Diversity, frequency, and geographic distribution of facultative bacterial endosymbionts in introduced aphid pests

Facultative bacterial endosymbionts in insects have been under intense study during the last years. Endosymbionts can modify the insect＇s phenotype, conferring adap- tive advantages under environmental stress. This seems particularly relevant for a group of worldwide agricultural aphid pests, because endosymbionts modify key fitness-related traits, including host plant use, protection against natural enemies and heat tolerance. Aimed to understand the role of facultative endosymbionts on the success of introduced aphid pests, the distribution and abundance of 5 facultative endosymbionts （Hamiltonella defensa, Regiella insecticola, Serratia symbiotica, Rickettsia and Spiroplasma） were stud- ied and compared in 4 cereal aphids （Sitobion avenae, Diuraphis noxia, Metopolophium dirhodum and Schizaphis graminium） and in the pea aphid Acyrthosiphon pisum complex from 2 agroclimatic zones in Chile. Overall, infections with faeultative endosymbionts exhibited a highly variable and characteristic pattern depending on the aphid species/host race and geographic zone, which could explain the success of aphid pest populations after their introduction. While S. symbiotica and H. defensa were the most frequent endosym- bionts carried by the A. pisum pea-race and A. pisum alfalfa-race aphids, respectively, the most frequent facultative endosymbiont carried by all cereal aphids was R. insecticola. Interestingly, a highly variable composition of endosymbionts carried by S. avenae was also observed between agroclimatic zones, suggesting that endosymbionts are responding differentially to abiotic variables （temperature and precipitations）. In addition, our findings constitute the first report of bacterial endosymbionts in cereal aphid species not screened before, and also the first report of aphid endosymbionts in Chile.

Selective and stable elimination of endosymbionts from multiple-infected whitefly Bemisia tabaci by feeding on a cotton plant cultured in antibiotic solutions

The maternally heritable endosymbiont provides many ecosystem functions.Antibiotic elimination of a specific symbiont and establishment of experimental host lines lacking certain symbionts enable the roles of a given symbiont to be explored.The whitefly Bemisia tabaci(Gennadius)in China harbors obligate symbiont Portiera infecting each in-dividual,as well as facultative symbionts,such as Hamiltonella,Rickettsia and Cardinium,with co-infections occurring relatively frequently.So far no studies have evaluated the selectivity and efficacy of a specific symbiont elimination using antibiotics in whiteflies co-infected with different symbionts.Furthermore,no success has been achieved in estab-lishing certain symbiont-free B.tabaci lines.In this study,we treated Hamiltonella infected B.tabaci line,Hamiltonella-Rickettsia-co infected line and Hamiltonella-Cardinium co-infected line by feeding B.tabaci adults with cotton plants cultured in water containing ri-fampicin,ampicillin or a mixture of them,aiming to selectively curing symbiont infections and establishing stable symbiont-free lines.We found ampicillin selectively eliminated Cardinium without afecting Portiera,Hamiltonella and Rickettsia,although they coex-isted in the same host body.Meanwhile,all of the symbionts considered in our study can be removed by rifampicin.The reduction of facultative symbionts occurred at a much quicker pace than obligate symbiont Portiera during rifampicin treatment.Also,we measured the stability of symbiont elimination in whitefly successive generations and established Rickettsia-infected and Cardinium-infected lines which are absent in natural populations.Our results provide new protocols for selective elimination of symbionts co-existing in a host and establishment of different symbiont-infected host lines.

Ecological factors influencing the beneficial endosymbionts of the hemlock woolly adelgid (Hemiptera:Adelgidae)

