Publications

4481

The citrus disease Huanglongbing (HLB) is highly destructive in many citrus-growing regions of the world. The putative causal agent of this disease, ‘Candidatus Liberibacter asiaticus’, is difficult to culture, and Koch's postulates have not yet been fulfilled. As a result, efforts have focused on obtaining the genome sequence of ‘Ca. L. asiaticus’ in order to give insight on the physiology of this organism. In this work, three next-generation high-throughput sequencing platforms, 454, Solexa, and SOLiD, were used to obtain metagenomic DNA sequences from phloem tissue of Florida citrus trees infected with HLB. A culture-independent, polymerase chain reaction (PCR)-independent analysis of 16S ribosomal RNA sequences showed that the only bacterium present within the phloem metagenome was ‘Ca L. asiaticus’. No viral or viroid sequences were identified within the metagenome. By reference assembly, the phloem metagenome contained sequences that provided 26-fold coverage of the ‘Ca. L. asiaticus’ contigs in GenBank. By the same approach, phloem metagenomic data yielded less than 0.2-fold coverage of five other alphaproteobacterial genomes. Thus, phloem metagenomic DNA provided a PCR-independent means of verifying the presence of ‘Ca L. asiaticus’ in infected tissue and strongly suggests that no other disease agent was present in phloem. Analysis of these metagenomic data suggest that this approach has a detection limit of one ‘Ca. Liberibacter’ cell for every 52 phloem cells. The phloem sample sequenced here is estimated to have contained 1.7 ‘Ca. Liberibacter’ cells per phloem cell.

ABSTRACTAs polyphagous, holometabolous insects, tephritid fruit flies (Diptera: Tephritidae) provide a unique habitat for endosymbiotic bacteria, especially those microbes associated with the digestive system. Here we examine the endosymbiont of the olive fly [Bactrocera oleae(Rossi) (Diptera: Tephritidae)], a tephritid of great economic importance. “CandidatusErwinia dacicola” was found in the digestive systems of all life stages of wild olive flies from the southwestern United States. PCR and microscopy demonstrated that “Ca.Erwinia dacicola” resided intracellularly in the gastric ceca of the larval midgut but extracellularly in the lumen of the foregut and ovipositor diverticulum of adult flies. “Ca.Erwinia dacicola” is one of the few nonpathogenic endosymbionts that transitions between intracellular and extracellular lifestyles during specific stages of the host's life cycle. Another unique feature of the olive fly endosymbiont is that unlike obligate endosymbionts of monophagous insects, “Ca.Erwinia dacicola” has a G+C nucleotide composition similar to those of closely related plant-pathogenic and free-living bacteria. These two characteristics of “Ca.Erwinia dacicola,” the ability to transition between intracellular and extracellular lifestyles and a G+C nucleotide composition similar to those of free-living relatives, may facilitate survival in a changing environment during the development of a polyphagous, holometabolous host. We propose that insect-bacterial symbioses should be classified based on the environment that the host provides to the endosymbiont (the endosymbiont environment).

Intracellular bacterial symbionts are known from various insect groups, particularly from those feeding on unbalanced diets, where the bacteria provide essential nutrients to the host. In the case of reed beetles (Coleoptera: Chrysomelidae, Donaciinae), however, the endosymbionts appear to be associated with specialized “glands” that secrete a material used for the beetles’ unusual water-tight cocoon. These glands were discovered over a century ago, but the bacteria they contain have yet to be characterized and placed in a phylogenetic context. Here, we describe the ultrastructure of two endosymbiotic species (“ Candidatus Macropleicola appendiculatae” and “ Candidatus Macropleicola muticae”) that reside in cells of the Malpighian tubules of the reed beetle species Macroplea appendiculata and Macroplea mutica , respectively. Fluorescent in situ hybridization using oligonucleotides targeting the 16S rRNA gene specific to Macroplea symbionts verified the localization of the symbionts in these organs. Phylogenetic analysis of 16S rRNA placed “Candidatus Macropleicola” in a clade of typically endosymbiotic Enterobacteriaceae (γ-proteobacteria). Finally, we discuss the evidence available for the hypothesis that the beetle larvae use a secretion produced by the bacteria for the formation of an underwater cocoon.