ABSTRACT
The DSIR-HA-1179 coleopteran cell line has been identified as a susceptible and permissive host for the in vitro replication of the Oryctes nudivirus, which can be used as a biopesticide against the coconut rhinoceros beetle, pest of palms. The major challenge to in vitro large-scale Oryctes nudivirus production is ensuring process economy. This rests, among other requisites, on the use of low-cost culture media tailored to the nutritional and metabolic needs of the cell line, both in uninfected and infected cultures. The aim of the present study was to characterize the nutritional demands and the metabolic characteristics of the DSIR-HA-1179 cell line during growth and subsequent infection with Oryctes nudivirus in the TC-100 culture medium. Serum-supplementation of the culture medium was found to be critical for cell growth, and addition of 10% fetal bovine serum v/v led to a maximum viable cell density (16.8 × 105 cells ml-1) with a population doubling time of 4.2 d. Nutritional and metabolic characterization of the cell line revealed a trend of glucose and glutamine consumption but minimal uptake of other amino acids, negligible production of lactate and ammonia, and the accumulation of alanine, both before and after infection. The monitoring of virus production kinetics showed that the TC-100 culture medium was nutritionally sufficient to give a peak yield of 7.38 × 107 TCID50 ml-1 of OrNV at the 6th day post-infection in attached cultures of DSIR-HA-1179 cells in 25 cm2 T-flasks. Knowledge of the cell line's nutritional demands and virus production kinetics will aid in the formulation of a low-cost culture medium and better process design for large-scale OrNV production in future.
Subject(s)
Coleoptera/cytology , Coleoptera/virology , DNA Viruses/pathogenicity , Insect Viruses/pathogenicity , Amino Acids/metabolism , Animals , Cell Culture Techniques , Cell Line , Cell Proliferation , Coleoptera/metabolism , Culture Media/pharmacology , DNA Viruses/growth & development , Insect Viruses/growth & development , Kinetics , SerumABSTRACT
BACKGROUND: Here we present a holistic screening of collapsing colonies from three professional apiaries in Spain. Colonies with typical honey bee depopulation symptoms were selected for multiple possible factors to reveal the causes of collapse. RESULTS: Omnipresent were Nosema ceranae and Lake Sinai Virus. Moderate prevalences were found for Black Queen Cell Virus and trypanosomatids, whereas Deformed Wing Virus, Aphid Lethal Paralysis Virus strain Brookings and neogregarines were rarely detected. Other viruses, Nosema apis, Acarapis woodi and Varroa destructor were not detected. Palinologic study of pollen demonstrated that all colonies were foraging on wild vegetation. Consequently, the pesticide residue analysis was negative for neonicotinoids. The genetic analysis of trypanosomatids GAPDH gene, showed that there is a large genetic distance between Crithidia mellificae ATCC30254, an authenticated cell strain since 1974, and the rest of the presumed C. mellificae sequences obtained in our study or published. This means that the latter group corresponds to a highly differentiated taxon that should be renamed accordingly. CONCLUSION: The results of this study demonstrate that the drivers of colony collapse may differ between geographic regions with different environmental conditions, or with different beekeeping and agricultural practices. The role of other pathogens in colony collapse has to bee studied in future, especially trypanosomatids and neogregarines. Beside their pathological effect on honey bees, classification and taxonomy of these protozoan parasites should also be clarified.
Subject(s)
Beekeeping/methods , Bees , Colony Collapse , Insect Viruses/pathogenicity , Nosema/pathogenicity , Trypanosomatina/pathogenicity , Animals , Bees/microbiology , Bees/parasitology , Bees/virology , Colony Collapse/microbiology , Colony Collapse/parasitology , Colony Collapse/virology , Ecosystem , Feeding Behavior , Host-Parasite Interactions , Host-Pathogen Interactions , Insect Viruses/genetics , Insect Viruses/isolation & purification , Nosema/genetics , Nosema/isolation & purification , Phylogeny , Pollen , Population Dynamics , Ribotyping , Spain , Trypanosomatina/genetics , Trypanosomatina/isolation & purificationABSTRACT
A new virus was isolated from the pea aphid, Acyrthosiphon pisum, and tentatively named Acyrthosiphon pisum virus (APV). The isometric virus particles were approximately 31 nm in diameter and contained a single-stranded RNA molecule of approximately 10 kb. Four structural proteins were observed with molecular masses of approximately 23.3, 24.2, 34.5, and 66.2 kDa. The 34.5-kDa capsid protein was the most abundant product in purified virions. Computer-assisted analysis revealed no significant homology between an internal sequence of 37 amino acids of the 34.5-kDa protein of APV and other polypeptides of viral origin. APV was not immunologically related to other ssRNA viruses from hemipteroid insects, such as aphid lethal paralysis virus, Rhopalosiphum padi virus, and Nezara viridula virus type 1. Immunolocalization on ultrathin sections of 3-day-old nymphs of A. pisum showed that APV antigen was predominantly present in the epithelial cells of the digestive tract. Virus particles were also observed associated with the microvilli of the intestine. Occasionally, muscle cells and mycetocyte cells were found infected. Purified APV, fed to 1-day-old A. pisum nymphs, significantly reduced the growth of the aphid and increased the time needed to reach maturity.