Bacterial endosymbionts of sap-sucking insects provide their host with a num- ber of beneficial qualities,including the supply of nutrition,defense against parasitoids, and protection from heat stress.Damage to these bacterial associates can therefore have a negative impact on the fitness of their insect host.We evaluated observational and experi- mental factors regarding the normative hemlock woolly adelgid (Adelges tsugae Annand) (Hemiptera:Adelgidae)to help understand the roles of its three recently identified symbionts,including under heat stress conditions.The prevalence of A.tsugae's facultafive symbiont (Serratia symbiotica)was examined at different spatial scales to determine how variable infection rates are for this symbiont.There was no significant difference found in infection rates between adelgids on a tree,within a plot,or within a state.However, significantly more adelgids in Georgia (95%)had S.symbiotica compared to those in New York (68%).Microsatellite genotyping of the adelgids found that this difference was most likely not the result of a second introduction ofA.tsugae into eastern North America.Comparison orS.symbiotica proportions between first and fourth instars showed that symbiont absence did not affect the ability ofA.tsugae to survive aestivation.Evaluations of sym- biont densities within each adelgid found that when S.symbiotica was absent,the density of obligate symbionts was significantly higher.Exposure to heat stress (32.5℃)was not consistently correlated with changes in symbiont densities over a 4-d period.Overall,we have shown that symbiont prevalence and densities vary within the broad population of A.tsugae in eastern North America,with potentially significant effects upon the ecology of this important pest.

Recent advances in molecular biology research of a rice pest, the brown planthopper

The brown planthopper, Nilaparvata lugens St?l, has become a major threat in tropical Asian and China since the rice green revolution of the 1960 s. Currently, insecticide application remains the primary choice for controlling this rice insect pest, but heavy use of insecticides poses dangerous risks to beneficial natural enemies and pollinators, and stimulates N. lugens reproductivity, and has caused a resurgence of the pest in the major rice-planting regions throughout Asia. Achieving the long-lasting goal of sustainable management of N. lugens requires understanding of the molecular basis of outbreaks of the pest and the development of environment-friendly pest-control strategies. Here, we review the recent molecular advances in N. lugens research on the aspects of its endosymbionts, virus transmission, insecticide resistance, and interaction between N. lugens and rice plants. We also put forward further research directions that may shed some lights on management of the rice pest.

Molecular Evidences for the Biosynthesis of Pederin by Endosymbiont

Pederin belongs to a group of antitumor compounds found in terrestrial beetles and marine sponges. It is apparently used by some members of the rove beetle Paederus as a chemical defense against predators. A recent cluster analysis of the putative pederin biosynthesis gene （ped） strongly suggests that pederin is produced by bacterial symbionts. This paper reviewed the criteria for proving symbiontic origin of bioactive metabolite, indirect and molecular evidences for pederin bacterial origin, as well as three sets ofped clusters and putative biosynthesis process of pederin.

Isolation and Identification of Symbiotic Bacteria from <i>Brasenia schreberi</i>and Analysis of their Polysaccharide Producing Characteristics and Antioxidant Activity

Brasenia schreberi (water shield), a perennial freshwater aquatic plant of the family Nymphaeaceae, is rich in polysaccharides with positive biological activity and potential application value. As an indicator plant of the environment in wetlands, B. schreberi original and wild water environment faced increasing deterioration and has been listed as a critically endangered species in several countries of East Asia. Seeking an alternative method to produce B. schreberi mucilage polysaccharide is of top priority. The morphological structure of the stem and leaf of B. schreberi was studied. Well-developed glandular cells and aerenchyma tissue were observed in the stem and leaves of water shield, which were related to its aquatic habitat and polysaccharide secretion and then three endophytic bacteria were successfully isolated and purified from its stem and leaf and identified as Micromonospora sp. YG-1, Xylariaceae sp. strain UT-X and Psychrobacter pulmonis strain T-15. Symbiotic bacteria from B. schreberi produced polysaccharides with similar spectral peaks and chemical functional groups to those of B. chinensis. Further, analysis of hydroxyl free radicals showed that symbiotic bacteria in water shield had significant antioxidant activity against hydroxyl free radicals. This study provides a theoretical and practical basis for further study using symbiotic bacteria to produce polysaccharides to overcome the environmental limitations of the growth and development of B. schreberi.

Prevalence of Wolbachia in 10 Tenebrionidae stored-product insects and spatiotemporal infection dynamics in Tribolium confusum(Jaquelin Du Val)(Coleoptera:Tenebrionidae)

