comparing across stages, we found that ontogenic immune priming at the larval stage conferred maximum protection against infection. Finally, we found that various forms of priming showed sex‐specific associations that may represent tradeoffs or shared mechanisms. These results indicate the importance of sex‐, life stage‐, and population‐specific selective pressures that can cause substantial divergence in priming responses even within a species. Our work highlights the necessity of further work to understand the mechanistic basis of this variability.     

昆虫免疫致敏研究进展

通常认为昆虫缺少获得性免疫(acquired immunity)且完全依赖天然免疫系统(innate immune defense system)来应对病原微生物的感染。然而越来越多的研究表明,昆虫等无脊椎动物早期的病原菌感染经历能够增强后期遭遇病原感染时的免疫力,这种现象称为免疫致敏(immune priming)。类似于脊椎动物的获得性免疫,一些昆虫在致敏后可以展现出极大程度的特异性和记忆性,致敏保护效应甚至可以达到种或菌株水平的特异性,并且可以跨代传递。昆虫在体内缺乏获得性免疫分子元件的基础上,仍然可以实现免疫的记忆性和特异性,说明昆虫的天然免疫系统存在独特的机制来调控该过程。本文综述了昆虫免疫致敏和跨代传递的研究进展,探讨了昆虫免疫致敏发生的特定条件及影响因素,并对昆虫免疫致敏和跨代传递的潜在调控机理进行了阐述。此外,免疫致敏本身可能是耗能的过程,本文也从致敏可塑的角度探讨了致敏反应的适应性代价。最后,对昆虫免疫致敏未来的研究方向以及在害虫防治中的应用前景进行了展望。     

Fitness in invasive social wasps: the role of variation in viral load,immune response and paternity in predicting nest size and reproductive output

Within any one habitat, the relative fitness of organisms in a population can vary substantially. Social insects like the common wasp are among the most successful invasive animals, but show enormous variation in nest size and other fitness‐related traits. Some of this variation may be caused by pathogens such as viruses that can have serious consequences in social insects, which range from reduced productivity to colony death. Both individual immune responses and colony‐level traits such as genetic diversity are likely to influence effects of pathogen infections on colony fitness. Here we investigate how infections with Kashmir Bee Virus (KBV), immune response and intracolony genetic diversity (due to queen polyandry) affect nest size in the invasive common wasp Vespula vulgaris. We show that KBV is highly prevalent in wasps and expression of antiviral immune genes is significantly increased with higher viral loads across individuals. Patriline membership within a nest did not influence KBV susceptibility or immune response. A permutational MANCOVA revealed that polyandry, viral load and expression of the immune gene Dicer were significant predictors of variation in nest size. High intracolony genetic diversity due to polyandry has previously been hypothesized to improve colony‐level resistance to parasites and pathogens. Consistent with this hypothesis, we observed genetically diverse colonies to be significantly larger and to produce more queens, although this effect was not driven by the pathogen we investigated. Invasive wasps clearly suffer from pathogens and expend resources, as indicated here by elevated immune gene expression, toward reducing pathogen‐impact on colony fitness.     

Non‐pathogenic aquatic bacteria activate the immune system and increase predation risk in damselfly larvae

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Transstadial immune activation in a mosquito: Adults that emerge from infected larvae have stronger antibacterial activity in their hemocoel yet increased susceptibility to malaria infection

Larval and adult mosquitoes mount immune responses against pathogens that invade their hemocoel. Although it has been suggested that a correlation exists between immune processes across insect life stages, the influence that an infection in the hemocoel of a larva has on the immune system of the eclosed adult remains unknown. Here, we used Anopheles gambiae to test whether a larval infection influences the adult response to a subsequent bacterial or malaria parasite infection. We found that for both female and male mosquitoes, a larval infection enhances the efficiency of bacterial clearance following a secondary infection in the hemocoel of adults. The adults that emerge from infected larvae have more hemocytes than adults that emerge from naive or injured larvae, and individual hemocytes have greater phagocytic activity. Furthermore, mRNA abundance of immune genes—such as cecropin A, Lysozyme C1, Stat‐A, and Tep1—is higher in adults that emerge from infected larvae. A larval infection, however, does not have a meaningful effect on the probability that female adults will survive a systemic bacterial infection, and increases the susceptibility of females to Plasmodium yoelii, as measured by oocyst prevalence and intensity in the midgut. Finally, immune proficiency varies by sex; females exhibit increased bacterial killing, have twice as many hemocytes, and more highly express immune genes. Together, these results show that a larval hemocoelic infection induces transstadial immune activation—possibly via transstadial immune priming—but that it confers both costs and benefits to the emerged adults.     

