A novel mitochondrial genome architecture in thrips (Insecta: Thysanoptera): extreme size asymmetry among chromosomes and possible recent control region duplication.

BACKGROUND: Multipartite mitochondrial genomes are very rare in animals but have been found previously in two insect orders with highly rearranged genomes, the Phthiraptera (parasitic lice), and the Psocoptera (booklice/barklice). RESULTS: We provide the first report of a multipartite mitochondrial genome architecture in a third order with highly rearranged genomes: Thysanoptera (thrips). We sequenced the complete mitochondrial genomes of two divergent members of the Scirtothrips dorsalis cryptic species complex. The East Asia 1 species has the single circular chromosome common to animals while the South Asia 1 species has a genome consisting of two circular chromosomes. The fragmented South Asia 1 genome exhibits extreme chromosome size asymmetry with the majority of genes on the large, 14.28 kb, chromosome and only nad6 and trnC on the 0.92 kb mini-circle chromosome. This genome also features paralogous control regions with high similarity suggesting a very recent origin of the nad6 mini-circle chromosome in the South Asia 1 cryptic species. CONCLUSIONS: Thysanoptera, along with the other minor paraenopteran insect orders should be considered models for rapid mitochondrial genome evolution, including fragmentation. Continued use of these models will facilitate a greater understanding of recombination and other mitochondrial genome evolutionary processes across eukaryotes.

Direct Evidence of Egestion and Salivation of Xylella fastidiosa Suggests Sharpshooters Can Be "Flying Syringes".

Xylella fastidiosa is unique among insect-transmitted plant pathogens because it is propagative but noncirculative, adhering to and multiplying on the cuticular lining of the anterior foregut. Any inoculation mechanism for X. fastidiosa must explain how bacterial cells exit the vector's stylets via the food canal and directly enter the plant. A combined egestion-salivation mechanism has been proposed to explain these unique features. Egestion is the putative outward flow of fluid from the foregut via hypothesized bidirectional pumping of the cibarium. The present study traced green fluorescent protein-expressing X. fastidiosa or fluorescent nanoparticles acquired from artificial diets by glassy-winged sharpshooters, Homalodisca vitripennis, as they were egested into simultaneously secreted saliva. X. fastidiosa or nanoparticles were shown to mix with gelling saliva to form fluorescent deposits and salivary sheaths on artificial diets, providing the first direct, conclusive evidence of egestion by any hemipteran insect. Therefore, the present results strongly support an egestion-salivation mechanism of X. fastidiosa inoculation. Results also support that a column of fluid is transiently held in the foregut without being swallowed. Evidence also supports (but does not definitively prove) that bacteria were suspended in the column of fluid during the vector's transit from diet to diet, and were egested with the held fluid. Thus, we hypothesize that sharpshooters could be true "flying syringes," especially when inoculation occurs very soon after uptake of bacteria, suggesting the new paradigm of a nonpersistent X. fastidiosa transmission mechanism.

ESTIMATING BACTERIAL DIVERSITY IN SCIRTOTHRIPS DORSALIS (THYSANOPTERA: THRIPIDAE) VIA NEXT GENERATION SEQUENCING.

The last 2 decades have produced a better understanding of insect-microbial associations and yielded some important opportunities for insect control. However, most of our knowledge comes from model systems. Thrips (Thysanoptera: Thripidae) have been understudied despite their global importance as invasive species, plant pests and disease vectors. Using a culture and primer independent next-generation sequencing and metagenomics pipeline, we surveyed the bacteria of the globally important pest, Scirtothrips dorsalis Hood. The most abundant bacterial phyla identified were Actinobacteria and Proteobacteria and the most abundant genera were Propionibacterium, Stenotrophomonas, and Pseudomonas. A total of 189 genera of bacteria were identified. The absence of any vertically transferred symbiont taxa commonly found in insects is consistent with other studies suggesting that thrips primarilly acquire resident microbes from their environment. This does not preclude a possible beneficial/intimate association between S. dorsalis and the dominant taxa identified and future work should determine the nature of these associations.

Improved real-time PCR detection of 'Candidatus Liberibacter asiaticus' from citrus and psyllid hosts by targeting the intragenic tandem-repeats of its prophage genes.

