Potential role for gut microbiota in cell wall digestion and glucoside detoxification in Tenebrio molitor larvae

Tenebrio molitor larvae were successfully reared free of cultivatable gut lumen bacteria, yeasts and fungi using two approaches; aseptic rearing from surface sterilized eggs and by feeding larvae with antibiotic-containing food. Insects were reared on a rich-nutrient complete diet or a nutrient-poor refractory diet. A comparison of digestive enzyme activities in germ free and conventional insects containing a gut microbiota did not reveal gross differences in enzymes that degrade cell walls from bacteria (lysozyme), fungi (chitinase and laminarinase) and plants (cellulase and licheninase). This suggested that microbial-derived enzymes are not an essential component of the digestive process in this insect. However, more detailed analysis of T. molitor midgut proteins using an electrophoretic separation approach showed that some digestive enzymes were absent and others were newly expressed in microbiota-free larvae. Larvae reared in antibiotic-containing refractory wheat bran diet performed poorly in comparison with controls. The addition of saligenin, the aglycone of the plant glucoside salicin, has more deleterious effects on microbiota-free larvae than on the conventionally reared larvae, suggesting a detoxifying role of midgut microbiota. Analysis of the volatile organic compounds released from the faecal pellets of the larvae shows key differences in the profiles from conventionally reared and aseptically reared larvae. Pentadecene is a semiochemical commonly found in other beetle species. Here we demonstrate the absence of pentadecene from aseptically reared larvae in contrast to its presence in conventionally reared larvae. The results are discussed in the light of the hypothesis that microbial products play subtle roles in the life of the insect, they are involved in the digestion of refractory food, detoxification of secondary plant compounds and modify the volatile profiles of the insect host.     

A meta-analysis of the effects of galling insects on host plant secondary metabolites

The idea that galling insects actively manipulate host plant chemistry has been previously documented but has not been quantified across a range of galler and host plant taxa. We present the first quantitative review of the relationship between insect galling and levels of secondary metabolites in host plants. Using meta-analytic techniques, we examined this relationship across 40 galler and host plant species combinations. We found that galling insects are associated with significantly higher levels of tannins and phenolics; however, no difference was found for volatiles. Hymenoptera, Diptera and Hemiptera were associated with higher levels of secondary metabolites; however, only Hymenoptera was significant. The climatic zone of the study area did not explain significant differences in gall-induced secondary metabolites. Overall the results show that the ability of galling insects to manipulate host plant secondary chemistry is widespread across insect and plant taxa. The evolutionary success of galling insects may be in part due to this unique ability.     

Microbial Community Profiling to Investigate Transmission of Bacteria Between Life Stages of the Wood-Boring Beetle, Anoplophora glabripennis

Many insects harbor specific bacteria in their digestive tract, and these gut microbiota often play important roles in digestion and nutrient provisioning. While it is common for a given insect species to harbor a representative gut microbial community as a population, how this community is acquired and maintained from generation to generation is not known for most xylophagous insects, except termites. In this study, we examined acquisition of gut microbiota by the wood-feeding beetle, Anoplophora glabripennis, by identifying and comparing microbial community members among different life stages of the insect and with microbes it encounters in the environment. Automated ribosomal intergenic spacer analysis was employed to compare bacterial communities present in the egg and larval stages of A. glabripennis as well as with microbes found in the oviposition site and the surrounding woody tissue. Multivariate analyses were used to identify relationships between sample type and specific bacterial types (operational taxonomic units). From this analysis, bacteria that were derived from the environment, the oviposition site, and/or the egg were identified and compared with taxa found in larvae. Results showed that while some larval microbes were derived from environmental sources, other members of the larval microbial community appear to be vertically transmitted. These findings could lead to a better understanding of which microbial species are critical for the survival of this insect and to development of techniques that could be used to alter this community to disrupt the digestive physiology of the host insect as a biological control measure.     

