Aseptic rearing procedure for the stinkbug <Emphasis Type="Italic">Plautia stali</Emphasis> (Hemiptera: Pentatomidae) by sterilizing food-derived bacterial contaminants

The stinkbug Plautia stali Scott is a notorious agricultural pest whose posterior midgut hosts specific bacteria essential for its growth and survival, highlighted as an experimental model for symbiosis studies. Some symbiotic bacteria of P. stali are cultivable, found free-living in and acquired from the environment, and, furthermore, some free-living environmental bacteria are potentially capable of establishing symbiotic association with P. stali. In this context, it is expected that such environmental bacteria may occasionally contaminate and infect the experimental insects maintained in the laboratory, which could potentially affect the functional analyses of the symbiosis. Here we report that such contamination events do occur under a laboratory rearing conditions for P. stali. When symbiont-deprived newborn nymphs from surface-sterilized eggs were reared in sterilized plastic containers with autoclaved water, most of them died as nymphs presumably as a result of aposymbiosis, but only a small fraction could attain adulthood and the adult insects were all infected with γ-proteobacteria allied to Pantoea and Enterobacter. A variety of bacteria, mainly Bacillus and also Pantoea and Enterobacter, were detected from peanuts and soybeans provided as food for P. stali. Autoclaving of peanuts and soybeans eradicated these bacteria but negatively affected the host survival, whereas ethanol sterilization of peanuts and soybeans removed Pantoea and Enterobacter, but not Bacillus, without negative effects on the host survival. On the basis of these results, we established a practical procedure for aseptic rearing of P. stali, which will enable reliable and strict analyses of host–symbiont interactions in the model symbiotic system.     

The ectomycorrhizae of <Emphasis Type="Italic">Lactarius rimosellus</Emphasis> and <Emphasis Type="Italic">Lactarius acatlanensis</Emphasis> with the endangered <Emphasis Type="Italic">Fagus grandifolia</Emphasis> var. <Emphasis Type="Italic">mexicana</Emphasis>

Gut bacterium Pantoea sp. is one of the predominant bacterial species in the larval gut of the diamondback moth, Plutella xylostella. The phenotypic characters of Pantoea sp. were investigated with BIOLOG phenotype MicroArray (PM) in this study. Totally 950 different metabolic phenotypes were tested using the PM plates 1–10. Results exhibited that Pantoea sp. was able to metabolize 37.37 % of the tested carbon sources, 91.32 % of nitrogen sources, 100 % of sulfur sources, and 98.31 % of phosphorus sources. Most informative utilization patterns for carbon sources of Pantoea sp. were organic acids and carbohydrates, and for nitrogen were various amino acids. The bacterium had 94 different biosynthetic pathways. It had a wide range of adaptabilities, and could still metabolize in osmolytes with up to 9 % sodium chloride, 6 % potassium chloride, 5 % sodium sulfate, 20 % ethylene glycol, 4 % sodium formate, 4 % urea, 5 % sodium lactate, 200 mmol/L sodium phosphate (pH 7.0), 100 mmol/L ammonium sulfate (pH 8.0), 100 mmol/L sodium nitrate, and 100 mmol/L sodium nitrite, respectively. It also exhibited active metabolism under pH values between 4.5 and 10. Pantoea sp. showed active decarboxylase activities while poor deaminase activities in the presence of various amino acids. The phenotypic characterization of Pantoea sp. increased our knowledge of the bacterium, in particular its interactions with insect hosts and the adaptability in gut environments, and showed us some possible approaches to controlling diamondback moth through decreasing Pantoea sp. density.     

Diversity of endophytic fungal and bacterial communities in <Emphasis Type="Italic">Ilex paraguariensis</Emphasis> grown under field conditions

