Publications

4474

Abstract Bat flies of the family Nycteribiidae are known for their extreme morphological and physiological traits specialized for ectoparasitic blood-feeding lifestyle on bats, including lack of wings, reduced head and eyes, adenotrophic viviparity with a highly developed uterus and milk glands, as well as association with endosymbiotic bacteria. We investigated Japanese nycteribiid bat flies representing 4 genera, 8 species and 27 populations for their bacterial endosymbionts. From all the nycteribiid species examined, a distinct clade of gammaproteobacteria was consistently detected, which was allied to endosymbionts of other insects such as Riesia spp. of primate lice and Arsenophonus spp. of diverse insects. In adult insects, the endosymbiont was localized in specific bacteriocytes in the abdomen, suggesting an intimate host–symbiont association. In adult females, the endosymbiont was also found in the cavity of milk gland tubules, which suggests uterine vertical transmission of the endosymbiont to larvae through milk gland secretion. In adult females of Penicillidia jenynsii, we discovered a previously unknown type of symbiotic organ in the Nycteribiidae: a pair of large bacteriomes located inside the swellings on the fifth abdominal ventral plate. The endosymbiont genes consistently exhibited adenine/thymine biased nucleotide compositions and accelerated rates of molecular evolution. The endosymbiont genome was estimated to be highly reduced, ∼0.76 Mb in size. The endosymbiont phylogeny perfectly mirrored the host insect phylogeny, indicating strict vertical transmission and host–symbiont co-speciation in the evolutionary course of the Nycteribiidae. The designation ‘Candidatus Aschnera chinzeii’ is proposed for the endosymbiont clade.

Carrot (Daucus carota) plants with symptoms resembling those associated with the carrot psyllid Trioza apicalis and the bacterium “Candidatus Liberibacter solanacearum” (1–4) were observed in 70 to 80% of commercial fields and experimental plots in southeastern Norway from late July to mid-September of 2011; all cultivars grown were affected with approximately 10 to 100% symptomatic plants per field. T. apicalis, a pest of carrot in northern and central Europe, including Norway, can cause as much as 100% crop loss and is associated with “Ca. L. solanacearum” (1–4). Symptoms on affected plants include leaf curling, yellow and purple discoloration of leaves, stunted growth of shoots and roots, and proliferation of secondary roots. Carrot plant samples were collected from five T. apicalis-infested fields in Ostfold, Vestfold, Oppland, and Hedmark counties. Total DNA was extracted from petiole and root tissues of 54 plants, including 27 symptomatic plants and 27 asymptomatic plants from four cultivars (Namdal, Panther, Romance, and Yukon) with the cetyltrimethylammonium bromide (CTAB) buffer extraction method (2,3). DNA samples were tested by PCR assay using primer pairs OA2/OI2c and CL514F/R to amplify a portion of 16S rDNA and rplJ/rplL ribosomal protein genes, respectively, of “Ca. L. solanacearum” (2,3). A 1,168-bp 16S rDNA fragment was detected in the DNA from 22 (81.5%) symptomatic plants and a 668-bp rplJ/rplL fragment was amplified from the DNA of 26 (96.3%) symptomatic and 5 (18.5%) asymptomatic plants, indicating the presence of liberibacter. No liberibacter was detected in the asymptomatic carrot plants with the primer pair OA2/OI2c. Amplicons from the DNA of four carrot root samples with each primer pair were cloned (pCR2.1-TOPO; Invitrogen, Carlsbad, CA) and three clones of each of the eight amplicons were sequenced (MCLAB, San Francisco, CA). BLAST analysis of the 16S rDNA consensus sequence from the carrot root tissues (GenBank Accession No. JN863097) showed 100% identity to those of “Ca. L. solanacearum” previously amplified from carrot (GU373048 and GU373049) and T. apicalis (GU477254 and GU477255) in Finland (2,3). The rplJ/rplL consensus sequence from the carrots (GenBank Accession No. JN863098) was 99% identical to the sequences of rplJ/rplL “Ca. L. solanacearum” ribosomal protein gene from carrots in Finland (GU373050 and GU373051). To our knowledge, this is the first report of “Ca. L. solanacearum” associated with carrot in Norway. This bacterial species has caused millions of dollars in losses to potato and several other solanaceous crops in North and Central America and New Zealand (1). This plant pathogen has also been reported from carrots and T. apicalis in Finland, where it has caused significant economic damage to carrot crops (2–4). References: (1) J. E. Munyaneza. Southwest. Entomol. 35:471, 2010. (2) J. E. Munyaneza et al. Plant Dis. 94:639, 2010. (3) J. E. Munyaneza et al. J. Econ. Entomol. 103:1060, 2010. (4) A. Nissinen et al. Entomol. Exp. Appl. 125:277, 2007.

