Provision of riboflavin to the host aphid,Acyrthosiphon pisum,by endosymbiotic bacteria,Buchnera

Differential cDNA display and quantitative RT-PCR suggested that the riboflavin synthase complex of the aphid endosymbiont, Buchnera, is active only when the symbiotic system is maintained and well organized in young hosts. Since this finding suggested the provision of riboflavin by Buchnera, we examined the effect of dietary riboflavin on the performance of symbiotic and aposymbiotic aphids using chemically-defined diets. Our results indicate: (1) dietary riboflavin is slightly detrimental to young, symbiotic aphids; (2) dietary riboflavin is essential to aposymbiotic aphids; (3) dietary riboflavin remarkably improves the performance of aposymbiotic aphids. These results strongly suggest that young, symbiotic aphids are provided with riboflavin by their endosymbionts, Buchnera.     

Do aphids infected with entomopathogenic fungi continue to produce and respond to alarm pheromone?

The response of pea aphids, Acyrthosiphon pisum, to aphid alarm pheromone was not modified by infection with Beauveria bassiana. Approximately 50% of uninfected and infected aphids responded to synthetic alarm pheromone. The simulated attack of aphids infected with B. bassiana did not elicit a response in uninfected aphids. Preliminary air entrainment experiments of both uninfected aphids and aphids at different stages of B. bassiana (generalist pathogen) or P. neoaphidis (obligate pathogen of aphids) demonstrated that B. bassiana infected aphids produced less alarm pheromone than uninfected aphids and, conversely, P. neoaphidis infected aphids produced more alarm pheromone than uninfected aphids. These results are discussed with particular emphasis on the different life history strategies of these two pathogens. We hypothesise that the obligate, specialist pathogen, P. neoaphidis, is under greater selection pressure to increase pathogen transmission and survival resulting in modified host behaviour, than the generalist pathogen, B. bassiana.     

Phosphocarrier proteins in an intracellular symbiotic bacterium of aphids.

A GroEL homolog produced by Buchnera, an intracellular symbiotic bacterium of aphids, is not only a molecular chaperone but also a novel phosphocarrier protein, suggesting that this protein plays a role in a signal transducing system specific to bacteria living in an intracellular environment. This prompted us to look into phosphocarrier proteins of Buchnera that may be shared in common with other bacteria. As a result, no evidence was obtained for the presence of sensor kinases of the two-component system in Buchnera, which are found in many bacteria. It is possible that the lack of sensor kinases is compensated for by the mulitifunctional GroEL homolog in this symbiotic bacteria. In contrast, we successfully identified three phosphotransferase system genes, ptsH, ptsI, and crr in Buchnera, and provide evidence for their active expression. While the deduced amino acid sequences of these gene products, histidine-containing phosphocarrier protein, Enzyme I, and Enzyme III were similar to their counterparts in Escherichia coli, the predicted isoelectric points of the Buchnera proteins were strikingly higher. It was also suggested that Buchnera Enzyme I, when produced in E. coli, is able to accept the phosphoryl group from phosphoenolpyruvate, but not from ATP.     

THE ROLE OF BACTERIAL SYMBIONTS IN AMINO ACID COMPOSITION OF BLACK BEAN APHIDS

To evaluate the role of bacterial symbionts (Buchnera spp.) in the black bean aphids (Aphis craccivora Koch), the aphids were treated with the antibiotic, rifampicin, to eliminate their intracellular symbiotic bacteria. Analysis of protein and amino acid concentration in 7‐day‐old of aposymbiotic aphids showed that the total protein content per mg fresh weight was significantly reduced by 29%, but free amino acid titers were increased by 17%. The ratio of the essential amino acids was in general only around 20% essential amino acids in phloem sap of broad bean, whereas it was 44% and 37% in symbiotic and aposymbiotic aphids, respectively, suggesting that the composition of the free amino acids was unbalanced. For example, the essential amino acid, threonine represented 21.6% of essential amino acids in symbiotic aphids, but it was only 16.7% in aposymbiotic aphids. Likewise, two nonessential amino acids, tyrosine and serine, represented 8.9% and 5.6% of total amino acids in symbiontic aphids, respectively, but they enhanced to 21.1% and 13.6% in aposymbiotic aphids. It seems likely that the elevated free amino acid concentration in aposymbiotic aphids was caused by the limited protein anabolism as the result of the unbalanced amino acid composition.     

