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.

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.

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.

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.

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.