Insect symbionts Wolbachia used for pest population control has focused on vector pest species and agricultural insects while rare reports in Chinese stored-product insect samples.In this paper,we surveyed the prevalence of Wolbachia using a PCR detection method in ten Tenebrionidae stored-product insects.Subsequently,the spatiotemporal Wolbachia infection dynamics in Tribolium confusum and Wolbachia elimination patterns using tetracycline treatment were investigated in detail by TaqMan®probe real-time quantitative PCR,and host reproductive fitness parameters were compared.T.confusum was the only Wolbachia infected species in all the surveyed species.Wolbachia infection density consistently increased with the development of T.confusumand plateaued at 3.7×107 wsp copies per individual insect at the young adult stage.Wolbachia densities in females were higher than that in males with a significant difference at the pupae stage and varied among different tissues and organs.Uninfected female beetles were completely incapable of producing mature progenies when crossed with Wolbachia infected males.Embryogenesis and egg hatch rate were specifically inhibited after Wolbachia elimination,while other traits,including the number of eggs,pupation rate and sex ratio,remained unaffected by tetracycline treatment.Our results showthat the TaqMan®probe qPCR is a reliable detection method for quantifying the density of Wolbachia as compared to qualitative detection of wsp gene by PCR and relatively quantified by real-time qPCR.The fitness results indicated that Wolbachia infection was not an obligate symbiont and benefited the host confused flour beetle.

Bacterial Community in Different Populations of Rice Brown Planthopper Nilaparvata lugens(Stl)

The structures of bacterial communities in the brown planthopper （BPH） Nilaparvata lugens （St^l） from different geographic and resistant virulent populations were analyzed by using denatured gradient gel electrophoresis （DGGE）. Results showed that the bacterial communities in BPH nymph from the first to the fifth instars varied with nymphal growth and development. The bacterial communities in the first-instar BPH nymph were similar to those in adults. Nine geographic BPH populations were divided into three groups based on the cluster analysis of DGGE fingerprint. The first group was from the Philippines; the second group was from Thailand and Hainan, Yunnan and Zhejiang provinces of China; and the third group was from Vietnam and Guangxi, Hunan and Jiangxi provinces of China. BPH populations adapted to different resistant rice varieties. The BPH populations from Mudgo （with resistant gene Bphl） and ASD7 （with resistant gene bph2） differed with those of the susceptible rice variety TNI.

Hemipteran and dipteran pests: Effectors and plant host immune regulators

Hemipteran and dipteran insects have behavioral,cellular and chemical strategies for evading or coping with the host plant defenses making these insects particularly destructive pests worldwide. A critical component of a host plant＇s defense to herbivory is innate immunity. Here we review the status of our understanding of the receptors that contribute to perception of hemipteran and dipteran pests and highlight the gaps in our knowledge in these early events in immune signaling. We also highlight recent advances in identification of the effectors that activate pattern-triggered immunity and those involved in effector-triggered immunity.

Genetic variation and potential coinfection of Wolbachia among widespread Asian citrus psyllid (Diaphorina citri Kuwayama) populations

Wolbachia can profoundly influence the survival, reproduction, and defenses of insect hosts. These interactions could potentially be harnessed for managing pests or insecttransmitted diseases. Diaphorina citri Kuwayama is a phloem-feeding pest capable of transmitting the putative causal agent of citrus greening, Candidatus Liberibacter asiaticus (CLas). Like many insects, D. citri is also infected with Wolbachia (wDi). Recent studies indicate that the relative abundance of wDi could be associated with the abundance of CLas, and that wDi may contribute to regulating expression of phage lytic cycle genes in CLas, suggesting the need for better understanding of wDi biology in general. This study investigated the genetic diversity of wDi among D. citri in populations spanning eleven countries and two U.S. territories. Six Wolbachia genes, wsp, coxA.fbpA.ftsZ, gatB, and hep A, were sequenced and compared across samples. Two prevalent wDi strains were identified across the samples, and screening of clone libraries revealed possible coinfection of wDi strains in specific populations. D. citri mitochondrial cytochrome oxidase subunit I gene (mtCOI) were more divergent between D. citri populations that were infected with different wDi strains or had different infection statuses (single infection vs. coinfection). While we could not eliminate the possibility that maternal transmission may contribute to such patterns, it is also possible that wDi may induce cytoplasmic incompatibility in their host. These fin dings should contribute to the understanding of wDi population ecology, which may facilitate manipulation of this endosymbiont for management of citrus greening disease worldwide.