白蚁共生真菌——蚁巢伞属研究概况

蚁巢伞属真菌是一类极具市场开发价值的野生食用真菌。本文对蚁巢伞属真菌的分类、与白蚁的共生关系、活性成分和作用、人工培养研究以及产生的相关木质纤维素降解酶类等方面进行综述,总结了近年来该属真菌的研究现状,指出了研究过程中出现的分类混乱、重名异名现象严重等问题,展望了未来有望开发菌丝体相关产品及深化出菇机制以逐步实现蚁巢伞人工栽培的研究方向,以期为未来的研究提供依据。     

Endosymbiosis of Aphids with Microorganisms: a Model Case of Dynamic Endosymbiotic Evolution

Abstract Almost all aphids harbor prokaryotic intracellular symbionts in the cytoplasm of mycetocytes, huge cells in the abdomen specialized for this purpose. The aphids and their intracellular symbionts are in close mutualistic association and unable to live without their partner. The intracellular symbionts of various aphids are of a single origin; they are descendants of a prokaryote that was acquired by the common ancestor of the present aphids. The date of establishment of the symbiotic association is estimated to be 160–280 million years ago using 16S rRNA molecular clock calibrated by aphid fossils. Molecular phylogeny indicates that the intracellular symbiont belongs to a group of gut bacteria, suggesting the possibility that it was derived from a gut microbe of aphids. While the in-tracellular symbionts are universal and highly conserved amongst aphids, other types of symbiotic microorganisms are also present. In various aphids, bacterial “secondary” intracellular symbionts are found in addition to the standard symbionts. They are thought to be acquired many times in various lineages independently. Some Cerataphidini aphids do not have intracellular symbiotic system but harbor yeast-like extracellular symbionts in the hemocoel. In a lineage of this group, symbiont replacement from intracellular prokaryote to extracellular yeast must have occurred. The diversity of the endosymbiotic system of aphids illuminates a dynamic aspect of endosymbiotic evolution.     

Age-dependent regulation of protein synthesis in an aphid endosymbiont by the host insect

In an old (45–50 days) aphid, the endosymbionts synthesize protein species which are not produced in the young (13–20 days) ones. Symbionin, the only protein synthesized in vivo by the endosymbionts of pea aphids, was not produced to any great extent in old hosts. It seems that control of the host over the endosymbiont is weakened in old insects, and that some of the symbiont's own genes begin to be expressed.     

The assimilation and allocation of nutrients by symbiotic and aposymbiotic pea aphids, Acyrthosiphon pisum

The failure of a nutritionally balanced diet to ameliorate the impact of symbiont disruption in the pea aphid Acyrthosiphon pisum (Harris) was investigated using two approaches. The assimilation of dietary nutrients by aphids was investigated using chemically-defined diets containing ³ H-labelled inulin and ¹⁴C-labelled sucrose or amino acids. Symbiotic aphids (i.e., aphids containing their bacteria) had a high sucrose demand and assimilated 72% of sucrose ingested in the diet, whereas the assimilation of sucrose by aposymbiotic aphids (in which the bacteria had been disrupted), was significantly reduced to 47%. The assimilation of individual dietary amino acids by symbiotic aphids varied between 61 and 92%, and there was no impact on the feeding or assimilation rate when the aphids were fed a phloem sap-like diet containing a reduced amount of essential amino acids. Consequently, the absolute amount of each essential amino acid assimilated by symbiotic aphids feeding on a phloem sap-like diet was reduced by 36–59%. Aposymbiotic aphids consistently assimilated a lower proportion of ingested amino acids, and lysine in particular was poorly assimilated from the diet. In a second experiment, the allocation of free amino acids in the haemocoel to aphid embryos was investigated following microinjection of ¹⁴C-labelled amino acids. After 2 h, radiolabel could be detected at varying levels from the embryo complement of both symbiotic and aposymbiotic aphids, indicating rapid but selective uptake by the embryos. The essential amino acids phenylalanine and lysine were incorporated into the protein fraction of embryo tissues, but the rate of incorporation per unit biomass was approximately 4-fold higher in the embryos of aposymbiotic aphids, possibly reflecting increased demand due to the lack of amino acid provisioning from the symbiotic bacteria.     

