Autochthonous bacterial flora indicated by PCR-DGGE of 16S rRNA gene fragments from the alimentary tract of Costelytra zealandica (Coleoptera: Scarabaeidae)

Aims: To locate and identify putative autochthonous bacteria within the grass grub gut that may have a role in symbiosis. Methods and Results: Polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) fingerprinting was used to investigate bacterial diversity in the grass grub larval gut. The microbial community profiles from five geographically distinct populations were compared and the influence of feeding was analysed. Bacterial community in the midgut was highly variable between locations and was affected by feeding. The hindgut contained a more diverse but stable bacterial community that was less affected by external conditions. Forty-seven distinct DGGE bands, representing different bacterial genotypes, could be distinguished from all samples, with 34 different bands occurring in the hindgut. The 22 most common bands were isolated and DNA was sequenced. Sequence analysis revealed that most bacteria (16/22) were affiliated to the Clostridiales with the predominant bacteria affiliated to the genus Clostridium. The remaining bacteria were aligned to the Betaproteobacteria, Deltaproteobacteria and Bacteroidetes. Conclusions: The grass grub larva has an autochthonous microflora with predominance of Clostridium spp. in the hindgut. Significance and Impact of the Study: Occurrence of an autocthonous microflora in the grass grub hindgut suggests a symbiotic relationship which could help explain the ability of larval scarabs to feed on recalcitrant organic matter.     

Regulatory impact on insect biotechnology and pest management

The application of insect biotechnology is promising for the development of environmentally compatible pest management solutions. As we have refined and enhanced genetic engineering techniques in several insect species that cause significant economic loss and public health injury, it has become clear that insect biotechnology will move forward as one of the key tools of pest management in agriculture and in the human environment. Well characterized genetic elements can be manipulated toward specific aims and maintain a viable insect, albeit one with diminished capacity to exchange genetic material, vector a virus or bacterium, or complete its life cycle. Despite this degree of knowledge and precision, there remain unanswered questions regarding environmental fate, release and public acceptance of this technology. The uncertainty surrounding any novel technology inevitably increases the level of regulatory scrutiny associated with its use. Although the term “insect biotechnology” has many connotations, it certainly includes the genetic modification of symbiotic or commensally associated microbes as a means of delivering a trait (e.g. a toxin) to manage plant and human diseases and insect pests. The distinction between this paratransgenic approach and direct genetic modification of insect pests is an important one biologically as well as from a regulatory standpoint. The regulatory framework for microbial applications to agriculture is in many instances in place; however, we must strive to forge the development of guidelines and regulations that will foster deployment of insect biotechnologies.     

Influence of diet on the structure and function of the bacterial hindgut community of crickets

The effect of diets varying in carbohydrate and protein content on the structure and function of the hindgut microbiota of crickets was evaluated by determining bacterial densities, fermentation activity, and guanine plus cytosine (G + C) profiles of the DNA extracted from the microbial hindgut community. DNA isolated from the gut community was fractionated and quantified according to G + C content as a comprehensive, coarse-level measure of the composition and structure of the community. The bacterial densities measured by direct counts were not significantly different among the four diets. The crickets were initially reared in the laboratory on cricket chow, which resulted in a hindgut community dominated by bacteria with a G + C content between 32% and 57%. Crickets shifted to an alfalfa diet showed a similar hindgut community G + C profile, although microbial populations with DNA between 35% and 45% G + C were more abundant in alfalfa- than chow-fed crickets. The apparent complexity of the gut community was reduced in crickets fed beet-pulp and protein-based diets compared to those fed chow and alfalfa, and was dominated by populations with a low percentage G + C content. Hindgut communities in crickets fed pulp and protein diets also showed a decrease in hydrogen and carbon dioxide production, suggesting that these diets affected the biochemical activity of the hindgut community. The protein-based diet resulted in a decrease in the rate of evolution of volatile fatty acids, while the ratio of butyrate production to acetate and propionate production was significantly higher in these crickets. Our results show the emergence of a new microbial community structure concomitant with changes in microbial biochemical activity due to shifts in the cricket's dietary regime.     