The composition and diversity of the endophytic community associated with yerba mate (Ilex paraguariensis) was investigated using culture-depending methods. Fungi were identified based on their micromorphological characteristics and internal transcribed spacer rDNA sequence analysis; for bacteria 16S rDNA sequence analysis was used. Fungal and bacterial diversity did not show significant differences between organ age. The highest fungal diversity was registered during fall season and the lowest in winter. Bacterial diversity was higher in stems and increased from summer to winter, in contrast with leaves, which decreased. The most frequently isolated fungus was Fusarium, followed by Colletotrichum; they were both present in all the sampling seasons and organ types assayed. Actinobacteria represented 57.5 % of all bacterial isolates. The most dominant bacterial taxa were Curtobacterium and Microbacterium. Other bacteria frequently found were Methylobacterium, Sphingomonas, Herbiconiux and Bacillus. Nitrogen fixation and phosphate solubilization activity, ACC deaminase production and antagonism against plant fungal pathogens were assayed in endophytic bacterial strains. In the case of fungi, strains of Trichoderma, Penicillium and Aspergillus were assayed for antagonism against pathogenic Fusarium sp. All microbial isolates assayed showed at least one growth promoting activity. Strains of Bacillus, Pantoea, Curtobacterium, Methylobacterium, Brevundimonas and Paenibacillus had at least two growth-promoting activities, and Bacillus, Paenibacillus and the three endophytic fungi showed high antagonistic activity against Fusarium sp. In this work we have made a wide study of the culturable endophytic community within yerba mate plants and found that several microbial isolates could be considered as potential inoculants useful for improving yerba mate production.     

Nutritional symbionts of a putative vector, <Emphasis Type="Italic">Xyleborus bispinatus</Emphasis>, of the laurel wilt pathogen of avocado, <Emphasis Type="Italic">Raffaelea lauricola</Emphasis>

Ambrosia beetles subsist on fungal symbionts that they carry to, and cultivate in, their natal galleries. These symbionts are usually saprobes, but some are phytopathogens. Very few ambrosial symbioses have been studied closely, and little is known about roles that phytopathogenic symbionts play in the life cycles of these beetles. One of the latter symbionts, Raffaelea lauricola, causes laurel wilt of avocado, Persea americana, but its original ambrosia beetle partner, Xyleborus glabratus, plays an uncertain role in this pathosystem. We examined the response of a putative, alternative vector of R. lauricola, Xyleborus bispinatus, to artificial diets of R. lauricola and other ambrosia fungi. Newly eclosed, unfertilized females of X. bispinatus were reared in no-choice assays on one of five different symbionts or no symbiont. Xyleborus bispinatus developed successfully on R. lauricola, R. arxii, R. subalba and R. subfusca, all of which had been previously recovered from field-collected females of X. bispinatus. However, no development was observed in the absence of a symbiont or on another symbiont, Ambrosiella roeperi, recovered from another ambrosia beetle, Xylosandrus crassiusculus. In the no-choice assays, mycangia of foundress females of X. bispinatus harbored significant colony-forming units of, and natal galleries that they produced were colonized with, the respective Raffaelea symbionts; with each of these fungi, reproduction, fecundity and survival of the beetle were positively impacted. However, no fungus was recovered from, and reproduction did not occur on, the A. roeperi and no symbiont diets. These results highlight the flexible nature of the ambrosial symbiosis, which for X. bispinatus includes a fungus with which it has no evolutionary history. Although the “primary” symbiont of the neotropical X. bispinatus is unclear, it is not the Asian R. lauricola.     

Analysis of the gut-specific microbiome from field-captured tsetse flies,and its potential relevance to host trypanosome vector competence

Background

The tsetse fly (Glossina sp.) midgut is colonized by maternally transmitted and environmentally acquired bacteria. Additionally, the midgut serves as a niche in which pathogenic African trypanosomes reside within infected flies. Tsetse’s bacterial microbiota impacts many aspects of the fly’s physiology. However, little is known about the structure of tsetse’s midgut-associated bacterial communities as they relate to geographically distinct fly habitats in east Africa and their contributions to parasite infection outcomes. We utilized culture dependent and independent methods to characterize the taxonomic structure and density of bacterial communities that reside within the midgut of tsetse flies collected at geographically distinct locations in Kenya and Uganda.

Results

Using culture dependent methods, we isolated 34 strains of bacteria from four different tsetse species (G. pallidipes, G. brevipalpis, G. fuscipes and G. fuscipleuris) captured at three distinct locations in Kenya. To increase the depth of this study, we deep sequenced midguts from individual uninfected and trypanosome infected G. pallidipes captured at two distinct locations in Kenya and one in Uganda. We found that tsetse’s obligate endosymbiont, Wigglesworthia, was the most abundant bacterium present in the midgut of G. pallidipes, and the density of this bacterium remained largely consistent regardless of whether or not its tsetse host was infected with trypanosomes. These fly populations also housed the commensal symbiont Sodalis, which was found at significantly higher densities in trypanosome infected compared to uninfected flies. Finally, midguts of field-captured G. pallidipes were colonized with distinct, low density communities of environmentally acquired microbes that differed in taxonomic structure depending on parasite infection status and the geographic location from which the flies were collected.