A plant pathogen modulates the effects of secondary metabolites on the performance and immune function of an insect herbivore

Host plant chemical composition critically shapes the performance of insect herbivores feeding on them. Some insects have become specialized on plant secondary metabolites, and even use them to their own advantage such as defense against predators. However, infection by plant pathogens can seriously alter the interaction between herbivores and their host plants. We tested whether the effects of the plant secondary metabolites, iridoid glycosides (IGs), on the performance and immune response of an insect herbivore are modulated by a plant pathogen. We used the IG‐specialized Glanville fritillary butterfly Melitaea cinxia, its host plant Plantago lanceolata, and the naturally occurring plant pathogen, powdery mildew Podosphaera plantaginis, as model system. Pre‐diapause larvae were fed on P. lanceolata host plants selected to contain either high or low IGs, in the presence or absence of powdery mildew. Larval performance was measured by growth rate, survival until diapause, and by investment in immunity. We assessed immunity after a bacterial challenge in terms of phenoloxidase (PO) activity and the expression of seven pre‐selected insect immune genes (qPCR). We found that the beneficial effects of constitutive leaf IGs, that improved larval growth, were significantly reduced by mildew infection. Moreover, mildew presence downregulated one component of larval immune response (PO activity), suggesting a physiological cost of investment in immunity under suboptimal conditions. Yet, feeding on mildew‐infected leaves caused an upregulation of two immune genes, lysozyme and prophenoloxidase. Our findings indicate that a plant pathogen can significantly modulate the effects of secondary metabolites on the growth of an insect herbivore. Furthermore, we show that a plant pathogen can induce contrasting effects on insect immune function. We suspect that the activation of the immune system toward a plant pathogen infection may be maladaptive, but the actual infectivity on the larvae should be tested.     

What physiological processes permit insects to eat Eucalyptus leaves?

Many species of insects eat Eucalyptus foliage despite its relatively low nutritional value and the many plant secondary metabolites (PSMs) present, for example, terpenes, phenols and formylated phloroglucinols (FPGs). Formylated phloroglucinols are a new class of PSMs that act as antifeedants for possums and koalas. What physiological processes are present that permit insects to eat eucalypt foliage and how do PSMs influence insect feeding or digestion? Some trees seem to be repeatedly infested with eucalypt‐feeding insects, possibly as a result of previous chemosensory cues remaining from parental selection of a plant. Avoidance or storage of PSMs permit jarrah leafminers (Perthida glyphopa) and sawflies (Perga sp.) to consume eucalypt foliage without dealing with the majority of these compounds. Some PSMs can be metabolized by polysubstrate membrane oxidases as found in caterpillars or sawflies that feed on eucalypts. High midgut pH may be advantageous for nutrient extraction and PSM metabolism, and midgut pH ranges between 8.5 and 8.9 for caterpillars of Hyalarcta huebneri. Plant secondary metabolites may not be absorbed as a result of the combined presence of the peritrophic matrix and endogenous surfactants. Excretion of PSMs can be as metabolites or intact compounds. Both putative metabolites and sideroxylonal‐A, an FPG, are present in the faeces of larvae of the case moth, H. huebneri. The presence of sideroxylonal‐A in the food had an effect on the presence of 5‐hydroxytryptamine (5HT) in the central nervous system of caterpillars, as larvae fed leaves with a high concentration of sideroxylonal‐A had relatively more 5HT in the brain and central nervous system ganglia than larvae fed leaves containing a low concentration. Further work is necessary to clarify how PSMs are handled by eucalypt‐feeding insects and what effect FPGs have on feeding and digestion.     

