Co-infection and localization of secondary symbionts in two whitefly species

<p>Abstract</p> <p>Background</p> <p>Whiteflies are cosmopolitan phloem-feeding pests that cause serious damage to many crops worldwide due to direct feeding and vectoring of many plant viruses. The sweetpotato whitefly <it>Bemisia tabaci </it>(Gennadius) and the greenhouse whitefly <it>Trialeurodes vaporariorum </it>(Westwood) are two of the most widespread and damaging whitefly species. To complete their unbalanced diet, whiteflies harbor the obligatory bacterium <it>Portiera aleyrodidarum. B. tabaci </it>further harbors a diverse array of secondary symbionts, including <it>Hamiltonella, Arsenophonus, Cardinium, Wolbachia, Rickettsia </it>and <it>Fritschea</it>. <it>T. vaporariorum </it>is only known to harbor <it>P. aleyrodidarum </it>and <it>Arsenophonus</it>. We conducted a study to survey the distribution of whitefly species in Croatia, their infection status by secondary symbionts, and the spatial distribution of these symbionts in the developmental stages of the two whitefly species.</p> <p>Results</p> <p><it>T. vaporariorum </it>was found to be the predominant whitefly species across Croatia, while only the Q biotype of <it>B. tabaci </it>was found across the coastal part of the country. <it>Arsenophonus </it>and <it>Hamiltonella </it>were detected in collected <it>T. vaporariorum </it>populations, however, not all populations harbored both symbionts, and both symbionts showed 100% infection rate in some of the populations. Only the Q biotype of <it>B. tabaci </it>was found in the populations tested and they harbored <it>Hamiltonella</it>, <it>Rickettsia, Wolbachia </it>and <it>Cardinium</it>, while <it>Arsenophonus </it>and <it>Fritschea </it>were not detected in any <it>B. tabaci </it>populations. None of the detected symbionts appeared in all populations tested, and multiple infections were detected in some of the populations. All endosymbionts tested were localized inside the bacteriocyte in both species, but only <it>Rickettsia </it>and <it>Cardinium </it>in <it>B. tabaci </it>showed additional localization outside the bacteriocyte.</p> <p>Conclusions</p> <p>Our study revealed unique co-infection patterns by secondary symbionts in <it>B. tabaci </it>and <it>T. vaporariorum</it>. Co-sharing of the bacteriocyte by the primary and different secondary symbionts is maintained through vertical transmission via the egg, and is unique to whiteflies. This system provides opportunities to study interactions among symbionts that co-inhabit the same cell in the same host: these can be cooperative or antagonistic, may affect the symbiotic contents over time, and may also affect the host by competing with the primary symbiont for space and resources.</p

Transmission of a Protease-Secreting Bacterial Symbiont Among Pea Aphids via Host Plants

Aphids are economically important pest insects that damage plants by phloem feeding and the transmission of plant viruses. Their ability to feed exclusively on nutritionally poor phloem sap is dependent on the obligatory symbiotic bacterium Buchnera aphidicola, but additional facultative symbionts may also be present, a common example of which is Serratia symbiotica. Many Serratia species secrete extracellular enzymes, so we hypothesised that S. symbiotica may produce proteases that help aphids to feed on plants. Molecular analysis, including fluorescence in situ hybridization (FISH), revealed that S. symbiotica colonises the gut, salivary glands and mouthparts (including the stylet) of the pea aphid Acyrthosiphon pisum, providing a mechanism to transfer the symbiont into host plants. S. symbiotica was also detected in plant tissues wounded by the penetrating stylet and was transferred to naïve aphids feeding on plants containing this symbiont. The maintenance of S. symbiotica by repeated transmission via plants may explain the high frequency of this symbiont in aphid populations. Proteomic analysis of the supernatant from a related but cultivable S. symbiotica strain cultured in liquid medium revealed the presence of known and novel proteases including metalloproteases. The corresponding transcripts encoding these S. symbiotica enzymes were detected in A. pisum and in plants carrying the symbiont, although the mRNA was much more abundant in the aphids. Our data suggest that enzymes from S. symbiotica may facilitate the digestion of plant proteins, thereby helping to suppress plant defense, and that the symbionts are important mediators of aphid–plant interactions