Conclusions

The results of this study will enhance our understanding of the tripartite relationship between tsetse, its microbiota and trypanosome vector competence. This information may be useful for developing novel disease control strategies or enhancing the efficacy of those already in use.

     

Inverse PCR for subtyping of <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> carrying IS<Emphasis Type="Italic">Aba</Emphasis>1

Acinetobacter baumannii has been prevalent in nosocomial infections, often causing outbreaks in intensive care units. ISAba1 is an insertion sequence that has been identified only in A. baumannii and its copy number varies among strains. It has been reported that ISAba1 provides a promoter for bla_OXA-51-like, bla_OXA-23-like, and bla_ampC, which are associated with the resistance of A. baumannii to carbapenems and cephalosporins. The main purpose of this study was to develop a novel inverse PCR method capable of typing A. baumannii strains. The method involves three major steps: cutting of genomic DNA with a restriction enzyme, ligation, and PCR. In the first step, bacterial genomic DNA was digested with DpnI. In the second step, the digested genomic DNAs were ligated to form intramolecular circular DNAs. In the last step, the ligated circular DNAs were amplified by PCR with primers specific for ISAba1 and the amplified PCR products were electrophoresed. Twenty-two clinical isolates of A. baumannii were used for the evaluation of the inverse PCR (iPCR) typing method. Dendrogram analysis revealed two major clusters, similar to pulsed-field gel electrophoresis (PFGE) results. Three ISAba1-associated genes — bla_ampC, bla_OXA-66-like, and csuD — were amplified and detected in the clinical isolates. This novel iPCR typing method is comparable to PFGE in its ability to discriminate A. baumannii strains, and is a promising molecular epidemiological tool for investigating A. baumannii carrying ISAba1.     

Interseasonal and interspecies diversities of <Emphasis Type="Italic">Symbiodinium</Emphasis> density and effective photochemical efficiency in five dominant reef coral species from Luhuitou fringing reef,northern South China Sea

Although it is well established that different coral species have different susceptibilities to thermal stress, the reasons behind this variation are still unclear. In this study, 384 samples across five dominant coral species were collected seasonally between September 2013 and August 2014 at Luhuitou fringing reef in Sanya, Hainan Island, northern South China Sea, and their algal symbiont density and effective photochemical efficiency (Φ _PSII) were measured. The results indicated that both the Symbiodinium density and Φ _PSII of corals were subject to significant interspecies and seasonal variations. Stress-tolerant coral species, including massive Porites lutea and plating Pavona decussata, had higher symbiont densities but lower Φ _PSII compared to the vulnerable branching species of Acropora over the course of all four seasons. Seasonally, coral symbiont densities were the lowest during winter, while during the same period, Φ _PSII of corals was at the highest point. Further analysis suggested that dissolved inorganic nutrients and upwelling in the reef area were probably responsible for the observed seasonal variations in symbiont density. The fact that Porites lutea has the lowest Φ _PSII during all four seasons is likely related to their symbionts’ lower capacity to provide required photosynthates for calcification. These results suggest that a coral’s thermal tolerance is primarily and positively dependent on its symbiont density and is less related to its effective photochemical efficiency.     

Increased trehalose biosynthesis improves <Emphasis Type="Italic">Mesorhizobium ciceri</Emphasis> growth and symbiosis establishment in saline conditions

