Identification of Hepatotropic Viruses from Plasma Using Deep Sequencing: A Next Generation Diagnostic Tool

We conducted an unbiased metagenomics survey using plasma from patients with chronic hepatitis B, chronic hepatitis C, autoimmune hepatitis (AIH), non-alcoholic steatohepatitis (NASH), and patients without liver disease (control). RNA and DNA libraries were sequenced from plasma filtrates enriched in viral particles to catalog virus populations. Hepatitis viruses were readily detected at high coverage in patients with chronic viral hepatitis B and C, but only a limited number of sequences resembling other viruses were found. The exception was a library from a patient diagnosed with hepatitis C virus (HCV) infection that contained multiple sequences matching GB virus C (GBV-C). Abundant GBV-C reads were also found in plasma from patients with AIH, whereas Torque teno virus (TTV) was found at high frequency in samples from patients with AIH and NASH. After taxonomic classification of sequences by BLASTn, a substantial fraction in each library, ranging from 35% to 76%, remained unclassified. These unknown sequences were assembled into scaffolds along with virus, phage and endogenous retrovirus sequences and then analyzed by BLASTx against the non-redundant protein database. Nearly the full genome of a heretofore-unknown circovirus was assembled and many scaffolds that encoded proteins with similarity to plant, insect and mammalian viruses. The presence of this novel circovirus was confirmed by PCR. BLASTx also identified many polypeptides resembling nucleo-cytoplasmic large DNA viruses (NCLDV) proteins. We re-evaluated these alignments with a profile hidden Markov method, HHblits, and observed inconsistencies in the target proteins reported by the different algorithms. This suggests that sequence alignments are insufficient to identify NCLDV proteins, especially when these alignments are only to small portions of the target protein. Nevertheless, we have now established a reliable protocol for the identification of viruses in plasma that can also be adapted to other patient samples such as urine, bile, saliva and other body fluids.     

AN EMPIRICAL PROTOCOL FOR WHOLE-CELL IMMUNOFLUORESCENCE OF MARINE PHYTOPLANKTON1

An immunofluorescence protocol for intracellular antigens of phytoplankton was developed empirically. Paraformaldehyde (4%) was used to fix samples for 6 h at 4° C; fixed samples can be stored in cold (–20°C) methanol for at least 1 month. We used Triton X-100, Nonidet P-40, and dimethyl sulfoxide to permeabilize the cells for antibody penetration across the cell wall and plasma membrane. After immunolabeling at room temperature, the samples were mounted on slides and could be stored at 4° C for up to 6 months without significant decay of the fluorescence. The staining was examined with an epifluorescence microscope; semiquantitative (percentage of cells with positive staining) and quantitative (staining intensity measured with an imaging system) analyses were performed. Reproducible staining was achieved for nuclear, chloroplastic, cytoplasmic, and cell surface antigens in species of Chlorophyceae, Prymnesiophyceae, Bacillariophyceae, and Dinophyceae including some field-collected samples. This protocol is advantageous to previously published protocols in that it allows relatively simple and long-term storage of fixed samples and allows staining of multiple antigens for the same sample. Although improvement on cell permeabilization is required for some species, the present protocol could prove useful for physiological or ecological studies when frequent sampling is required and immediate processing of the samples is not possible.     

Targets of an invasive species: oviposition preference and larval performance of Cactoblastis cactorum (Lepidoptera: Pyralidae) on 14 North American opuntioid cacti