Challenges of metamorphosis in invertebrate hosts: maintaining parasite resistance across life‐history stages

1. Insects lack the acquired immune system of vertebrates, but there is some evidence that insect immunity can be primed against an encountered pathogen to mitigate the intensity of future infections within a life stage. 2. Many invertebrates have multiple life‐history stages separated by complete metamorphosis, but different life stages can often be infected by the same pathogens, and the potential loss of immune priming during metamorphosis could therefore have detrimental effects on the host. Evidence that invertebrate immune priming can persist through metamorphosis is still missing, and consequently it is unclear how host–parasite interactions change across different life‐history stages in the context of infection history. 3. By experimentally manipulating the infection history of the flour beetle Tribolium confusum, we show that intestinal gregarine parasite infections during the larval stage reduced parasite load in adults, demonstrating that a host‐controlled mechanism for parasite resistance can persist through complete metamorphosis in insects. 4. Infections reduced larval developmental rates and increased host mortality but only during the crucial metamorphic stage, indicating that parasites impact multiple life stages. In general, our results demonstrate that invertebrates can show surprisingly robust immune priming despite dramatic physiological changes and protect hosts across completely different life‐history stages.     

Elevated ozone induces jasmonic acid defense of tomato plants and reduces midgut proteinase activity in Helicoverpa armigera

As a consequence of membrane lipid peroxidation, foliar defense compounds are changed by elevated ozone (O₃), which in turn affects the palatability and performance of insect herbivores. The induced defense of two tomato [Solanum esculentum L. (Solanaceae)] genotypes, namely jasmonic acid (JA) pathway‐deficient mutant spr2 and its wild‐type control, was studied in response to cotton bollworm, Helicoverpa armigera Hübner (Lepidoptera: Noctuidae), as well as the digestive adaptation of these insects under elevated O₃ in open‐top field chambers. Our data indicated that elevated O₃ increased foliar JA and salicylic acid (SA) levels simultaneously and up‐regulated proteinase inhibitors (PIs) and lipoxidase activities in wild‐type plants, regardless of H. armigera infestation. In contrast, only the O₃+H. armigera treatment increased free SA levels in spr2 plants, but did not affect JA level or PI activities. Additionally, the lower activity of midgut digestive enzymes, including active alkaline trypsin‐like enzyme and chymotrypsin‐like enzyme, was observed in the midgut of cotton bollworms after they consumed wild‐type plants treated for 2 h with elevated O₃. With temporary increases at 8 h, all four digestive enzymes of interest in the insect midgut dropped when they were fed with wild‐type plants under elevated O₃ treatment. Increases in atmospheric O₃ are thought to increase JA signaling and consequently reduce the activities of midgut digestive enzymes in H. armigera, therefore enhancing plant resistance against insect herbivores.     

Caspase‐1 inflammasome activity in patients with Staphylococcus aureus bacteremia

The inflammasome is a multiprotein complex that mediates caspase‐1 activation with subsequent maturation of the proinflammatory cytokines IL‐1β and IL‐18. The NLRP3 inflammasome is known to be activated by Staphylococcus aureus, one of the leading causes of bacteremia worldwide. Inflammasome activation and regulation in response to bacterial infection have been found to be of importance for a balanced host immune response. However, inflammasome signaling in vivo in humans initiated by S. aureus is currently sparsely studied. This study therefore aimed to investigate NLRP3 inflammasome activity in 20 patients with S. aureus bacteremia (SAB), by repeated measurement during the first week of bacteremia, compared with controls. Caspase‐1 activity was measured in monocytes and neutrophils by flow cytometry detecting FLICA (fluorescent‐labeled inhibitor of caspase‐1), while IL‐1β and IL‐18 was measured by Luminex and ELISA, respectively. As a measure of inflammasome priming, messenger RNA (mRNA) expression of NLRP3, CASP1 (procaspase‐1), and IL1B (pro‐IL‐1β) was analyzed by quantitative PCR. We found induced caspase‐1 activity in innate immune cells with subsequent release of IL‐18 in patients during the acute phase of bacteremia, indicating activation of the inflammasome. There was substantial interindividual variation in caspase‐1 activity between patients with SAB. We also found an altered inflammasome priming with low mRNA levels of NLRP3 accompanied by elevated mRNA levels of IL1B. This increased knowledge of the individual host immune response in SAB could provide support in the effort to optimize management and treatment of each individual patient.     