Cicer arietinum (chickpea) is a legume very sensitive to salinity, and so are most of its rhizobial symbionts belonging to the species Mesorhizobium ciceri. We observed that exogenous trehalose (i.e., added to the growth medium) can significantly improve growth of M. ciceri strain Rch125 under moderate salinity. In order to test if endogenous trehalose (i.e., synthesized by the cell) could also enhance salt tolerance, strain Rch125 was genetically modified with various trehalose biosynthesis genes from Sinorhizobium meliloti 1021 (otsA, treS, treY) and Mesorhizobium loti MAFF 303099 (otsAB). We found that overexpression of otsA or otsAB, but not treS or treY, significantly improved M. ciceri Rch125 growth in saline media. This growth improvement correlated with enhanced trehalose accumulation in otsA- and otsAB-modified cells, suggesting that increased trehalose synthesis via trehalose-6-phosphate can enhance bacterial salt tolerance. Chickpea plants inoculated with M. ciceri Rch125 derivatives carrying extra otsAB or otsA genes formed more nodules and accumulated more shoot biomass than wild type inoculated plants when grown in the presence of NaCl. These results support the notion that improved salt tolerance of the bacterial symbiont can alleviate the negative effects of salinity on chickpeas, and that such improvement in M. ciceri can be achieved by manipulating trehalose metabolism.     

Expression of <Emphasis Type="Italic">Tlr2</Emphasis>, <Emphasis Type="Italic">Defa</Emphasis>, and <Emphasis Type="Italic">Muc2</Emphasis> genes in rat duodenum epithelial cells during prolonged stomach hypoacidity and after hypoacidity correction by multiprobiotics

Expression of Tlr2, Defa, and Muc2 genes in epithelial cells of rat duodenum during prolonged stomach hypoacidity (hypochlorhydria) and after hypoacidity correction by multiprobiotics has been investigated. Elevation of Tlr2, Muc2, and Defa gene expression levels upon the intensification of lipid peroxidation processes in the epithelial cells of the villi and crypts of the rat duodenum under hypoacidic conditions has been demonstrated. Administration of a multiprobiotic under the same conditions downregulated the expression of the aforementioned genes in epithelial cells of the villi and crypts, bringing it to a nearly normal level, and exerted a similar effect on the levels of lipid peroxidation products. The data obtained may be indicative of the involvement of Tlr2, Muc2, and Defa genes in the development of duodenal inflammation induced by dysbiotic changes occurring in prolonged hypochlorhydria.     

Detection of symbionts and virus in the whitefly <Emphasis Type="Italic">Bemisia tabaci</Emphasis> (Hemiptera: Aleyrodidae), vector of the <Emphasis Type="Italic">Mungbean yellow mosaic India virus</Emphasis> in Central India

Legume crops in Central India, the main soybean production area of the country, may suffer from yellow mosaic disease caused by the Mungbean yellow mosaic India virus (MYMIV). MYMIV is transmitted by the sweet potato whitefly, Bemisia tabaci (Gennadius), which is a species complex composed of various genetic groups. This vector species harbors different endosymbionts among regional strains and among individuals. To elucidate fundamental aspects of this virus vector in the state of Madhya Pradesh, the infection status of the symbionts and the virus in whiteflies was studied. A polymerase chain reaction (PCR) survey of the whiteflies collected in Madhya Pradesh found four secondary endosymbionts, Arsenophonus, Hemipteriphilus, Wolbachia, and Cardinium, in addition to the primary endosymbiont Portiera. Arsenophonus and Hemipteriphilus were highly infected but the infection rates of Wolbachia and Cardinium were low. MYMIV was detected in whitefly populations collected from various host plants in Madhya Pradesh. The whitefly populations belonged to the Asia I and II genetic groups; several different Asia II populations were also distributed. Specific relations were not observed among symbiont infection status, virus infection, and the whitefly genetic groups in the populations of Madhya Pradesh, though Cardinium was highly detected in the Asia II-1 group. New primers, which can be used for PCR template validation and for discriminating two phylogenetically close endosymbionts, were designed.     

Investigation of the midgut structure and ultrastructure in <Emphasis Type="Italic">Cimex lectularius</Emphasis> and <Emphasis Type="Italic">Cimex pipistrelli</Emphasis> (Hemiptera: Cimicidae)

Cimicidae are temporary ectoparasites, which means that they cannot obtain food continuously. Both Cimex species examined here, Cimex lectularius (Linnaeus 1758) and Cimex pipistrelli (Jenyns 1839), can feed on a non-natal host, C. lectularius from humans on bats, C. pipistrelli on humans, but never naturally. The midgut of C. lectularius and C. pipistrelli is composed of three distinct regions—the anterior midgut (AMG), which has a sack-like shape, the long tube-shaped middle midgut (MMG), and the posterior midgut (PMG). The different ultrastructures of the AMG, MMG, and PMG in both of the species examined suggest that these regions must fulfill different functions in the digestive system. Ultrastructural analysis showed that the AMG fulfills the role of storing food and synthesizing and secreting enzymes, while the MMG is the main organ for the synthesis of enzymes, secretion, and the storage of the reserve material. Additionally, both regions, the AMG and MMG, are involved in water absorption in the digestive system of both Cimex species. The PMG is the part of the midgut in which spherites accumulate. The results of our studies confirm the suggestion of former authors that the structure of the digestive tract of insects is not attributed solely to diet but to the basic adaptation of an ancestor.     