Cactoblastis cactorum Berg (Lepidoptera: Pyralidae), the cactus moth, is a well-known biological control agent of prickly pear cactus (Cactaceae: Opuntia Miller). The arrival of the moth in Florida and its subsequent spread through the southeastern United States poses a threat to opuntioid diversity in North America. Of particular concern are the ecological and economic impacts the moth could have in the southwestern United States and Mexico, where both native and cultivated Opuntia species are important resources. It is unknown which species would best support larval development if the moth were to spread further westward in North America. This study aimed to determine if ovipositing females demonstrate preferences for any of 14 common opuntioids native to or naturalized in Mexico and the southwestern United States; which of these opuntioids best support larval development; and if oviposition preference correlates with larval performance, as predicted by simple adaptive models. Results from a field experiment showed that female moths preferred O. engelmannii Salm-Dyck ex Engelmann variety linguiformis (Griffiths) Parfitt and Pinkava and O. engelmannii variety engelmannii for oviposition. A generalized linear model showed number of cladodes and degree of spininess to be significant predictors of oviposition activity. Results from a no-choice larval survival experiment showed Consolea rubescens (Salm-Dyck ex de Candolle.) Lemaire and O. streptacantha Lemaire to be the best hosts. Epidermal toughness was a significant predictor of most larval fitness parameters. In general, oviposition preference was not correlated with larval performance. A lack of co-evolutionary history between C. cactorum and North American opuntioid species may help explain this disconnect.     

Improvement of the sterile insect technique for codling moth Cydia pomonella (Linnaeus) (Lepidoptera Tortricidae) to facilitate expansion of field application

The codling moth Cydia pomonella (L.) (Lepidoptera Tortricidae) is a key pest of pome fruit (apple, pear and quince) and walnut orchards in most temperate regions of the world. Efforts to control the codling moth in the past mostly relied on the use of broad spectrum insecticide sprays, which has resulted in the development of insecticide resistance, and the disruption of the control of secondary pests. In addition, the frequent reliance and use of these insecticides are a constant threat to the environment and human health. Consequently, there have been increased demands from the growers for the development of codling moth control tactics that are not only effective but also friendly to the environment. In that respect, the sterile insect technique (SIT) and its derivative, inherited sterility (IS), are, together with mating disruption and granulosis virus, among the options that offer great potential as cost‐effective additions to available control tactics for integration in area‐wide integrated pest‐management approaches. In support of the further development of the SIT/IS for codling moth control, the Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture implemented a 5‐year Coordinated Research Project (CRP) entitled ‘Improvement of codling moth SIT to facilitate expansion of field application’. Research focussed on sterile codling moth quality and management (e.g. mobility and life‐history traits in relation to rearing strategy, dispersal, flight ability, radiosensitivity and mating compatibility) and a better understanding of the basic genetics of codling moth to assist the development of genetic sexing strains (e.g. cytogenetics, the development of dominant conditional lethal mutations, molecular characterization of the sex chromosomes, sex identification in embryos and cytogenetic markers). The results of the CRP are presented in this special issue.     

The Loss of Species: Mangrove Extinction Risk and Geographic Areas of Global Concern

Mangrove species are uniquely adapted to tropical and subtropical coasts, and although relatively low in number of species, mangrove forests provide at least US $1.6 billion each year in ecosystem services and support coastal livelihoods worldwide. Globally, mangrove areas are declining rapidly as they are cleared for coastal development and aquaculture and logged for timber and fuel production. Little is known about the effects of mangrove area loss on individual mangrove species and local or regional populations. To address this gap, species-specific information on global distribution, population status, life history traits, and major threats were compiled for each of the 70 known species of mangroves. Each species'' probability of extinction was assessed under the Categories and Criteria of the IUCN Red List of Threatened Species. Eleven of the 70 mangrove species (16%) are at elevated threat of extinction. Particular areas of geographical concern include the Atlantic and Pacific coasts of Central America, where as many as 40% of mangroves species present are threatened with extinction. Across the globe, mangrove species found primarily in the high intertidal and upstream estuarine zones, which often have specific freshwater requirements and patchy distributions, are the most threatened because they are often the first cleared for development of aquaculture and agriculture. The loss of mangrove species will have devastating economic and environmental consequences for coastal communities, especially in those areas with low mangrove diversity and high mangrove area or species loss. Several species at high risk of extinction may disappear well before the next decade if existing protective measures are not enforced.