Response of Solanum tuberosum to Myzus persicae infestation at different stages of foliage maturity

Young leaves of the potato Solanum tuberosum L. cultivar Kardal contain resistance factors to the green peach aphid Myzuspersicae （Sulzer） （Hemiptera： Aphididae） and normal probing behavior is impeded. However, M. persicae can survive and reproduce on mature and senescent leaves of the cv. Kardal plant without problems. We compared the settling ofM. persicae on young and old leaves and analyzed the impact of aphids settling on the plant in terms of gene expression. Settling, as measured by aphid numbers staying on young or old leaves, showed that after 21 h significantly fewer aphids were found on the young leaves. At earlier time points there were no difference between young and old leaves, suggesting that the young leaf resistance factors are not located at the surface level but deeper in the tissue. Gene expression was measured in plants at 96 h postinfestation, which is at a late stage in the interaction and in compatible interactions this is long enough for host plant acceptance to occur. In old leaves of cv. Kardal （compatible interaction）, M. persicae infestation elicited a higher number of differentially regulated genes than in young leaves. The plant response to aphid infestation included a larger number of genes induced than repressed, and the proportion of induced versus repressed genes was larger in young than in old leaves. Several genes changing expression seem to be involved in changing the metabolic state of the leaf from source to sink.     

Distinction of water-soluble constituents between natural and cultured Cordyceps by capillary electrophoresis

Cordyceps is an expensive traditional Chinese medicine, which has anti-tumor activity and significant effects on the immune system. In Southeast Asia, Cordyceps is commonly sold in capsule form as a health food product. Most of these products are derived from cultured Cordyceps mycelia. Because of the price difference, some manufacturers claim their products are from natural Cordyceps. In order to distinguish among various types of Cordyceps in the market, the profiles of water-soluble constituents derived from different sources of Cordyceps were determined by capillary electrophoresis (CE). Both natural and cultured Cordyceps showed three peak clusters migrated at 5–7, 9–11 and 12–13 min, and the height and resolution of these peak clusters were rather distinct. Peak cluster at 9–11 min was identified as adenosine, guanosine and uridine, and shared a similarity between natural and cultured products. In contrast, the peak cluster at 5–7 min was characteristic of natural Cordyceps, regardless of hosts and sources. By using the peak characteristics of CE profiles of different Cordyceps samples, hierarchical clustering analysis was performed. The result shows that those samples of natural Cordyceps were grouped together distinct from the cultured and commercial products. Thus, the CE profiles could serve as fingerprints for the quality control of Cordyceps.     

脱共生作用对黑豆蚜氨基酸代谢的影响(英文)

为了解共生菌对黑豆蚜蛋白质、氨基酸代谢的影响 ,用利福平处理黑豆蚜以除去其细胞内共生细菌 ,产生脱共生蚜虫。结果表明 ,被脱去共生菌的蚜虫与未经抗生素处理的正常蚜虫相比 ,7日龄时 ,脱共生蚜虫每毫克鲜重的总蛋白含量降低了 2 9% ,每毫克鲜重的游离氨基酸含量提高了 17%。对黑豆蚜取食的蚕豆苗韧皮部组织中必需氨基酸所占的比例进行分析后发现 ,蚕豆苗韧皮部组织中的必需氨基酸含量仅占 2 0 % ,而有共生菌的黑豆蚜组织中必需氨基酸已达到 4 4% ,脱共生后降低到 37% ,这些结果证明了黑豆蚜的胞内共生菌为其寄主提供了部分必需氨基酸。通过对游离氨基酸组成的分析发现 ,在测定的 17种氨基酸中 ,必需氨基酸中的苏氨酸在共生蚜虫中所占的比例为 2 1 6 % ,在脱共生蚜虫中仅为 16 7%。同样 ,非必需氨基酸中的酪氨酸和丝氨酸 ,在共生蚜虫中分别占总游离氨基酸的 8 9%和 5 6 % ,而在脱共生蚜虫中却分别升高到 2 1 1%和 13 6 %。这些结果表明 ,各种氨基酸比例的失调 ,造成了脱共生蚜虫蛋白质合成受阻和部分游离氨基酸的积累 ,并因此导致蚜虫发育和繁殖的失调。     