Digestive proteolysis organization in two closely related Tenebrionid beetles: red flour beetle (Tribolium castaneum) and confused flour beetle (Tribolium confusum)

The spectra of Tribolium castaneum and T. confusum larval digestive peptidases were characterized with respect to the spatial organization of protein digestion in the midgut. The pH of midgut contents in both species increased from 5.6–6.0 in the anterior to 7.0–7.5 in the posterior midgut. However, the pH optimum of the total proteolytic activity of the gut extract from either insect was pH 4.1. Approximately 80% of the total proteolytic activity was in the anterior and 20% in the posterior midgut of either insect when evaluated in buffers simulating the pH and reducing conditions characteristic for each midgut section. The general peptidase activity of gut extracts from either insect in pH 5.6 buffer was mostly due to cysteine peptidases. In the weakly alkaline conditions of the posterior midgut, the serine peptidase contribution was 31 and 41% in T. castaneum and T. confusum, respectively. A postelectrophoretic peptidase activity assay with gelatin also revealed the important contribution of cysteine peptidases in protein digestion in both Tribolium species. The use of a postelectrophoretic activity assay with p‐nitroanilide substrates and specific inhibitors revealed a set of cysteine and serine endopeptidases, 8 and 10 for T. castaneum, and 7 and 9 for T. confusum, respectively. Serine peptidases included trypsin‐, chymotrypsin‐, and elastase‐like enzymes, the latter being for the first time reported in Tenebrionid insects. These data support a complex system of protein digestion in the Tribolium midgut with the fundamental role of cysteine peptidases. © 2009 Wiley Periodicals, Inc.     

Toxic activity of a protein complex purified from Xenorhabdus nematophila HB310 to Plutella xylostella larvae

Abstract Xenorhabdus nematophila, a Gram‐negative proteobacterium belonging to the family Enterobacteriaceae and associated symbiotically with soil entomopathogenic nematodes, Steinernema carpocapsae, is pathogenic to a wide range of insects. A protein complex with insecticidal activity was isolated from the cells of X. nematophila HB310 strain using methods of salting out and native polyacrylamide gel electrophoresis (PAGE). Seven polypeptides ranging 50～250 kDa were well separated from the protein complex (named Xnpt) by sodium dodecyl sulfate (SDS)‐PAGE, five of which are identified as XptA2, xptC1, XptB1, GroEL and hypothetical protein by matrix‐assisted laser desorption‐time‐of‐flight mass spectrometry (MALDI‐TOFMS). Xnpt showed high oral virulence to larvae of diamondback moth (DBM), Plutella xylostella L. (Lepidoptera, Plutellidae) as its median lethal concentration (LC₅₀) against second and third instar larvae were 331.45 ng/mL and 553.59 ng/mL at 72 h, respectively. The histological analysis of Xnpt‐fed DBM larvae showed extensive histopathological effects on the midgut. Biochemical analysis indicated that Xnpt markedly inhibited the activities of three important enzymes in the midgut. Overall, our data showed that the protein complex isolated from X. nematophila HB310 induced the antifeedant and death of insects by destroying midgut tissues and inhibiting midgut proteases activities.     

Inhibition of Helicoverpa armigera gut pro‐proteinase activation in response to synthetic protease inhibitors

Protease inhibitors play an important role in host plant defence against herbivores. However, insects have the ability to elevate the production of proteinases or resort to production of a diverse array of proteinases to offset the effect of proteinase inhibitors. Therefore, we studied the inhibition of pro‐proteinase(s) activation in the midgut of the polyphagous pest Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in response to protease inhibitors to develop appropriate strategies for the control of this pest. Gelatin coating present on X‐ray film was used as a substrate to detect electrophoretically separated pro‐proteinases and proteinases of H. armigera gut extract on native‐ and sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. Six activated pro‐proteinase bands were detected in H. armigera gut lumen, which were partially purified and characterized using substrate assays. Activated H. armigera midgut pro‐proteinase(s) showed activity maxima at pH 8 and 10, and exhibited optimal activity at 40 °C. The activation of H. armigera gut pro‐proteinase isoforms was observed in the fraction eluted on benzamidine‐sepharose 4B column. Purification and substrate assay studies revealed that 23–70 kDa polypeptides were likely the trypsin/chymotrypsin‐like pro‐proteinases. Larvae of H. armigera fed on a cocktail of synthetic inhibitors (antipain, aprotinin, leupeptin, and pefabloc) showed maximum activation of pro‐proteinases compared with the larvae fed on individual inhibitors. The implications of these results for developing plants expressing proteinase inhibitors for conferring resistance to H. armigera are discussed.     