Mode of action of Bacillus thuringiensis δ-endotoxin: General characteristics of intoxicated Bombyx larvae

The general pathology induced by δ-endotoxin in terms of larval behavior and hemolymph chemistry has been widely studied in the so-called Type I insect, Bombyx mori. The succession of symptoms is divided into four arbitrary stages: Stage 0, appearance and locomotion normal, no feeding; Stage 1, slightly sluggish; Stage 2, extremely sluggish; and Stage 3, complete paralysis. The action of δ-endotoxin is highly specific to the midgut since contractile movement of both foregut and hindgut continues long after all locomotor activity and heartbeat have stopped. Immediately after the silkworm stops feeding and blood pH sharply rises, there is an associated abrupt rise in the K⁺ concentration of hemolymph. Thereafter, the rise in K⁺ is linear while the rise in pH is not. In vivo measurements have not yielded the same simple linear dependence of pH on K⁺ concentrations that is found in in vitro mixtures of hemolymph and midgut juice. Ligation experiments showed that the same pathological sequence (rise of pH and K⁺ concentration, and general paralysis) follows whether the toxin has unrestricted access to the entire midgut or only part of it (anterior or posterior). From the results of injections of midgut juice or various salt solutions into hemocoel, we came to the conclusions that the blood pH and the symptoms are not necessarily parallel and the intact midgut and Malpighian tubules have strong functions for ion regulation.     

肠道微生物与昆虫的共生关系

昆虫肠道栖息着大量的微生物。随着近年来研究肠道微生物的方法不断进步,尤其是基于16S rDNA的分子生物学方法的应用,人们对肠道微生物的了解逐渐加深。昆虫肠道对于微生物的拓殖存在一定的选择作用。肠道微生物对昆虫寄主的作用包括提供营养、利用拓殖抗性抵抗外来微生物侵袭、参与多重营养关系、引起昆虫免疫反应。长期进化过程中肠道微生物与昆虫发展出紧密的共生关系,微生物发展出一系列手段适应昆虫肠道环境。文章从以上几个方面对近年来的研究进展进行总结,并对昆虫肠道微生态学的实践意义和将来可能的研究热点进行展望。     

Transgenic rice established to express corn cystatin exhibits strong inhibitory activity against insect gut proteinases

Corn cystatin (CC), a phytocystatin, shows a wide inhibitory spectrum against various cysteine proteinases. We produced transgenic rice plants by introducing CC cDNA under CaMV 35S promoter as a first step to obtain a rice plant with insecticidal activity. This attempt was based on the observation that many insect pests, especially Coleoptera, have cysteine proteinases, probably digestive enzymes, and also that oryzacystatin, an intrinsic rice cystatin, shows a narrow inhibition spectrum and is present in ordinary rice seeds in insufficient amounts to inhibit the cysteine proteinases of rice insect pests. The transgenic rice plants generated contained high levels of CC mRNA and CC protein in both seeds and leaves, the CC protein content of the seed reaching ca. 2% of the total heat soluble protein. We also recovered CC activity from seeds and found that the CC fraction efficiently inhibited both papain and cathepsin H, whereas the corresponding fraction from non-transformed rice seeds showed much lower or undetectable inhibitory activities against these cysteine proteinases. Furthermore, CC prepared from transgenic rice plants showed potent inhibitory activity against proteinases that occur in the gut of the insect pest, Sitophilus zeamais.     

基于VOSviewer和CiteSpace的昆虫肠道微生物领域可视化分析

【背景】近年来,昆虫肠道微生物领域受到科研工作者的广泛关注,相应发表了大量的科研论文和著作。然而目前尚缺乏对昆虫肠道微生物全面、系统的文献计量分析。【目的】了解国内外昆虫肠道微生物领域的历史、研究热点和新兴趋势。【方法】以昆虫肠道微生物为搜索主题对中国知网(China National Knowledge Infrastructure, CNKI)数据库和Web of Science (WOS)数据库进行检索,利用文献计量工具VOSviewer和Cite Space对关键词进行分析。【结果】1991-2022年间该领域全球研究发文量整体呈现上升趋势,国内外对昆虫肠道微生物领域的关注点和研究方向逐渐扩大。关键词聚类锚定了3个新兴研究领域：昆虫饮食(insect meal)、次生代谢物(secondary metabolites)和生物降解(biodegradation)。【结论】基于文献计量学研究的发现提供了昆虫肠道微生物领域的现状和趋势,可能有助于确定该领域的热点话题和探索新的方向。     