Commercial production of microalgae: ponds, tanks, tubes and fermenters

The commercial culture of microalgae is now over 30 years old with the main microalgal species grown being Chlorella and Spirulina for health food, Dunaliella salina for β-carotene, Haematococcus pluvialis for astaxanthin and several species for aquaculture. The culture systems currently used to grow these algae are generally fairly unsophisticated. For example, Dunaliella salina is cultured in large (up to approx. 250 ha) shallow open-air ponds with no artificial mixing. Similarly, Chlorella and Spirulina also are grown outdoors in either paddle-wheel mixed ponds or circular ponds with a rotating mixing arm of up to about 1 ha in area per pond. The production of microalgae for aquaculture is generally on a much smaller scale, and in many cases is carried out indoors in 20–40 l carboys or in large plastic bags of up to approximately 1000 l in volume. More recently, a helical tubular photobioreactor system, the BIOCOIL™, has been developed which allows these algae to be grown reliably outdoors at high cell densities in semi-continuous culture. Other closed photobioreactors such as flat panels are also being developed. The main problem facing the commercialisation of new microalgae and microalgal products is the need for closed culture systems and the fact that these are very capital intensive. The high cost of microalgal culture systems relates to the need for light and the relatively slow growth rate of the algae. Although this problem has been avoided in some instances by growing the algae heterotrophically, not all algae or algal products can be produced this way.     

Rpfan37在刺槐共生结瘤过程中的功能探究

目的:研究刺槐中与磷脂酰肌醇转运蛋白有较高同源性的基因Rpfan37的功能,为探究相关基因参与豆科植物与根瘤菌共生结瘤过程提供新的思路。方法:通过前期研究,建立豆科植物刺槐与共生根瘤菌互作的抑制差减杂交反交文库,筛选疑似与共生结瘤相关的基因。利用PCR技术快速克隆经实时荧光定量PCR技术(qRT-PCR)分析基因在不同接菌时间及不同植物组织的表达。构建RNA干扰(RNAi)重组载体,转农杆菌介导转化植物根部,接种根瘤菌后验证该基因在刺槐共生结瘤过程的功能。结果:基因表达分析显示,在接菌与未接菌的刺槐根中,处理后第15天,Rpfan37表达均显著上调,但接菌与未接菌处理对该基因表达无显著影响;在成熟的根瘤中,该基因仅为低水平表达。RNAi转化植株的鲜重、株高、根长及结瘤数较对照组显著降低。在显微镜下观察到RNAi植株根毛发育异常;与对照相比,RNAi转化植株形成的根毛卷曲、根毛侵染线及根瘤原基数目均显著降低。根瘤石蜡切片结果显示RNAi植株根瘤中的侵染细胞与对照相比明显减少,分析豆血红蛋白表达发现,RNAi植株中根瘤发育成熟过程明显受阻。结论:在豆科植物刺槐中发现的相关基因Rpfan37能够参与刺槐共生结瘤过程,为研究磷脂酰肌醇转运蛋白在共生结瘤过程中的作用提供了新的理论基础。     

Precise molecular weight determination of PCR products of the rRNA intergenic spacer region using electrospray quadrupole mass spectrometry for differentiation of B. subtilis and B. atrophaeus, closely related species of bacilli

Assessment of 16S–23S rRNA intergenic spacer region (ISR) sequence variability is an important supplement to 16S rRNA sequencing for differentiating closely related bacterial species. Species differentiation can also be achieved by determination of approximate size of PCR (polymerase chain reaction) products of ISRs, based on their relative electrophoretic mobility on agarose gels. Closely-related species can have ISR PCR products that are similar in size. More precise molecular weight (M.W.) determination of these products might allow improved discrimination of such species. Electrospray quadrupole mass spectrometry (ESI-Q-MS) has the potential to provide such precision. For ESI-Q-MS analysis, size limitation of PCR products is currently limited to around 130 base pairs (bp). Bacillus subtilis and Bacillus atrophaeus are two closely related species with few distinguishing phenotypic characteristics. B. subtilis has recently been sub-divided into two subgroups, W23 (type strain, W23) and 168 (type strain, 168). PCR products amplified from the ISR including the 5′ terminal end of the 23S rRNA and a conserved portion of the ISR were analyzed by ESI-Q-MS. A 119 or 120 bp PCR product was produced for B. atrophaeus strains. However, strains of B. subtilis subgroups W23 and 168 each produced 114 bp products. In summary, a mass spectrometry method was developed for differentiation of B. subtilis and B. atrophaeus. Also, the genetic similarity of B. subtilis subgroups W23 and 168 was confirmed. Accurate determination of the molecular weight of PCR products from the 16S–23S rRNA intergenic spacer region using electrospray quadrupole mass spectrometry has great potential as a general technique for characterizing closely related bacterial species.     