昆虫共生菌的垂直传播

共生菌普遍存在于昆虫体内,它们能够为宿主昆虫提供生长发育所必需的氨基酸、固醇类等营养物质,还能提高昆虫适应高温、寄生虫、病毒等不利环境因素的能力,昆虫则为共生菌提供稳定的生存环境和营养物质,昆虫与共生菌相互依存。多数情况下,共生菌通过垂直传播在宿主代次间进行传播,即共生菌由母代传递给子代。结合最近几年相关研究,本文综述了不同昆虫共生菌的垂直传播模式。除极少数肠道共生菌通过污染卵壳被宿主幼虫取食得以垂直传播外,垂直传播的共生菌多为经卵传播。根据侵染时期的不同,共生菌经卵传播模式多数可分为以下4种：侵染宿主昆虫幼虫中的生殖干细胞、侵染宿主昆虫年轻雌成虫中的生殖干细胞、侵染宿主昆虫雌成虫中的成熟卵母细胞以及侵染宿主昆虫囊胚期胚胎。其中,有些共生菌是以共生菌菌胞整体侵染的方式进入到宿主卵巢。另外,少数肠道共生菌也通过卵巢进行垂直传播,此类共生菌先侵染卵巢侧输卵管并在侧输卵管聚集,待卵排放至侧输卵管时再进入到卵中。在文中,我们也探讨了昆虫共生菌垂直传播过程中的细胞机制和免疫机制,包括共生菌避开宿主免疫反应、共生菌通过内吞作用进入卵巢以及不同共生菌间的协同作用等。     

Yeast-like microorganisms in the scale insect Kermes quercus (Insecta,Hemiptera, Coccomorpha: Kermesidae). Newly acquired symbionts?

Scale insects, like other plant sap-consumers, are host to symbiotic microorganisms which provide them with the substances missing from their diet. In contrast to most scale insects, Kermes quercus (Linnaeus) was regarded as asymbiotic. Our histological and ultrastructural observations show that in the body of the feeding stages of K. quercus collected in two locations (Warsaw and Cracow), numerous yeast-like microorganisms occur. These microorganisms were localized in the cytoplasm of fat body cells. The yeast-like microorganisms were observed neither in other organs of the host insect nor in the eggs. These microorganisms did not cause any damage to the structure of the ovaries and the course of oogenesis of the host insect. The females infected by them produced about 1300 larvae. The lack of these microorganisms in the cytoplasm of eggs indicates that they are not transmitted transovarially from mother to offspring. Molecular analyses indicated that the microorganisms which reside in the body of K. quercus are closely related to the entomopathogenic fungi Cordyceps and Ophiocordyceps, which belong to the Sordariomycetes class within the Ascomycota. The role of yeast-like microorganisms to their host insects remains unknown; however, it has been suggested that they may represent newly acquired symbionts.     

Insect endosymbionts: manipulators of insect herbivore trophic interactions?

Throughout their evolutionary history, insects have formed multiple relationships with bacteria. Although many of these bacteria are pathogenic, with deleterious effects on the fitness of infected insects, there are also numerous examples of symbiotic bacteria that are harmless or even beneficial to their insect host. Symbiotic bacteria that form obligate or facultative associations with insects and that are located intracellularly in the host insect are known as endosymbionts. Endosymbiosis can be a strong driving force for evolution when the acquisition and maintenance of a microorganism by the insect host results in the formation of novel structures or changes in physiology and metabolism. The complex evolutionary dynamics of vertically transmitted symbiotic bacteria have led to distinctive symbiont genome characteristics that have profound effects on the phenotype of the host insect. Symbiotic bacteria are key players in insect–plant interactions influencing many aspects of insect ecology and playing a key role in shaping the diversification of many insect groups. In this review, we discuss the role of endosymbionts in manipulating insect herbivore trophic interactions focussing on their impact on plant utilisation patterns and parasitoid biology.     

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.     

Feeding by Hessian fly [Mayetiola destructor (Say)] larvae does not induce plant indirect defences

Abstract.  1. Recent research has addressed the function of herbivore-induced plant volatiles in attracting natural enemies of feeding herbivores. While many types of insect herbivory appear to elicit volatile responses, those triggered by gall insects have received little attention. Previous work indicates that at least one gall insect species induces changes in host-plant volatiles, but no other studies appear to have addressed whether gall insects trigger plant indirect defences.
2. The volatile responses of wheat to feeding by larvae of the Hessian fly Mayetiola destructor (Say) (Diptera: Cecidomyiidae) were studied to further explore indirect responses of plants to feeding by gall insects. This specialist gall midge species did not elicit a detectable volatile response from wheat plants, whereas a generalist caterpillar triggered volatile release. Moreover, Hessian fly feeding altered volatile responses to subsequent caterpillar herbivory.
3. These results suggest that Hessian fly larvae exert a degree of control over the defensive responses of their host plants and offer insight into plant-gall insect interactions. Also, the failure of Hessian fly larvae to elicit an indirect defensive response from their host plants may help explain why natural enemies, which often rely on induced volatile cues, fail to inflict significant mortality on M. destructor populations in the field.     