'Candidatus Liberibacter asiaticus' (CLas) is a Gram-negative α-proteobacterium, and the prominent species of Liberibacter associated with a devastating worldwide citrus disease known as huanglongbing (HLB). This fastidious bacterium resides in phloem sieve cells of host plants and is vectored by the Asian citrus psyllid (Diaphorina citri). Due to its uneven distribution in planta and highly variable bacterial titers, detection of HLB bacteria can be challenging. Here we demonstrated a new utility of nearly identical tandem-repeats of two CLas prophage genes for real-time PCR by SYBR Green 1 (LJ900fr) and TaqMan(®) (LJ900fpr). When compared with conventional 16S rDNA-based real-time PCR, targeting the repeat sequence reduced the relative detectable threshold by approximately 9 and 3 real-time PCR cycles for LJ900fr and LJ900fpr, respectively. Additionally, both LJ900 methods detected CLas from otherwise non-detectable samples by other methods. CLas was also detected from globally derived samples including psyllids, various citrus varieties, periwinkle, dodder, and orange jasmine, suggesting the new detection method can be applicable worldwide. Additionally, we demonstrated the presence of the hyv(I)/hyv(II) repeat sequence within the 'Ca. Liberibacter americanus' strain. The method thereby provides sensitive HLB detection with broad application for scientific, regulatory, and citrus grower communities.

The Scirtothrips dorsalis Species Complex: Endemism and Invasion in a Global Pest.

Invasive arthropods pose unique management challenges in various environments, the first of which is correct identification. This apparently mundane task is particularly difficult if multiple species are morphologically indistinguishable but accurate identification can be determined with DNA barcoding provided an adequate reference set is available. Scirtothrips dorsalis is a highly polyphagous plant pest with a rapidly expanding global distribution and this species, as currently recognized, may be comprised of cryptic species. Here we report the development of a comprehensive DNA barcode library for S. dorsalis and seven nuclear markers via next-generation sequencing for identification use within the complex. We also report the delimitation of nine cryptic species and two morphologically distinguishable species comprising the S. dorsalis species complex using histogram analysis of DNA barcodes, Bayesian phylogenetics, and the multi-species coalescent. One member of the complex, here designated the South Asia 1 cryptic species, is highly invasive, polyphagous, and likely the species implicated in tospovirus transmission. Two other species, South Asia 2, and East Asia 1 are also highly polyphagous and appear to be at an earlier stage of global invasion. The remaining members of the complex are regionally endemic, varying in their pest status and degree of polyphagy. In addition to patterns of invasion and endemism, our results provide a framework both for identifying members of the complex based on their DNA barcode, and for future species delimiting efforts.

Formation of Stylet Sheaths in aere (in air) from eight species of phytophagous hemipterans from six families (Suborders: Auchenorrhyncha and Sternorrhyncha).

Stylet sheath formation is a common feature among phytophagous hemipterans. These sheaths are considered essential to promote a successful feeding event. Stylet sheath compositions are largely unknown and their mode of solidification remains to be elucidated. This report demonstrates the formation and solidification of in aere (in air) produced stylet sheaths by six hemipteran families: Diaphorina citri (Psyllidae, Asian citrus psyllid), Aphis nerii (Aphididae, oleander/milkweed aphid), Toxoptera citricida (Aphididae, brown citrus aphid), Aphis gossypii (Aphididae, cotton melon aphid), Bemisia tabaci biotype B (Aleyrodidae, whitefly), Homalodisca vitripennis (Cicadellidae, glassy-winged sharpshooter), Ferrisia virgata (Pseudococcidae, striped mealybug), and Protopulvinaria pyriformis (Coccidae, pyriform scale). Examination of in aere produced stylet sheaths by confocal and scanning electron microscopy shows a common morphology of an initial flange laid down on the surface of the membrane followed by continuous hollow core structures with sequentially stacked hardened bulbous droplets. Single and multi-branched sheaths were common, whereas mealybug and scale insects typically produced multi-branched sheaths. Micrographs of the in aere formed flanges indicate flange sealing upon stylet bundle extraction in D. citri and the aphids, while the B. tabaci whitefly and H. vitripennis glassy-winged sharpshooter flanges remain unsealed. Structural similarity of in aere sheaths are apparent in stylet sheaths formed in planta, in artificial diets, or in water. The use of 'Solvy', a dissolvable membrane, for intact stylet sheath isolation is reported. These observations illustrate for the first time this mode of stylet sheath synthesis adding to the understanding of stylet sheath formation in phytophagous hemipterans and providing tools for future use in structural and compositional analysis.