Stages of Infection during the Tripartite Interaction between Xenorhabdus nematophila, Its Nematode Vector, and Insect Hosts

Bacteria of the genus Xenorhabdus are mutually associated with entomopathogenic nematodes of the genus Steinernema and are pathogenic to a broad spectrum of insects. The nematodes act as vectors, transmitting the bacteria to insect larvae, which die within a few days of infection. We characterized the early stages of bacterial infection in the insects by constructing a constitutive green fluorescent protein (GFP)-labeled Xenorhabdus nematophila strain. We injected the GFP-labeled bacteria into insects and monitored infection. We found that the bacteria had an extracellular life cycle in the hemolymph and rapidly colonized the anterior midgut region in Spodoptera littoralis larvae. Electron microscopy showed that the bacteria occupied the extracellular matrix of connective tissues within the muscle layers of the Spodoptera midgut. We confirmed the existence of such a specific infection site in the natural route of infection by infesting Spodoptera littoralis larvae with nematodes harboring GFP-labeled Xenorhabdus. When the infective juvenile (IJ) nematodes reached the insect gut, the bacterial cells were rapidly released from the intestinal vesicle into the nematode intestine. Xenorhabdus began to escape from the anus of the nematodes when IJs were wedged in the insect intestinal wall toward the insect hemolymph. Following their release into the insect hemocoel, GFP-labeled bacteria were found only in the anterior midgut region and hemolymph of Spodoptera larvae. Comparative infection assays conducted with another insect, Locusta migratoria, also showed early bacterial colonization of connective tissues. This work shows that the extracellular matrix acts as a particular colonization site for X. nematophila within insects.     

Amber-colored excreta: a source of arrestment pheromone in firebrats, Thermobia domestica

Female, male, and juvenile firebrats, Thermobia domestica (Packard) (Thysanura: Lepismatidae), employ a pheromone that arrests conspecifics on contact. Paper shelters placed in a T. domestica colony accumulate fecal excreta (= frass) and other insect‐derived debris. Such shelters elicit arrestment by conspecifics. However, the definitive source of the arrestment pheromone was not known. We tested the hypothesis that one or more debris components from a T. domestica shelter constitute the source of the arrestment pheromone. In dual‐choice, still‐air olfactometer experiments, scales, exuviae, antennae, caudal filaments, gregarine parasite cysts, and silk (each intact or macerated) retrieved from shelters and separated for experiments, as well as saliva, hemolymph, and fat body extracted from insects all failed to arrest female T. domestica. Similarly, paper that had been fed upon by insects did not elicit an arrestment response, eliminating insect‐altered cellulose as the arrestant pheromone. In contrast, insect‐exposed glass significantly arrested females. Moreover, females were significantly arrested by (i) loose, insect‐derived debris brushed from shelters, (ii) a frass mixture manually separated from loose debris, and (iii) specific amber‐type frass manually separated from the frass mixture. These results lead us to conclude that amber‐type frass constitutes the source of at least part of the T. domestica arrestment pheromone.     

Immunity of the greater wax moth Galleria mellonella

Investigation of insect immune mechanisms provides important information concerning innate immunity, which in many aspects is conserved in animals. This is one of the reasons why insects serve as model organisms to study virulence mechanisms of human pathogens. From the evolutionary point of view, we also learn a lot about host–pathogen interaction and adaptation of organisms to conditions of life. Additionally, insect‐derived antibacterial and antifungal peptides and proteins are considered for their potential to be applied as alternatives to antibiotics. While Drosophila melanogaster is used to study the genetic aspect of insect immunity, Galleria mellonella serves as a good model for biochemical research. Given the size of the insect, it is possible to obtain easily hemolymph and other tissues as a source of many immune‐relevant polypeptides. This review article summarizes our knowledge concerning G. mellonella immunity. The best‐characterized immune‐related proteins and peptides are recalled and their short characteristic is given. Some other proteins identified at the mRNA level are also mentioned. The infectious routes used by Galleria natural pathogens such as Bacillus thuringiensis and Beauveria bassiana are also described in the context of host–pathogen interaction. Finally, the plasticity of G. mellonella immune response influenced by abiotic and biotic factors is described.