Gene expression of serine and cysteine proteinase inhibitors during cereal leaf beetle larvae feeding on wheat: the role of insect-associated microorganisms

Plants and insects have been coexisting for more than 350 million years. During this time, both have evolved many strategies to successfully exploit or respond to reciprocal adaptation and defense reactions. Plants tend to minimize the damage caused by pest feeding, while pests tend to manipulate plant response by suppressing plant defense mechanisms or developing strategies to overcome plant defense systems. Plants recognize insect pests by the wounding that they cause and elicitors present in pest oral secretions (saliva and/or regurgitant). These elicitors or insect-associated microorganisms can modulate plant response to the benefit of their insect hosts. In this article, we have undertaken an analysis of gene expression in serine and cysteine proteinase inhibitors (SerPI and CysPI, respectively) in wheat (Triticum aestivum) plants exposed to cereal leaf beetle (CLB, Oulema melanopus, Coleoptera, Chrysomelidae) larvae feeding, and the impact of microbes associated with CLB on the expression levels of these genes. Using three wheat varieties and antibiotic-treated and untreated CLB larvae, we found that SerPI plays a more important role than CysPIs in plant defense against CLB larvae. Additionally, higher levels of SerPI gene expression were observed in systemic leaves in comparison to the wounded leaves (local response). Each of the tested wheat varieties reacted in a specific way to the particular treatment. Moreover, the presence of microbial components associated with insects influenced plant response to CLB larvae feeding.     

昆虫聚集信息素

昆虫聚集信息素是昆虫重要的信息化学物质之一,对昆虫的聚集行为有重要意义。近三十年来,国外鉴定了多种昆虫聚集信息素,主要成分为一些烃、醇、醛、酮、酯、酸、酸酐、胺以及腈类化合物,但其在有害生物可持续治理中的应用潜能尚未充分利用;昆虫聚集信息素的来源多样,除蛹外,多个虫态均有聚集信息素释放,有些学者甚至把一些寄主释放的挥发物作为聚集信息素的组分;同种昆虫,不同生理状态,其聚集信息素可以完全不同或同一信息化学物质的功能不同;但是,并非所有昆虫的聚集行为均为聚集信息素调节,利他素、性信息素以及报警信息素等其它信息化学物质均能导致一些昆虫的聚集。本文综述了5目17科55种昆虫的聚集信息素。     

Insect species interactions and resource effects in treeholes: are helodid beetles bottom-up facilitators of midge populations?

The insect community living in central Pennsylvania treeholes in autumn consists primarily of larvae of two species of helodid beetles, Prionocyphon discoideus and Helodes pulchella, and larvae of one species of ceratopogonid midge,Culicoides guttipennis. We manipulated treehole volume and the densities of these insects in laboratory microcosms. We hypothesized that: (1) helodid beetle larvae, which are shredders, would enhance growth and survival of ceratopogonid midge larvae (deposit feeders) in a processing chain commensalism, and (2) the quantity of resources expressed as water volume plus leaf litter would affect helodids and protozoans directly. Intraspecific competition was not found in midges, nor was interspecific competition between the two helodid species. Protozoan population densities decreased or grew slower in the presence of insects and in smaller microcosms. Development time and adult wing length of the midge (C. guttipennis) were affected by both total microcosm volume and insect species combination. Under resource limitation, midges grew larger in the presence of helodids, and in general, midges were larger in treatments with higher ratios of helodids to midges. Water chemistry in the microcosms was affected both temporally and by insect presence. Hydrogen ion levels decreased over time, and microcosms with no insects had lower hydrogen ion levels. Specific conductance increased in all treatments over time, and microcosms with no insects had lower conductivity than most treatments. Helodid larvae have a positive effect on midges, possibly due to a processing chain facilitation. If helodids are keystone decomposers in this system, their presence could affect resource availability and affect other organisms in the community. Similar processing chain commensalisms occur in other phytotelmata. These types of interactions may therefore be important in the structure and function of detritus-based communities. Received: 31 January 1996 /Accepted: 7 August 1996     

Feeding by Hessian Fly (Mayetiola destructor [Say]) Larvae on Wheat Increases Levels of Fatty Acids and Indole-3-Acetic Acid but not Hormones Involved in Plant-Defense Signaling