Diversity and Phylogenetic Analyses of Bacterial Symbionts in Three Whitefly Species from Southeast Europe

Bemisia tabaci (Gennadius), Trialeurodes vaporariorum (Westwood), and Siphoninus phillyreae (Haliday) are whitefly species that harm agricultural crops in many regions of the world. These insects live in close association with bacterial symbionts that affect host fitness and adaptation to the environment. In the current study, we surveyed the infection of whitefly populations in Southeast Europe by various bacterial symbionts and performed phylogenetic analyses on the different symbionts detected. Arsenophonus and Hamiltonella were the most prevalent symbionts in all three whitefly species. Rickettsia was found to infect mainly B. tabaci, while Wolbachia mainly infected both B. tabaci and S. phillyreae. Furthermore, Cardinium was rarely found in the investigated whitefly populations, while Fritschea was never found in any of the whitefly species tested. Phylogenetic analyses revealed a diversity of several symbionts (e.g., Hamiltonella, Arsenophonus, Rickettsia), which appeared in several clades. Reproductively isolated B. tabaci and T. vaporariorum shared the same (or highly similar) Hamiltonella and Arsenophonus, while these symbionts were distinctive in S. phillyreae. Interestingly, Arsenophonus from S. phillyreae did not cluster with any of the reported sequences, which could indicate the presence of Arsenophonus, not previously associated with whiteflies. In this study, symbionts (Wolbachia, Rickettsia, and Cardinium) known to infect a wide range of insects each clustered in the same clades independently of the whitefly species. These results indicate horizontal transmission of bacterial symbionts between reproductively isolated whitefly species, a mechanism that can establish new infections that did not previously exist in whiteflies

Diversity and Phylogenetic Analyses of Bacterial Symbionts in Three Whitefly Species from Southeast Europe

Bemisia tabaci (Gennadius), Trialeurodes vaporariorum (Westwood), and Siphoninus phillyreae (Haliday) are whitefly species that harm agricultural crops in many regions of the world. These insects live in close association with bacterial symbionts that affect host fitness and adaptation to the environment. In the current study, we surveyed the infection of whitefly populations in Southeast Europe by various bacterial symbionts and performed phylogenetic analyses on the different symbionts detected. Arsenophonus and Hamiltonella were the most prevalent symbionts in all three whitefly species. Rickettsia was found to infect mainly B. tabaci, while Wolbachia mainly infected both B. tabaci and S. phillyreae. Furthermore, Cardinium was rarely found in the investigated whitefly populations, while Fritschea was never found in any of the whitefly species tested. Phylogenetic analyses revealed a diversity of several symbionts (e.g., Hamiltonella, Arsenophonus, Rickettsia), which appeared in several clades. Reproductively isolated B. tabaci and T. vaporariorum shared the same (or highly similar) Hamiltonella and Arsenophonus, while these symbionts were distinctive in S. phillyreae. Interestingly, Arsenophonus from S. phillyreae did not cluster with any of the reported sequences, which could indicate the presence of Arsenophonus, not previously associated with whiteflies. In this study, symbionts (Wolbachia, Rickettsia, and Cardinium) known to infect a wide range of insects each clustered in the same clades independently of the whitefly species. These results indicate horizontal transmission of bacterial symbionts between reproductively isolated whitefly species, a mechanism that can establish new infections that did not previously exist in whiteflies

Proteomic Analysis of the Venom from the Ruby Ant <i>Myrmica rubra</i> and the Isolation of a Novel Insecticidal Decapeptide

Ants are a biodiverse group of insects that have evolved toxic venom containing many undiscovered bioactive molecules. In this study, we found that the venom of the ruby ant Myrmica rubra is a rich source of peptides. LC-MS analysis revealed the presence of 142 different peptides varying in molecular weight, sequence length, and hydrophobicity. One of the most abundant peaks was selected for further biochemical and functional characterization. Combined Edman degradation and de novo peptide sequencing revealed the presence of a novel decapeptide (myrmicitoxin) with the amino acid sequence NH2-IDPKLLESLA-CONH2. The decapeptide was named U-MYRTX-MRArub1 and verified against a synthetic standard. The amidated peptide was tested in a synthetic form to determine the antimicrobial activity towards the bacterial pathogens and insecticidal potential against pea aphids (Acyrthosiphon pisum). This peptide did not show antimicrobial activity but it significantly reduced the survival of aphids. It also increased the sensitivity of the aphids to two commonly used chemical insecticides (imidacloprid and methomyl). Since ant venom research is still in its infancy, the findings of this first study on venom peptides derived from M. rubra highlight these insects as an important and rich source for discovery of novel lead structures with potential application in pest control