A PCR-based analysis of plant DNA reveals the feeding preferences of <Emphasis Type="Italic">Apolygus lucorum</Emphasis> (Heteroptera: Miridae)

The mirid bug Apolygus lucorum (Meyer-Dür) (Heteroptera: Miridae) is a severe pest of cotton and other crops in China. The feeding preferences of this pest are unclear due to its frequent movement among different host plants and the inconspicuous signs of its feeding. Here, we present results of a field trial that used direct observation of bug densities and a PCR-based molecular detection assay to detect plant DNA in bugs to explore relationships between A. lucorum population abundance and its feeding preference between two host plants, Humulus scandens (Loureiro) Merrill and Medicago sativa L. The field-plot samples showed that A. lucorum adults generally prefer flowering host plants. Its density was significantly higher on flowering H. scandens than on seedlings of M. sativa, and a similarly higher bug density was observed on flowering M. sativa than on seedlings of H. scandens. In the laboratory, we designed two pairs of species-specific primers targeting the trnL-F region for H. scandens and M. sativa, respectively. The detectability of plant DNA generally decreased with time post-feeding, and the half-life of plant DNA detection (DS₅₀) in the gut was estimated as 6.26 h for H. scandens and 3.79 h for M. sativa with significant differences between each other. In mirid bugs exposed to seedlings of H. scandens and flowering M. sativa, the detection rate of M. sativa DNA was significantly higher than that of H. scandens. Meanwhile, in mirid bugs exposed to seedlings of M. sativa and flowering H. scandens, a significantly higher detection rate of H. scandens DNA was found. We developed a useful tool to detect the remaining plant food species specifically from the gut of A. lucorum in the current study. We provided direct evidence of its feeding preference between H. scandens and M. sativa at different growth stages, which strongly supported a positive correlation between population abundance and feeding preference of A. lucorum on different plants under field conditions. The findings provide new insights into the understanding of A. lucorum’s feeding preference, and are helpful for developing the strategies to control this pest.     

Effect of entomopathogenic fungus <Emphasis Type="Italic">Metarhizium robertsii</Emphasis> on non-target organisms,water bugs (Heteroptera: Corixidae,Naucoridae, Notonectidae)

The influence of the fungus Metarhizium robertsii Bischoff, Rehner and Humber on the mortality of four water bug species, Cymatia coleoptrata (Fabricius), Sigara assimilis (Fieber), Ilyocoris cimicoides cimicoides (Linnaeus), and Notonecta reuteri Hungerford, and bloodsucking mosquito Anopheles messeae Falleroni, was investigated under various concentrations of conidia and different treatment types. We found that the mortality of adults of the water bug species was similar or higher than that of A. messeae, with C. coleoptrata and S. assimilis being more susceptible to M. robertsii than N. reuteri, I. c. cimicoides, and the mosquito A. messeae. Treatment with dry conidia at concentrations of 5 × 10⁴ and 5 × 10⁵ conidia/ml caused higher mortality of the water bug species than did treatment at the same concentrations with conidia in an aqueous suspension. In contrast, higher concentrations (5 × 10⁶ conidia/ml) led to higher mortality after treatment with the aqueous suspension, relative to treatment with dry conidia. Our studies showed that water bugs exhibited the classical development of a mycosis with hemocoel colonization, mummification, and conidia formation on cadavers directly on the surface of the water. Possible changes in invertebrate communities in aquatic ecosystems after treatment with Metarhizium are discussed.     