Gall-inducing insects exert a unique level of control over the physiology of their host plants. This control can extend to host–plant defenses so that some, if not most, gall-inducing species appear to avoid or modify host plant defenses to effect production of their gall. Included among gall insects is Hessian fly (Mayetiola destructor [Say], Diptera: Cecidomyiidae), a damaging pest of wheat (Triticum aestivum L.) and an emerging model system for studying plant–insect interactions. We studied the dynamics of some defense-related phytohormones and associated fatty acids during feeding of first instar Hessian fly larvae on a susceptible variety of wheat. We found that Hessian fly larvae significantly elevated in their host plants’ levels of linolenic and linoleic acids, fatty acids that may be nutritionally beneficial. Hessian fly larvae also elevated levels of indole-3-acetic acid (IAA), a phytohormone hypothesized to be involved in gall formation, but not the defense-related hormones jasmonic (JA) and salicylic acids. Moreover, we detected in Hessian fly-infested plants a significant negative relationship between IAA and JA that was not present in control plants. Our results suggest that Hessian fly larvae may induce nutritionally beneficial changes while concomitantly altering phytohormone levels, possibly to facilitate plant-defense avoidance.     

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.     

Capsule-transmitted gut symbiotic bacterium of the Japanese common plataspid stinkbug, Megacopta punctatissima.

The Japanese common plataspid stinkbug, Megacopta punctatissima, deposits small brown particles, or symbiont capsules, on the underside of the egg mass for the purpose of transmission of symbiotic bacteria to the offspring. We investigated the microbiological aspects of the bacteria contained in the capsule, such as microbial diversity, phylogenetic placement, localization in vivo, and fitness effects on the host insect. Restriction fragment length polymorphism analysis of 16S ribosomal DNA clones revealed that a single bacterial species dominates the microbiota in the capsule. The bacterium was not detected in the eggs but in the capsules, which unequivocally demonstrated that the bacterium is transmitted to the offspring of the insect orally rather than transovarially, through probing of the capsule content. Molecular phylogenetic analysis showed that the bacterium belongs to the gamma-subdivision of the Proteobacteria. In adult insects the bacterium was localized in the posterior section of the midgut. Deprivation of the bacterium from the nymphs resulted in retarded development, arrested growth, abnormal body coloration, and other symptoms, suggesting that the bacterium is essential for normal development and growth of the host insect.     

植食性昆虫唾液效应子和激发子的研究进展

植食性昆虫与寄主植物通过协同进化形成了复杂的防御和反防御机制。本文系统综述了昆虫唾液效应子和激发子在植物与昆虫互作中的作用及机理。昆虫取食中释放的唾液激发子被植物识别而激活植物早期免疫反应,昆虫也能从口腔分泌效应子到植物体内抑制免疫;抗性植物则利用抗性(R)蛋白识别昆虫无毒效应子,启动效应子诱导的免疫反应,而昆虫又进化出多种方式来躲避植物R蛋白的识别。总之,在这场军备竞赛中,昆虫的唾液成分决定着昆虫能否取食成功。取食过程中,咀嚼式口器害虫分泌大量酶类到植物体内,而刺吸式害虫则分泌胶状和水样唾液到植物中,它们都利用激发子和效应子去调控植物的免疫防御反应。分析现已报道的昆虫效应子发现其作用机制各有不同,具体表现为影响植物早期防御信号,调控植物激素通路及其他通路,或靶向小分子RNA通路。本文还综述了昆虫激发子的最新进展,揭示激发子可以通过诱导释放植物次生代谢物以及调控激素水平、Ca²⁺内流和活性氧爆发增强植物抗性。最后对昆虫效应子的分泌特性、寄主特异性和多功能性作了分析,并对无毒效应子及其对应的植物R基因,以及激发子的模式识别受体的研究进行了展望。     

Capsule-Transmitted Gut Symbiotic Bacterium of the Japanese Common Plataspid Stinkbug, Megacopta punctatissima

The Japanese common plataspid stinkbug, Megacopta punctatissima, deposits small brown particles, or symbiont capsules, on the underside of the egg mass for the purpose of transmission of symbiotic bacteria to the offspring. We investigated the microbiological aspects of the bacteria contained in the capsule, such as microbial diversity, phylogenetic placement, localization in vivo, and fitness effects on the host insect. Restriction fragment length polymorphism analysis of 16S ribosomal DNA clones revealed that a single bacterial species dominates the microbiota in the capsule. The bacterium was not detected in the eggs but in the capsules, which unequivocally demonstrated that the bacterium is transmitted to the offspring of the insect orally rather than transovarially, through probing of the capsule content. Molecular phylogenetic analysis showed that the bacterium belongs to the γ-subdivision of the Proteobacteria. In adult insects the bacterium was localized in the posterior section of the midgut. Deprivation of the bacterium from the nymphs resulted in retarded development, arrested growth, abnormal body coloration, and other symptoms, suggesting that the bacterium is essential for normal development and growth of the host insect.     

