A Model for Design and Implementation of a Laboratory Information-Management System Specific for Molecular Pathology Laboratory Operations.

The Molecular Pathology Section, Cleveland Clinic (Cleveland, OH), has undergone enhancement of its testing portfolio and processes. An Excel 2013- and paper-based data-management system was replaced with a commercially available laboratory information-management system (LIMS) software application, a separate bioinformatics platform, customized test-interpretation applications, a dedicated sample-accessioning service, and a results-releasing software application. The customized LIMS solution manages complex workflows, large-scale data packets, and process automation. A customized approach was required because, in a survey of commercially available off-the-shelf software products, none met the diverse and complex needs of this molecular diagnostics service. The project utilized the expertise of clinical laboratorians, pathologists, genetics counselors, bioinformaticians, and systems analysts in partnering with software-engineering consultants to design and implement a solution. Concurrently, Agile software-building best practices were formulated, which may be emulated for scalable and cost-effective laboratory-authored software.

Bacterial associates of Hyalesthes obsoletus (Hemiptera: Cixiidae), the insect vector of bois noir disease, with a focus on cultivable bacteria.

The planthopper Hyalesthes obsoletus (Hemiptera: Cixiidae) is an important vector of phytoplasma diseases in grapevine. In the current study, the bacterial community compositions of symbionts of this insect were examined. Two dominant bacterial lineages were identified by mass sequencing: the obligate symbiont Candidatus Sulcia, and a facultative symbiont that is closely related to Pectobacterium sp. and to BEV, a cultivable symbiont of another phytoplasma vector, the leafhopper Euscelidius variegatus. In addition, one bacterium was successfully isolated in this study - a member of the family Xanthomonadaceae that is most closely related to the genus Dyella. This Dyella-like bacterium (DLB) was detected by FISH analysis in H. obsoletus guts but not ovaries, and its prevalence in H. obsoletus increased during the fall, suggesting that it was acquired by the host through feeding. We found that DLB inhibits Spiroplasma melliferum, a cultivable relative of phytoplasma, suggesting that it is a potential candidate for biological control against phytoplasma in grapevines.

Morphological and molecular characterization of a sexually reproducing colony of the booklouse Liposcelis bostrychophila (Psocodea: Liposcelididae) found in Arizona.

The booklouse, Liposcelis bostrychophila, is a worldwide pest of stored products. For decades, only thelytokous parthenogenetic reproduction was documented in L. bostrychophila. Male L. bostrychophila were first found in Hawaii in 2002. In 2009, a sexual strain was found in Arizona. We examined the morphology of both males and females of the Arizona strain and compared the Arizona sexual strain with the Hawaii sexual strain and the parthenogenetic strains of L. bostrychophila. The sexual and parthenogenetic strains show some differences in eye morphology. To examine the relationship between sexual and asexual lineages, we sequenced the mitochondrial 12S and 16S ribosomal RNA genes of males and females from the Arizona strain. Phylogenetic analyses of L. bostrychophila individuals revealed that: 1) the sexually reproducing colony found in Arizona contains two closely related mitochondrial DNA haplotypes--one present in only females and the other in both males and females; and 2) the Arizona sexual strain was most closely related to a parthenogenetic strain in Illinois. We detected Rickettsia in all of the parthenogenetic individuals we checked but not in any Arizona sexual individuals. Further evidence is required to establish whether the presence of Rickettsia is linked to asexual reproduction in Liposcelis.

Microbial associates of the vine mealybug Planococcus ficus (Hemiptera: Pseudococcidae) under different rearing conditions.

Sap-feeding insects harbor diverse microbial endosymbionts that play important roles in host ecology and evolution, including contributing to host pest status. The vine mealybug, Planococcus ficus, is a serious pest of grapevines, vectoring a number of pathogenic grape viruses. Previous studies have shown that virus transmission is abolished when mealybugs are raised in the laboratory on potato. To examine the possible role of microbial symbionts in virus transmission, the archaeal, bacterial, and fungal microbiota of field and laboratory P. ficus were characterized using molecular and classical microbiological methods. Lab and field colonies of P. ficus harbored different microbiota. While both were dominated by the bacterial obligate nutritional symbionts Moranella and Tremblaya, field samples also harbored a third bacterium that was allied with cluster L, a lineage of bacterial symbionts previously identified in aphids. Archaea were not found in any of the samples. Fungal communities in field-collected mealybugs were dominated by Metschnikowia and Cladosporium species, while those from laboratory-reared mealybugs were dominated by Alternaria and Cladosporium species. In conclusion, this study has identified a diverse set of microbes, most of which appear to be facultatively associated with P. ficus, depending on environmental conditions. The role of various members of the mealybug microbiome, as well as how the host plant affects microbial community structure, remains to be determined.

Novel Rickettsiella bacterium in the leafhopper Orosius albicinctus (Hemiptera: Cicadellidae).

Bacteria in the genus Rickettsiella (Coxiellaceae), which are mainly known as arthropod pathogens, are emerging as excellent models to study transitions between mutualism and pathogenicity. The current report characterizes a novel Rickettsiella found in the leafhopper Orosius albicinctus (Hemiptera: Cicadellidae), a major vector of phytoplasma diseases in Europe and Asia. Denaturing gradient gel electrophoresis (DGGE) and pyrosequencing were used to survey the main symbionts of O. albicinctus, revealing the obligate symbionts Sulcia and Nasuia, and the facultative symbionts Arsenophonus and Wolbachia, in addition to Rickettsiella. The leafhopper Rickettsiella is allied with bacteria found in ticks. Screening O. albicinctus from the field showed that Rickettsiella is highly prevalent, with over 60% of individuals infected. A stable Rickettsiella infection was maintained in a leafhopper laboratory colony for at least 10 generations, and fluorescence microscopy localized bacteria to accessory glands of the female reproductive tract, suggesting that the bacterium is vertically transmitted. Future studies will be needed to examine how Rickettsiella affects host fitess and its ability to vector phytopathogens.