昆虫肠道微生物的多样性、功能及应用

昆虫肠道微生物种类繁多、数量巨大,在与宿主长期的协同进化过程中,不仅形成极为多样的种群结构,也进化出多样的生物学功能,对宿主的营养、生理、发育、防御、抗逆等方面都产生显著影响。近年来,越来越多的昆虫肠道微生物的多样性和生物学特性被揭示,具有农业、能源和环保价值的众多微生物种类和活性基因得到了开发,展现出巨大的应用潜力。本文将从昆虫肠道微生物的多样性、生物学功能、应用三个方面对近年来的研究进展进行总结,并进行展望。     

Utilisation of protein by human gut bacteria

Abstract Mixed populations of human gut bacteria degraded cas casein by producing a variety of cell-bound and extracellular proteolytic enzymes. Casein was initially hydrolysed to TCA soluble peptides which were subsequently broken down to volatile fatty acids, ammonia, dicarboxylic acids and a range of phenolic compounds. Amino acids did not accumulate to any extent during casein breakdown, suggesting that the rate of peptide hydrolysis was the limiting step in protein utilisation by these bacteria. Similar fermentation products were produced from bovine serum albumin, however, the insoluble protein collagen was considerably more resistant to degradation by the colonic microflora, as evidenced by the reduced quantities of fermentation end-products formed.     

Gut Colonization by an Ice Nucleation Active Bacterium,Erwinia(Pantoea)ananasReduces the Cold Hardiness of Mulberry Pyralid Larvae

To evaluate the suitability of using ice nucleation active (INA) bacteria for the biological control of insect pests, the supercooling point (SCP) of larvae of mulberry pyralid,Glyphodes duplicalis,and silkworm,Bombyx mori,ingesting INA strains ofErwinia(Pantoea)ananasandPseudomonas syringaewas determined. Mean SCP of the guts of silkworm larvae ingesting INA strains ofE. ananasranged from −2.5 to −2.8°C, being 5°C higher than that in control treatments. Similarly, mean SCP of mulberry pyralid larvae ingesting INA strain ofE. ananas,which can grow well in the gut, was −4.7°C at 3 days after treatment, being 6.5°C higher than that in control treatments. On the other hand, mean SCP of the larvae-ingesting INA strain ofP. syringae,which cannot grow in the gut, was −9.0°C at 3 days after treatment, rising by only 2.5°C higher than that in the control treatments. In addition, more than 80% of the larvae of mulberry pyralid ingesting the INA strain ofE. ananasfroze and eventually died when exposed to −6°C for 18 h, while only 36% of the larvae ingesting the INA strain ofP. syringae,or approximately 20% of the control larvae, froze and died. Thus, the gut colonization by INA strains ofE. ananasreduced remarkably the cold hardiness of the insects. These findings suggest that INA strains ofE. ananascould be effective as a potential biological control agent of insect pests.     

The internal microbial flora of the elm bark beetle, Scolytus scolytus, at all stages of its development

Adult Scolytus scolytus emerged from the pupal stage with sterile guts although microorganisms were present within the pupal body. Gut sterility was maintained in a large proportion of newly formed adults while they were boring out of an elm log. The adult gut microbial flora is mainly acquired during subsequent feeding. Differences were found between the microbial gut flora of both larvae and adult S. scolytus, and of both male and female beetles.     

Novel strategies targeting pathogen transmission reduction in insect vectors: Tsetse-transmitted trypanosomiasis control

Insect vectors are essential for the transmission of important human diseases such as malaria, leishmaniasis, Chagas and sleeping sickness. Insects are also responsible for the transmission of agricultural diseases that affect livestock and crops. Traditionally, control of the vector populations has been an effective disease management strategy. Recently, vector control strategies have been fortified by research in insect biology and in insect–pathogen interactions as well as by the development of transgenic technologies. In addition to insect population reduction methods, disease control via selective elimination of pathogens in insects can now be explored. Here we explore the tsetse vectors of African trypanosomes and describe the application of recent knowledge gained in their symbiotic, reproductive and vectorial biology to develop novel disease control strategies.     