棉铃虫的紫云英-麦胚人工饲料

用不同人工饲料连续饲养棉铃虫Heliothis armtgera;通过试验比较,设计和发展了适合于大量饲养棉铃虫的紫云英-麦胚人工饲料。与自然饲料相比,用这种人工饲料饲养的棉铃虫发育快、存活率高、蛹大、成虫产卵多。已经用紫云英-麦胚人工饲料连续饲养棉铃虫16代,各代幼虫的存活率在85—97%之间,成虫平均产卵1,000多粒,孵化率不低于70%。6龄幼虫对这种人工饲料的利用和转化效率分别是26.7%和65.0%。     

Independent origins and horizontal transfer of bacterial symbionts of aphids

Many insect groups have obligate associations with primary endosymbionts: mutualistic bacteria that are maternally transmitted and derived from an ancient infection. Often, the same insects are hosts to 'secondary' bacterial symbionts which are maternally transmitted but relatively labile within host lineages. To explore the dynamics of secondary symbiont associations in aphids, we characterized bacteria infecting 15 species of macrosiphine aphids using DNA sequencing, diagnostic polymerase chain reaction (PCR), diagnostic restriction digests, phylogenetic analyses, and electron microscopy to examine aphids from nature and from laboratory colonies. Three types of bacteria besides Buchnera were found repeatedly; all three fall within the Enterobacteriaceae. The R-type has a 16S rDNA less than 0.1% different from that of the secondary symbiont previously reported from Acyrthosiphon pisum and is related to Serratia species. The T-type includes a symbiont previously reported from a whitefly; the U-type comprises a new cluster near the T-type. The T-type was found in every one of 40 Uroleucon ambrosiae clones collected throughout the United States. In contrast, A. pisum individuals were infected by any combination of the three symbiont types. Secondary symbionts were maternally transmitted for 11 months within laboratory-reared A. pisum clones and were present in sexually produced eggs. PCR screens for a bacteriophage, APSE-1, indicated its presence in both A. pisum and U. ambrosiae containing secondary symbionts. Electron microscopy of R-type and T-type bacteria in A. pisum and in U. ambrosiae revealed rod-shaped organisms that attain extremely high densities within a few bacteriocytes.     

Does mother know best? The preference–performance hypothesis and parent–offspring conflict in aboveground–belowground herbivore life cycles

1. A substantial amount of research on host‐plant selection by insect herbivores is focused around the preference–performance hypothesis (PPH). To date, the majority of studies have primarily considered insects with aboveground life cycles, overlooking insect herbivores that have both aboveground and belowground life stages, for which the PPH could be equally applicable. 2. This study investigated the factors influencing the performance of the root‐feeding vine weevil (Otiorhynchus sulcatus) larvae and whether this was linked to the oviposition behaviour of the maternal adult living aboveground. 3. Maternal insects feeding aboveground reduced root biomass by 34% and increased root carbon by 4%. Larvae feeding on plants subjected to aboveground herbivory had reduced mass. Irrespective of the presence of maternal herbivory, larval mass was positively correlated with root biomass. 4. Larval mass was also reduced by conspecific larvae, previously feeding on roots (19% reduction). However, the mechanism underpinning this effect remains unclear, as in contrast to maternal herbivory aboveground, prior larval feeding did not significantly affect root biomass or root carbon concentrations. 5. Maternal insects did not distinguish between plants infested with larvae and those that were free of larvae, in terms of their egg‐laying behaviour. Conversely, maternal insects tended to lay eggs on plants with smaller root systems, a behaviour that is likely to negatively affect offspring performance. 6. The PPH is not supported by our findings for the polyphagous vine weevil feeding on the host plant raspberry (Rubus idaeus), and in fact our results suggest that there is the potential for strong parent–offspring conflict in this system.