Species and endosymbiont diversity of Bemisia tabaci （Homoptera： Aleyrodidae） on vegetable crops in Senegal

Bemisia tabaci-transmitted geminiviruses are one of the major threats on cassava and vegetable crops in Africa. However, to date, few studies are available on the diversity orB. tabaci and their associated endosymbionts in Africa. More than 28 species have been described in the complex ofB. tabaci cryptic species; among them, 2 are invasive pests worldwide： MED and MEAM1. In order to assess the species diversity orB. tabaci in vegetable crops in Senegal, several samplings in different localities, hosts and seasons were collected and analyzed with nuclear （microsatellite） and mitochondrial （COI） markers. The bacterial endosymbiont community was also studied for each sample. Two species were detected： MED Q1 and MEAM1 B. Patterns of MED Q1 （dominance on most of the samples and sites, highest nuclear and mitochondrial diversity and broader secondary endosymbiont community： Hamiltonella, Cardinium, Wolbachia and Rickettsia）, point toward a predominant resident begomovirus vector group for MED Q 1 on market gardening crops. Furthermore, the lower prevalence of the second species MEAM1 B, its lower nuclear and mitochondrial diversity and a narrower secondary endosymbiont community （Hamiltonella/Rickettsia）, indicate that this genetic group is exotic and results from a recent invasion in this area.

Quenching autofluorescence in the alimentary canal tissues of Bactericera cockerelli(Hemiptera:Triozidae)for immunofluorescence labeling

Immunofluorescence has been widely used to localize microbes or specific molecules in insect tissues or cells.However,significant autofluorescence is frequently observed in tissues which can interfere with the fluorescent identification of target antigens,leading to inaccurate or even false positive fluorescent labeling.The alimentary canal of the potato psyllid,Bactericera cockerelliŠulc,exhibits intense autofluorescence,hindering the application of immunolocalization for the detection and localization of the economically important pathogen transmitted by this insect,“Candidatus Liberibacter solanacearum”(Lso).In the present study,we tested the use of irradiation,hydrogen peroxide(H2O2)and Sudan black B(SBB)treatments to reduce the autofluorescence in the B.cockerelli alimentary canal tissues.Furthermore,we assessed the compatibility of the above‐mentioned treatments with Lso immunolocalization and actin staining using phalloidin.Our results showed that the autofluorescence in the alimentary canal was reduced by irradiation,H2O2,or SBB treatments.The compatibility assays indicated that irradiation and H2O2 treatment both greatly reduced the fluorescent signal associated with Lso and actin.However,the SBB incubation preserved those target signals,while efficiently eliminating autofluorescence in the psyllid alimentary canal.Therefore,herein we propose a robust method for reducing the autofluorescence in the B.cockerelli alimentary canal with SBB treatment,which may improve the use of immunofluorescence labeling in this organism.This method may also have a wide range of uses by reducing the autofluorescence in other arthropod species.

The Japanese ladybirds, Coccinula crotchi and Coccinula sinensis, are infected with very closely related strains of male-killing Flavobacterium

Male-killing is 1 of 4 known strategies that inherited parasitic endosymbionts have evolved to manipulate their host＇s reproduction. In early male-killing, infected male offspring are killed early in embryogenesis. Within the Insecta, male-killing bacteria have been found in a wide range of hosts. The Coccinellidae families of beetles, better known as ladybirds, are particularly prone to male-killer invasion. In samples of the coccinellid, Coc- cinula crotchi, from Japan, a new male-killing bacterium was revealed by phenotypic assay. Molecular genetic analysis revealed the identity to be a tetracycline-sensitive Flavobac- terium that causes female-biased offspring sex ratio. Furthermore, that Flavobacterium strain was found to be closely related to the Flavobacterium causing male-killing in the congeneric Japanese coccinellid, Coccinula sinensis, which was collected from the same region. However, we found slightly different Flavobacterium strains infecting C. sinen- sis from regions with different environmental conditions. This may be an indication of horizontal transmission of male-killing Flavobacterium between these 2 ladybird spices. Finally, environmental conditions may affect the spread of male-killing bacteria among their hosts.