In September 2011, potato (Solanum tuberosum L.) tubers graded in a packing facility in south-central Idaho were observed with internal discolorations suggestive of zebra chip disease (ZC). Symptoms were observed in 1 to 2% of tubers of cv. Russet Norkotah and included brown spots and streaks especially in and near the vascular tissue. Some tubers also showed a dark and sunken stolon attachment typical of ZC (1). Initially, tissue samples were taken from seven symptomatic tubers and tested by PCR for “Candidatus Liberibacter solanacearum”, the bacterium associated with ZC. Primers specific for the 16S rDNA (primers CLipoF [4] and OI2c [3]) and the outer membrane protein (OMB 1482f and 2086r) (2) were used. Six of these samples were positive for the bacterium. The amplified 16S rDNA and OMB products from two symptomatic tubers of cv. Russet Norkotah were cloned and three clones of each were sequenced. The 16S sequences (1,071 bp; GenBank Accession Nos. JN848755 and JN848756) from the two tubers varied by one nucleotide and had 99 to 100% sequence identity to numerous “Ca. L. solanacearum” sequences in GenBank (e.g., Accession Nos. HM246509, FJ957897, and EU935004). Sequences of the two OMB clones (605 bp; GenBank Accession Nos. JN848757 and JN848758) had 97% sequence identity to the two “Ca. L. solanacearum” OMB sequences in GenBank (Accession Nos. CP002371 and FJ914617). Six of eight additional symptomatic field-collected cv. Russet Norkotah tubers corresponding to tubers collected in the packing facility were also positive for “Ca. L. solanacearum” by PCR. Additional severely symptomatic tubers of cvs. Russet Burbank, Yukon Gold, and raw cut French fries of Ranger Russet produced in south-central Idaho were subsequently tested by PCR and were found to be positive for “Ca. L. solanacearum” as well. On the basis of the symptoms, specific PCR amplification with two distinct primer pairs and DNA sequence analysis, zebra chip disease caused by “Ca. L. solanacearum” was determined to be present in Idaho. This disease has caused significant economic damage to potatoes in many regions, including Texas, Mexico, Central America, and New Zealand (1). Idaho is the largest potato-producing state in the United States, with over 150,000 ha planted this year, and therefore, ZC potentially poses a significant risk to agriculture in this state. References: (1) J. M. Crosslin et al. Online publication. doi:10.1094/PHP-2010-0317-01-RV, Plant Health Progress, 2010. (2) J. M. Crosslin et al. Southwest. Entomol. 36:125, 2011. (3) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (4) G. A. Secor. Plant Dis. 93:574, 2009.

It has long been known that armored scale insects harbor endosymbiotic bacteria inside specialized cells called bacteriocytes. Originally, these endosymbionts were thought to be fungal symbionts but they are now known to be bacterial and have been named Uzinura diaspidicola. Bacteriocyte and endosymbiont distribution patterns within host insects were visualized using in situ hybridization via 16S rRNA specific probes. Images of scale insect embryos, eggs and adult scale insects show patterns of localized bacteriocytes in embryos and randomly distributed bacteriocytes in adults. The symbiont pocket was not found in the armored scale insect eggs that were tested. The pattern of dispersed bacteriocytes in adult scale insects suggest that Uzinura and Blattabacteria may share some homologous traits that coincide with similar life style requirements, such as dispersal in fat bodies and uric acid recycling.

ABSTRACT We present the genome sequence of “ Candidatus Mycoplasma haemominutum” strain Birmingham 1, a low-pathogenicity feline hemoplasma strain.

Abstract Adelgids (Insecta: Hemiptera: Adelgidae) are known as severe pests of various conifers in North America, Canada, Europe and Asia. Here, we present the first molecular identification of bacteriocyte-associated symbionts in these plant sap-sucking insects. Three geographically distant populations of members of the Adelges nordmannianae/piceae complex, identified based on coI and ef1alpha gene sequences, were investigated. Electron and light microscopy revealed two morphologically different endosymbionts, coccoid or polymorphic, which are located in distinct bacteriocytes. Phylogenetic analyses of their 16S and 23S rRNA gene sequences assigned both symbionts to novel lineages within the Gammaproteobacteria sharing <92% 16S rRNA sequence similarity with each other and showing no close relationship with known symbionts of insects. Their identity and intracellular location were confirmed by fluorescence in situ hybridization, and the names ‘Candidatus Steffania adelgidicola’ and ‘Candidatus Ecksteinia adelgidicola’ are proposed for tentative classification. Both symbionts were present in all individuals of all investigated populations and in different adelgid life stages including eggs, suggesting vertical transmission from mother to offspring. An 85 kb genome fragment of ‘Candidatus S. adelgidicola’ was reconstructed based on a metagenomic library created from purified symbionts. Genomic features including the frequency of pseudogenes, the average length of intergenic regions and the presence of several genes which are absent in other long-term obligate symbionts, suggested that ‘Candidatus S. adelgidicola’ is an evolutionarily young bacteriocyte-associated symbiont, which has been acquired after diversification of adelgids from their aphid sister group.