The Gram-Positive Bacterium Leuconostoc pseudomesenteroides Shows Insecticidal Activity against Drosophilid and Aphid Pests

Insect pests reduce global crop yields by up to 20%, but the most effective control measures are currently based on environmentally hazardous chemical pesticides. An alternative, ecologically beneficial pest-management strategy involves the use of microbial pathogens (or active compounds and extracts derived from them) that naturally target selected insect pests. A novel strain of the bacterium Leuconostoc pseudomesenteroides showed promising activity in our preliminary tests. Here, we investigated its effects in more detail, focusing on drosophilid and aphid pests by testing the survival of two species representing the family Drosophilidae (Drosophila suzukii and D. melanogaster) and one representing the family Aphididae (Acyrthosiphon pisum). We used oral and septic infection models to administer living bacteria or cell-free extracts to adult flies and aphid nymphs. We found that infection with living bacteria significantly reduced the survival of our insect models, whereas the administration of cell-free extracts had a significant effect only in aphids. These results confirm that L. pseudomesenteroides has potential as a new biocontrol agent for sustainable pest management

The Transmission Efficiency of Tomato Yellow Leaf Curl Virus by the Whitefly Bemisia tabaci Is Correlated with the Presence of a Specific Symbiotic Bacterium Species▿

Tomato yellow leaf curl virus (TYLCV) (Geminiviridae: Begomovirus) is exclusively vectored by the whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). TYLCV transmission depends upon a 63-kDa GroEL protein produced by the vector's endosymbiotic bacteria. B. tabaci is a species complex comprising several genetically distinct biotypes that show different secondary-symbiont fauna. In Israel, the B biotype harbors Hamiltonella, and the Q biotype harbors Wolbachia and Arsenophonus. Both biotypes harbor Rickettsia and Portiera (the obligatory primary symbionts). The aim of this study was to determine which B. tabaci symbionts are involved in TYLCV transmission using B. tabaci populations collected in Israel. Virus transmission assays by B. tabaci showed that the B biotype efficiently transmits the virus, while the Q biotype scarcely transmits it. Yeast two-hybrid and protein pulldown assays showed that while the GroEL protein produced by Hamiltonella interacts with TYLCV coat protein, GroEL produced by Rickettsia and Portiera does not. To assess the role of Wolbachia and Arsenophonus GroEL proteins (GroELs), we used an immune capture PCR (IC-PCR) assay, employing in vivo- and in vitro-synthesized GroEL proteins from all symbionts and whitefly artificial feeding through membranes. Interaction between GroEL and TYLCV was found to occur in the B biotype, but not in the Q biotype. This assay further showed that release of virions protected by GroEL occurs adjacent to the primary salivary glands. Taken together, the GroEL protein produced by Hamiltonella (present in the B biotype, but absent in the Q biotype) facilitates TYLCV transmission. The other symbionts from both biotypes do not seem to be involved in transmission of this virus

Promoter activation in Δhfq mutants as an efficient tool for specialized metabolite production enabling direct bioactivity testing

Natural products (NPs) from microorganisms have been important sources for discovering new therapeutic and chemical entities. While their corresponding biosynthetic gene clusters (BGCs) can be easily identified by gene-sequence-similarity-based bioinformatics strategies, the actual access to these NPs for structure elucidation and bioactivity testing remains difficult. Deletion of the gene encoding the RNA chaperone, Hfq, results in strains losing the production of most NPs. By exchanging the native promoter of a desired BGC against an inducible promoter in Δhfq mutants, almost exclusive production of the corresponding NP from the targeted BGC in Photorhabdus, Xenorhabdus and Pseudomonas was observed including the production of several new NPs derived from previously uncharacterized non-ribosomal peptide synthetases (NRPS). This easyPACId approach (easy Promoter Activated Compound Identification) facilitates NP identification due to low interference from other NPs. Moreover, it allows direct bioactivity testing of supernatants containing secreted NPs, without laborious purification