Symbiotic bacteria associated with gut symbiotic organs and female genital accessory organs of the leaf beetle <Emphasis Type="Italic">Bromius obscurus</Emphasis> (Coleoptera: Chrysomelidae)

Leaf beetles (Coleoptera: Chrysomelidae) constitute one of the most species-rich insect families, and live exclusively on leaves or other plant parts. Early histological works described the presence of symbiotic bacteria in gut-associated symbiotic organs of some chrysomelid species, but their microbiological nature has been poorly characterized except for those associated with reed beetles of the subfamily Donaciinae. Here we investigated symbiotic bacteria of the leaf beetle Bromius obscurus (L.) belonging to the subfamily Eumolpinae. Specific bacterial 16S ribosomal RNA and gyrB gene sequences were consistently obtained from the symbiotic organs, which radially surround the foregut-midgut junction, of all adult males and females examined. In adult females, the same sequences were also obtained from a pair of genital accessory organs, which are presumably for vertical symbiont transmission. Whole mount in situ hybridization specifically detected the symbiont in the gut symbiotic organs endocellularly and also in the female genital accessory organs extracellularly. In the gut symbiotic organs, the endocellular symbiont cells were small and rosette-like or aggregated and granule-like, whereas in the female genital organs the extracellular symbiont cells were of a condensed form. Molecular phylogenetic analysis showed that the symbiont of B. obscurus constitutes a distinct lineage in the Gammaproteobacteria. Molecular evolutionary analysis has identified significantly accelerated molecular evolution and a highly adenine–thymine-biased nucleotide composition of the symbiont genes, presumably reflecting reductive evolution of the symbiont genome. These results suggest an intimate and stable host-symbiont association in B. obscurus, in which the symbiont may play some important, though hitherto unknown, biological roles in its herbivorous insect host.     

Linkages of <Emphasis Type="Italic">Firmicutes</Emphasis> and <Emphasis Type="Italic">Bacteroidetes</Emphasis> populations to methanogenic process performance

To identify potential linkages between specific bacterial populations and process performance in anaerobic digestion, the dynamics of bacterial community structure was monitored with high-throughput sequencing in triplicate anaerobic digesters treating animal waste. Firmicutes and Bacteroidetes were found as the two most abundant populations, however, with contrasting population dynamics in response to organic overloading. Firmicutes dominated the bacterial community during stable process performance at low organic loading rate, representing over 50 % of the bacterial abundance. In contrast, the onset of organic overloading raised the relative abundance of Bacteroidetes from 20 ± 2.6 to 44 ± 3.1 %. In addition to the significant negative correlation between the relative abundance of Firmicutes and Bacteroidetes, populations of Firmicutes and Bacteroidetes were found to be linked to process parameters including organic loading rate, volatile fatty acids concentration, and methane production. Therefore, the population abundance ratio of Firmicutes to Bacteroidetes (F/B ratio) was suggested as a potential indicator for process performance. The interactions between Firmicutes and Bacteroidetes populations could be exploited to develop strategies for the prevention of performance perturbation in anaerobic digestion processes.     

Identification of the di-<Emphasis Type="Italic">n</Emphasis>-butyl phthalate-biodegrading strains and the biodegradation pathway in strain LMB-1

DNA isolated from a greenhouse soil (Nanjing, Jiangsu Province, China) was suitable for PCR amplification of gene segment coding for the 16S rRNA. Diverse PCR products were characterized by cloning and sequencing, and analysis of bacterial colonies showed the presence over 26 phyla. The most bacteria belonged to Proteobacteria, Actinobacteria, Gemmatimonadetes, Acidobacteria and Planctomycetes. Furthermore, after the enrichment procedure of DBP-degrading microorganisms, 4 strains were isolated from the soil sample with di-n-butyl phthalate (DBP) biodegradability, and they were identified to be Rhizobium sp., Streptomyces sp., Pseudomonas sp. and Acinetobacter sp. Analysis of the degradation products by LC-MS led to identification of metabolites of DBP in strain LMB-1 (identified as Rhizobium sp.) which suggests that DBP was degraded through β-oxidation, demethylation, de-esterification and cleavage of aromatic ring.     

Are microbial symbionts involved in the speciation of the gall-inducing aphid, <Emphasis Type="Italic">Slavum wertheimae</Emphasis>?