Evaluation of five antibiotics on larval gut bacterial diversity of Plutella xylostella （Lepidoptera： Plutellidae）

Larvae of the diamondback moth, Plutella xylostella L. （Lepidoptera： Plutelli- dae）, have rich microbial communities inhabiting the gut, and these bacteria contribute to the fitness of the pest. In this study we evaluated the effects of five antibiotics （rifampicin, ampicillin, tetracycline, streptomycin sulfate and chloramphenicol） on the gut bacterial di- versity ofP xylostella larvae. We screened five different concentrations for each antibiotic in a leaf disc assay, and found that rifampicin and streptomycin sulfate at 3 mg/mL signif- icantly reduced the diversity of the bacterial community, and some bacterial species could be rapidly eliminated. The number of gut bacteria in the rifampicin group and strepto- mycin sulfate group decreased more rapidly than the others. With the increase of antibiotic concentration, the removal efficiency was improved, whereas toxic effects became more apparent. All antibiotics reduced larval growth and development, and eventually caused high mortality, malformation of the prepupae, and hindered pupation and adult emergence. Among the five antibiotics, tetracycline was the most toxic and streptomycin sulfate was a relatively mild one. Some dominant bacteria were not affected by feeding antibiotics alone. Denaturing gradient gel electrophoresis graph showed that the most abundant and diverse bacteria in P xylostella larval gut appeared in the cabbage feeding group, and diet change and antibiotics intake influenced gut flora abundance. Species diversity was significantly reduced in the artificial diet and antibiotics treatment groups. After feeding on the artificial diet with rifampicin, streptomycin sulfate and their mixture for 10 days, larval gut bacteria could not be completely removed as detected with the agarose gel electrophoresis method.

Characteristics of the draft genome of ＂Candidatus Arsenophonus nilaparvatae＂, a facultative endosymbiont of Nilaparvata lugens

There exists a kind of symbiotic bacterium named ＂Candidatus Arsenophonus nilaparvatae＂ in the brown planthopper （BPH）, Nilaparvata lugens. After being filtered and assembled from the BPH genome sequencing project, the genome sequence of this bacterial symbiont was obtained. After initial analysis based on the genome, we have found its potential role to synthesize B vitamins for the host. In order to better understand the lifestyle and the genomic changes of this symbiotic bacterium after the symbiotic relationship was established, we further report the characteristics of this draft genome. Compared with several other related bacteria, ＂Candidatus Arsenophonus nilaparvatae＂ has proven to be a facultative endosymbiont at the genomic level. Concurrently, the presence of fimbriae and flagella formation related genes indicates this maternally transmitted endosymbiont is most likely to retain the capacity to invade new hosts. Through further analysis of annotated gene sets, we also find evidence of genome reduction in its secretion system and metabolic pathways. These findings reflect its evolutionary trend to be an obligate one and enable a deeper study of microbe-insect interactions.

Sequencing and comparison of the Rickettsia genomes from the whitefly Bemisia tabaci Middle East Asia Minor I

The whitefly, Bemisia tabaci, harbors the primary symbiont ＇Candidatus Portiera aleyrodidarum＇ and a variety of secondary symbionts. Among these secondary symbionts, Rickettsia is the only one that can be detected both inside and outside the bacteriomes. Infection with Rickettsia has been reported to influence several aspects of the whitefly biology, such as fitness, sex ratio, virus transmission and resistance to pesticides. However, mechanisms underlying these differences remain unclear, largely due to the lack of genomic information of Rickettsia. In this study, we sequenced the genome of two Rickettsia strains isolated from the Middle East Asia Minor 1 （MEAM1） species of the B. tabaci complex in China and Israel. Both Rickettsia genomes were of high coding den- sity and AT-rich, containing more than 1000 coding sequences, much larger than that of the coexisted primary symbiont, Portiera. Moreover, the two Rickettsia strains isolated from China and Israel shared most of the genes with 100% identity and only nine genes showed sequence differences. The phylogenetic analysis using orthologs shared in the genus, inferred the proximity of Rickettsia in MEAM1 and Rickettsia bellii. Functional analysis revealed that Rickettsia was unable to synthesize amino acids required for complementing the whitefly nutrition. Besides, a type IV secretion system and a number of virulence- related genes were detected in the Rickettsia genome. The presence of virulence-related genes might benefit the symbiotic life of the bacteria, and hint on potential effects of Rickettsia on whiteflies. The genome sequences of Rickettsia provided a basis for further understanding the function of Rickettsia in whiteflies.