Abundance and distribution of the gut flora of the desert locust,Schistocerca gregaria

The abundance of bacteria in the gut of Schistocerca gregaria was determined. A large population of Enterobacteriaceae was found with numbers increasing posteriorly from foregut to rectal sac. In the hindgut, Enterobacter agglomerans was the dominant organism. Streptococci were also present but they were 10- to 100-fold less numerous than the Enterobacteriaceae. The distribution of the microflora was investigated using light microscopy and scanning electron microscopy. Bacteria in the anterior regions of the gut were restricted to the lumen and inside of the peritrophic membrane. However, in the hindgut, bacteria were also associated with the cuticular lining.     

Kin recognition in Drosophila: the importance of ecology and gut microbiota

The animal gut commonly contains a large reservoir of symbiotic microbes. Although these microbes have obvious functions in digestion and immune defence, gut microbes can also affect behaviour. Here, we explore whether gut microbiota has a role in kin recognition. We assessed whether relatedness, familiarity and food eaten during development altered copulation investment in three species of Drosophila with diverse ecologies. We found that a monandrous species exhibited true kin recognition, whereas familiarity determined kin recognition in a species living in dense aggregations. Finally, in a food generalist species, food eaten during development masked kin recognition. The effect of food type on copulation duration, in addition to the removal of this effect via antibiotic treatment, suggests the influence of bacteria associated with the gut. Our results provide the first evidence that varied ecologically determined mechanisms of kin recognition occur in Drosophila, and that gut bacteria are likely to have a key role in these mechanisms.     

胆汁酸功能及其与肠道细菌相互关系

胆汁酸具有多种重要生理功能. 近年研究发现,胆汁酸可作为信号分子与褐色脂肪细胞表面受体TGR5结合,可激活法尼酯衍生物X受体（nuclear receptor farnesoid X receptor, FXR）的表达. 通过激活这些不同的信号传导途径,胆汁酸可以分别起到调节体内能量代谢平衡、控制肥胖以及抑制肠道细菌过度增殖的作用. 胆汁酸与人及动物肠道细菌具有复杂的相互关系：肠道细菌对于胆汁酸的转化很重要,除了在胆汁酸的转化中发挥重要作用外,肠道微生物菌群还可以十分有效地水解已被胆汁酸清除的生物体内结合寄生物或异源物质,促进这些物质的活化或肠肝循环. 宿主拥有一些抑制细菌过度增殖的机制,这些机制包括快速转运以及利用抗菌肽、蛋白水解肽和胆汁酸等进行抑菌;而有些肠道细菌在进化中形成一些抗性机制可避免胆汁酸胁迫. 本文主要就胆汁酸控制肥胖以及抑制细菌过度增殖的机制和胆汁酸与肠道细菌相互关系进行了综述.     

What do we know about biological nitrogen fixation in insects? Evidence and implications for the insect and the ecosystem

Many insects feed on a low‐nitrogen diet,and the origin of their nitrogen supply is poorly understood.It has been hypothesized that some insects rely on nitrogen‐fixing bacteria(diazotrophs)to supplement their diets.Nitrogen fixation by diazotrophs has been extensively studied and convincingly demonstrated in termites,while evidence for the occurrence and role of nitrogen fixation in the diet of other insects is less conclusive.Here,we summarize the methods to detect nitrogen fixation in insects and review the available evidence for its occurrence(focusing on insects other than termites).We distinguish between three aspects of nitrogen fixation investigations:(i)detecting the presence of potential diazotrophs;(ii)detecting the activity of the nitrogen‐fixing enzyme;and(iii)detecting the assimilation of fixed nitrogen into the insect tissues.We show that although evidence from investigations of the first aspect reveals ample opportunities for interactions with potential diazotrophs in a variety of insects,demonstrations of actual biological nitrogen fixation and the assimilation of fixed nitrogen are restricted to very few insect groups,including wood‐feeding beetles,fruit flies,leafcutter ants,and a wood wasp.We then discuss potential implications for the insect's fitness and for the ecosystem as a whole.We suggest that combining these multiple approaches is crucial for the study of nitrogen fixation in insects,and argue that further demonstrations are desperately needed in order to determine the relative importance of diazotrophs for insect diet and fitness,as well as to evaluate their overall impact on the ecosystem.