Microbial symbionts have come to be recognized as agents in the speciation of their eukaryote hosts. In this study, we asked if bacterial symbionts are, or were in the past, involved in the speciation of the gall-inducing aphid Slavum wertheimae (Hemiptera: Aphididae). This aphid is specific to the tree Pistacia atlantica, which has a fragmented distribution among mesic and xeric habitats, leading to corresponding fragmentation of the aphid population. Previous studies revealed genetic differentiation among populations of the gall-inducing aphid, suggesting cryptic allopatric speciation. Pistacia atlantica trees show no such variation. By means of diagnostic PCR, we screened several populations of S. wertheimae from mesic and xeric sites in Israel for the presence of nine known aphid symbionts: Arsenophonus, Hamiltonella, Regiella, Rickettsia, Rickettsiella, Serratia, Spiroplasma, Wolbachia, and X-type, as well as Cardinium, known to be a reproductive manipulator. Only one symbiont, Wolbachia, was detected in S. wertheimae. Wolbachia was found in all the aphids of the mesic populations, compared to 26% in the aphids from the xeric populations. Multilocus Sequence typing of Wolbachia revealed new haplotypes in the fbpA and coxA genes in both the mesic and xeric populations. Phylogenetic analysis showed that Wolbachia of S. wertheimae is closely related to Wolbachia strains from assorted hosts, mostly lepidopterans, but only distantly related to Wolbachia strains from other aphid species. We conclude that the cryptic speciation of mesic and xeric populations of S. wertheimae was likely driven by geographical isolation rather than by Wolbachia.     

Diversity of the Most Commonly Reported Facultative Symbionts in Two Closely-Related Aphids with Different Host Ranges

Richness and abundance of facultative symbionts vary strongly with aphid species and genotype, symbiont strain, host plant, biogeography, and a number of abiotic factors. Despite indications that aphids in the same ecological niche show similar levels of facultative symbiont richness, existing reports do not consider the potential role of host plants on aphid microbial community. Little is known about how oligophagy and polyphagy may be influenced by secondary symbiont distribution, mainly because studies on secondary symbiont diversity are biased towards polyphagous aphids from the Northern Hemisphere. Here, we demonstrate the richness and abundance of the most common aphid-associated facultative symbionts in two tropical aphid species, the oligophagous Aphis (Toxoptera) citricidus (Kirkaldy) (Hemiptera: Aphididae) and the polyphagous Aphis aurantii (Boyer de Fonscolombe) (Hemiptera: Aphididae). Aphis citricidus is restricted to Citrus sp. host plants and closely related genera, whereas A. aurantii successfully exploits a wide variety of host plants from different families. Both were collected in the same ecological niche and our data basically indicated the same richness of secondary symbionts, but the abundance at which secondary symbionts occurred was very distinct between the two species. Spiroplasma was the most abundant facultative symbiont associated with A. citricidus and A. aurantii in the ecological niche studied. Single and multiple secondary symbiont infections were observed, but diversity of multiple infections was particularly high in A. citricidus. We discuss our findings and suggest hypotheses to explain causes and consequences of the differences in secondary symbiont diversity observed between these two aphid species.     

Impact of <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> inoculation on indigenous bacterial communities during agricultural waste composting

This research was conducted to distinguish between the separate effects of the Phanerochaete chrysosporium inoculation and sample property heterogeneity induced by different inoculation regimes on the indigenous bacterial communities during agricultural waste composting. P. chrysosporium was inoculated during different phases. The bacterial community abundance and structure were determined by quantitative PCR and denaturing gradient gel electrophoresis analysis, respectively. Results indicated a significant stimulatory effect of P. chrysosporium inoculation on the bacterial community abundance. The bacterial community abundance significantly coincided with pile temperature, ammonium, and nitrate (P?<?0.006). Variance partition analysis showed that the P. chrysosporium inoculation directly explained 20.5 % (P?=?0.048) of the variation in the bacterial communities, whereas the sample property changes induced by different inoculation regimes indirectly explained up to 35.1 % (P?=?0.002). The bacterial community structure was significantly related to pile temperature, water-soluble carbon (WSC), and C/N ratio when P. chrysosporium were inoculated. The C/N ratio solely explained 7.9 % (P?=?0.03) of the variation in community structure, whereas pile temperature and WSC explained 7.7 % (P?=?0.026) and 7.5 % (P?=?0.034) of the variation, respectively. P. chrysosporium inoculation affected the indigenous bacterial communities most probably indirectly through increasing pile temperature, enhancing the substrate utilizability, and changing other physico-chemical factors.