Annual nitrogen budget of a temperate coastal barrier salt-marsh system along a productivity gradient at low and high marsh elevation

An annual nitrogen budget was established for a temperate back barrier salt-marsh system along a productivity gradient at low and high marsh elevation. We measured plant biomass and nitrogen content in three plant compartments to deduce plant N-allocation patterns. Measurements were done along a successional sequence in a salt-marsh system. In addition, N-mineralization, wet and dry atmospheric N-deposition and sediment N-deposition were measured.

Plant-species dominance changed along the successional sequence. In early stages, Elymus farctus and Spergularia media formed a large part of total plant biomass. Festuca rubra and Puccinellia maritima were dominant at intermediate stages, whereas Elymus pycnanthus and Limonium vulgare were dominant at late stages of succession. Shoot biomass was highest in June, whereas litter biomass was highest in September and December. Root biomass formed by far the largest fraction of total plant biomass, especially at a low-marsh elevation.

Wet deposition of nitrate and ammonium was 1.7 g N m⁻² yr⁻¹, whereas throughfall deposition (dry and wet deposition) amounted to 2.1–3.6 g N m⁻² yr⁻¹, and was positively related to the height of an artificial plant canopy. Sediment organic nitrogen deposition rate was 0.3–5.4 g N m⁻² yr⁻¹, and negatively related to marsh elevation. Nitrogen mineralization rate increased from 2.5–2.8 g N m⁻² yr⁻¹ in young marshes towards 8.0–12.7 g N m⁻² yr⁻¹ at older marshes, depending on marsh elevation.

At a low-marsh elevation, plant N-availability depended equally on tidal N, atmospheric N and mineralized N, especially in young marshes, whereas the decomposition pathway became more important in older marshes. Tidal N contributed most to ecoystem N-accumulation rate at early successional stages, whereas atmospheric N was more important at later stages. Tidal influence was low at high-marsh elevation sites. Here, atmospheric deposition was the dominant exogenous nitrogen source both in young and old marshes.     

Symbiotic microbial studies in diverse populations of Aphis gossypii,existing on altered host plants in different localities during different times

Complex interactions between symbiotic bacteria and insects ultimately result in equilibrium in all aspects of life in natural insect populations. In this study, abundance of principal symbiotic bacteria was estimated using qPCR in 1553 individuals of aphids, Aphis gossypii. Aphids were sampled from primary and secondary host plants—hibiscus and cotton. Hibiscus aphids were collected from 24 different locations in April, September, and November, whereas cotton aphids were collected between 2015 and 2017 from areas with wide variations in climatic conditions. About 30%–45% aphids were recorded with the most dominant symbiont, Arsenophonus. The other symbionts were in low frequency, and about 7% of aphids were noted with Hamiltonella, Acinetobacter, and Microbacterium, and 3% of aphids were verified with Serratia and Pseudomonas. Aphids infected with Hamiltonella, Arsenophonus, and Serratia can influence Buchnera densities. Hamiltonella has positive interaction with densities of Arsenophonus and Serratia. Almost 100% coinfection of Hamiltonella and Arsenophonus was detected in Xinxiang aphids and 50% coinfection was reported in aphids from North China, while no coinfection was detected in Hainan aphids. These findings describe the prevalence pattern and richness of core community of symbiotic bacteria in naturally occurring populations of A. gossypii and provide new insights for the study of symbiotic bacteria.     

Resistance of young lambs to Haemonchus contortus infection, and its loss following anthelmintic treatment

1988. Resistance of young lambs to Haemonchus contortus infection, and its loss following anthelmintic treatment. International Journal for Parasitology 18: 1107–1109. A comparison of worm burdens of grazing Merino lambs that were untreated, treated once and allowed to graze for 2 weeks, or given suppressive anthelmintic treatment until their final 2 weeks on pasture, showed that resistance to reinfection with Ostertagia circumcincta and Trichostrongylus colubriformis did not develop until the lambs were about 8 months old. By contrast, there was clear evidence that untreated lambs, but not lambs given one or more treatments, had acquired substantial resistance to infection with Haemonchus contortus by an age of 4 months.     

Alteration with age of symbiosis of gene expression in aphid endosymbionts

Aphid endosymbionts in vivo in young hosts synthesized almost exclusively only one protein, symbionin. The synthesis of symbionin declined with age of the host and instead the endosymbiont began to express some of its own genes which were expressed in vitro but were repressed in vivo in young host. A prolonged treatment of young host with cycloheximide brought about a physiological state similar to that in old insect. Though in the very old insect symbionin was no longer produced by its endosymbiont, the host seemed to depend almost entirely upon the gene products of the endosymbiont.