Metamicrobiomics in herbivore beetles of the genus Cryptocephalus （Chrysomelidae）： toward the understanding of ecological determinants in insect symbiosis

The Cryptocephalus marginellus （Coleoptera： Chrysomelidae） complex is composed by six species that are supposed to have originated by events of allo- or parapatric speciation. In the present study we investigated the altemative hypotheses that the bacterial communities associated with six populations of this species complex are shaped by environmental factors, or reflect the proposed pattern of speciation. The microbiota associated with the six populations, from five species of the complex, have been characterized through 16S rRNA pyrotag sequencing. Based on a 97% sequence similarity threshold, data were clustered into 381 OTUs, which were analyzed using a variety of diversity indices. The microbiota of C. acquitanus and C. marginellus （Calanques） were the most diverse （over 100 OTUs）, while that from C. zoiai yielded less bacterial diversity （45 OTUs）. Taxonomic assignment revealed Proteobacteria, Tenericutes and Firmicutes as the dominant components of these beetles＇ microbiota. The most abtmdant genera were Ralstonia, Sphingomonas, Rickettsia, and Pseudomonas. Different strains of Rickettsia were detected in C. eridani and C. renatae. The analysis of β-diversity revealed high OTU turnover among the populations of C. marginellus complex, with only few shared species. Hierarchical clustering taking into account relative abundances of OTUs does not match the phylogeny of the beetles, therefore we hypothesize that factors other than phylogenetic constraints play a role in shaping the insects＇ microbiota. Environmental factors that could potentially affect the composition of bacterial communities were tested by fitting them on the results of a multi-dimensional scaling analysis. No significant correlations were observed towards the geographic distances or the host plants, while the composition of the microbiota appeared associated with altitude. The metabolic profiles of the microbiotas associated with each population were inferred from bacterial taxonomy, and interestingly, the obtained clustering pattern was consistent with the host phylogeny.

Antagonistic interaction between male-killing and cytoplasmic incompatibility induced by Cardinium and Wolbachia in the whitefly,Bemisia tabaci

Cardinium and Wolbachia are maternally inherited bacterial symbionts of arthropods that can manipulate host reproduction by increasing the fitness of infected females.Here,we report that Cardinium and Wolbachia coinfection induced male-killing and cytoplasmic incompatibility(CI)when they coexisted in a cryptic species of whitefly,Bemisia tabaci Asia II7.Cardinium and Wolbachia symbionts were either singly or simul-taneously localized in the bacteriocytes placed in the abdomen of B.tabaci nymphs and adults.Cardinium-Wolbachia coinfection induced male-killing and resulted in a higher female sex ratio in the intraspecific amphigenetic progeny of Asia II7 IcwH and IcwL lines;interestingly,male-illing induction was enhanced with increased Cardinium titer.Moreover,single infection of Wolbachia induced partial CI in the Asia II7 Iw line and resulted in reduced fecundity,higher embryonic mortality,and lower female sex ratio.The uninfected Asia II7 Iu line had significantly higher fecundity,lower embryonic and nymphal mortalities,and a lower level of CI than both the Wolbachia infected Asia II7 Iw line and the Cardinium--Wolbachia-coinfected Asia II7 IcwH line.Our findings indicate that Cardinium-Wolbachia coinfection induced male-killing,which may have had antag-onistic effects on Wolbachia-induced CI in the Asia II7 whiteflies.For the first time,our study revealed that B.tabaci Asia II7 reproduction is co-manipulated by Cardinium and Wolbachia endosymbionts.

Drosophila melanogaster brain invasion pathogenic Wolbachia in central nervous system of the fly

The pathogenic Wolbachia strain wMelPop rapidly over-replicates in the brain, muscles, and retina of Drosophila melanogaster, causing severe tissue degeneration and premature death of the host. The unique features of this endosymbiont make it an excellent tool to be used for biological control of insects, pests, and vectors of human diseases. To follow the dynamics of bacterial morphology and titer in the nerve cells we used transmission electron microscopy of 3-d-old female brains. The neurons and glial cells from central brain of the fly had different Wolbachia titers ranging from single bacteria to large accumulations, tearing cell apart and invading extracellular space. The neuropile regions of the brain were free of wMelPop. Wolbaehia tightly interacted with host cell organelles and underwent several morphological changes in nerve cells. Based on different morphological types of bacteria described we propose for the first time a scheme of wMelPop dynamics within the somatic tissue of the host.