Two plant bacteria, S. meliloti and Ca. Liberibacter asiaticus, share functional znuABC homologues that encode for a high affinity zinc uptake system.

The Znu system, encoded for by znuABC, can be found in multiple genera of bacteria and has been shown to be responsible for the import of zinc under low zinc conditions. Although this high-affinity uptake system is known to be important for both growth and/or pathogenesis in bacteria, it has not been functionally characterized in a plant-associated bacterium. A single homologue of this system has been identified in the plant endosymbiont, Sinorhizobium meliloti, while two homologous systems were found in the destructive citrus pathogen, Candidatus Liberibacter asiaticus. To understand the role of these protein homologues, a complementation assay was devised allowing the individual genes that comprise the system to be assayed independently for their ability to reinstate a partially-inactivated Znu system. Results from the assays have demonstrated that although all of the genes from S. meliloti were able to restore activity, only one of the two Ca. Liberibacter asiaticus encoded gene clusters contained genes that were able to functionally complement the system. Additional analysis of the gene clusters reveals that distinct modes of regulation may also exist between the Ca. Liberibacter asiaticus and S. meliloti import systems despite the intracellular-plant niche common to both of these bacteria.

Survey of endosymbionts in the Diaphorina citri metagenome and assembly of a Wolbachia wDi draft genome.

Diaphorina citri (Hemiptera: Psyllidae), the Asian citrus psyllid, is the insect vector of Ca. Liberibacter asiaticus, the causal agent of citrus greening disease. Sequencing of the D. citri metagenome has been initiated to gain better understanding of the biology of this organism and the potential roles of its bacterial endosymbionts. To corroborate candidate endosymbionts previously identified by rDNA amplification, raw reads from the D. citri metagenome sequence were mapped to reference genome sequences. Results of the read mapping provided the most support for Wolbachia and an enteric bacterium most similar to Salmonella. Wolbachia-derived reads were extracted using the complete genome sequences for four Wolbachia strains. Reads were assembled into a draft genome sequence, and the annotation assessed for the presence of features potentially involved in host interaction. Genome alignment with the complete sequences reveals membership of Wolbachia wDi in supergroup B, further supported by phylogenetic analysis of FtsZ. FtsZ and Wsp phylogenies additionally indicate that the Wolbachia strain in the Florida D. citri isolate falls into a sub-clade of supergroup B, distinct from Wolbachia present in Chinese D. citri isolates, supporting the hypothesis that the D. citri introduced into Florida did not originate from China.

Polar localization of the Coxiella burnetii type IVB secretion system.

Coxiella burnetii is a Gram-negative pleomorphic bacterium and the causative agent of Q fever. During infection, the pathogen survives and replicates within a phagosome-like parasitophorous vacuole while influencing cellular functions throughout the host cell, indicating a capacity for effector protein secretion. Analysis of the C. burnetii (RSA 493 strain) genome sequence indicates that C. burnetii contains genes with homology to the Legionella pneumophila Dot/Icm type IVB secretion system (T4BSS). T4BSSs have only been described in L. pneumophila and C. burnetii, marking it a unique virulence determinate. Characterization of bacterial virulence determinants ranging from autotransporter proteins to diverse secretion systems suggests that polar localization may be a virulence mechanism hallmark. To characterize T4BSS subcellular localization in C. burnetii, we analyzed C. burnetii-infected Vero cells by indirect immunofluorescent antibody (IFA) and immunoelectron microscopy (IEM). Using antibodies against the C. burnetii T4BSS homologs IcmT, IcmV, and DotH, IFA show that these proteins are localized to the poles of the bacterium. IEM supports this finding, showing that antibodies against C. burnetii IcmT and DotH preferentially localize to the bacterial cell pole(s). Together, these data demonstrate that the C. burnetii T4BSS localizes to the pole(s) of the bacterium during infection of host cells.