Transmission of Nephridial Bacteria of the Earthworm Eisenia fetida

The lumbricid earthworms (annelid family Lumbricidae) harbor gram-negative bacteria in their excretory organs, the nephridia. Comparative 16S rRNA gene sequencing of bacteria associated with the nephridia of several earthworm species has shown that each species of worm harbors a distinct bacterial species and that the bacteria from different species form a monophyletic cluster within the genus Acidovorax, suggesting that there is a specific association resulting from radiation from a common bacterial ancestor. Previous microscopy and culture studies revealed the presence of bacteria within the egg capsules and on the surface of embryos but did not demonstrate that the bacteria within the egg capsule were the same bacteria that colonized the nephridia. We present evidence, based on curing experiments, in situ hybridizations with Acidovorax-specific probes, and 16S rRNA gene sequence analysis, that the egg capsules contain high numbers of the bacterial symbiont and that juveniles are colonized during development within the egg capsule. Studies exposing aposymbiotic hatchlings to colonized adults and their bedding material suggested that juvenile earthworms do not readily acquire bacteria from the soil after hatching but must be colonized during development by bacteria deposited in the egg capsule. Whether this is due to the developmental stage of the host or the physiological state of the symbiont remains to be investigated.     

Yy1 Gene Dosage Effect and Bi-Allelic Expression of Peg3

In the current study, we tested the in vivo effects of Yy1 gene dosage on the Peg3 imprinted domain with various breeding schemes utilizing two sets of mutant alleles. The results indicated that a half dosage of Yy1 coincides with the up-regulation of Peg3 and Zim1, suggesting a repressor role of Yy1 in this imprinted domain. This repressor role of Yy1 is consistent with the observations derived from previous in vitro studies. The current study also provided an unexpected observation that the maternal allele of Peg3 is also normally expressed, and thus the expression of Peg3 is bi-allelic in the specific areas of the brain, including the choroid plexus, the PVN (Paraventricular Nucleus) and the SON (Supraoptic Nucleus) of the hypothalamus. The exact roles of the maternal allele of Peg3 in these cell types are currently unknown, but this new finding confirms the previous prediction that the maternal allele may be functional in specific cell types based on the lethality associated with the homozygotes for several mutant alleles of the Peg3 locus. Overall, these results confirm the repressor role of Yy1 in the Peg3 domain and also provide a new insight regarding the bi-allelic expression of Peg3 in mouse brain.     

PCR Amplification-Independent Methods for Detection of Microbial Communities by the High-Density Microarray PhyloChip

Environmental microbial community analysis typically involves amplification by PCR, despite well-documented biases. We have developed two methods of PCR-independent microbial community analysis using the high-density microarray PhyloChip: direct hybridization of 16S rRNA (dirRNA) or rRNA converted to double-stranded cDNA (dscDNA). We compared dirRNA and dscDNA communities to PCR-amplified DNA communities using a mock community of eight taxa, as well as experiments derived from three environmental sample types: chromium-contaminated aquifer groundwater, tropical forest soil, and secondary sewage in seawater. Community profiles by both direct hybridization methods showed differences that were expected based on accompanying data but that were missing in PCR-amplified communities. Taxon richness decreased in RNA compared to that in DNA communities, suggesting a subset of 20% in soil and 60% in groundwater that is active; secondary sewage showed no difference between active and inactive populations. Direct hybridization of dscDNA and RNA is thus a viable alternative to PCR-amplified microbial community analysis, providing identification of the active populations within microbial communities that attenuate pollutants, drive global biogeochemical cycles, or proliferate disease states.     

Effect of Simulated Acid Rain on Bursaphelenchus xylophilus Infection of Pine Seedlings

White, Scots, and Austrian 3-year-old pine seedlings were treated with conditions simulating acid rain and inoculated with the white pine specific pathotype of Bursaphelenchus xylophilus, VPSt-1. Oleoresin concentration increased slightly and carbohydrate concentration decreased in all seedlings treated with simulated acid rain (SAR). The changes were significantly increased after inoculation of SAR-treated white and Scots pine seedlings with VPSt-1. Wilting was delayed and nematode reproduction decreased in SAR-treated white pine seedlings inoculated with VPSt-1. SAR-treated Austrian pine seedlings were resistant to VPSt-1, but SAR-treated Scots pine seedlings lost tolerance to VPSt-1 and wilted 50-60 days after inoculation.     

Transformation Frequency of a mariner-Based Transposon in Rickettsia rickettsii

Transformation frequencies of a mariner-based transposon system in Rickettsia rickettsii were determined using a plaque assay system for enumeration and isolation of mutants. Sequence analysis of insertion sites in both R. rickettsii and R. prowazekii indicated that insertions were random. Transposon mutagenesis provides a useful tool for rickettsial research.     

Effect of Simulated Rainfall on Efficacy and Leaching of Two Formulations of Fenamiphos

Recoverable fenamiphos in the soil and residue in squash following different simulated rainfall treatments after nematicide application were determined in a 2-year study. Efficacy of fenamiphos also was evaluated. Fenamiphos treatments (3 SC and 15 G) were broadcast (6.7 kg a.i./ha) over plots and incorporated into the top 15 cm of soil immediately before planting ''Dixie Hybrid'' squash. Simulated rainfall treatments of 0, 2.5, and 5.0 cm water were applied 1 day after fenamiphos application. Soil samples from 0- to 8-cm, 8- to 15-cm, and 15- to 30-cm soil depths were collected 1 day after the simulated rainfall applications and analyzed for fenamiphos, fenamiphos sulfoxide (FSO), and fenamiphos sulfone (FSO₂). Squash was analyzed for total fenamiphos residue. Greater concentrations of fenamiphos were present in the 0- to 8-cm soil layer following application of 15 G than 3 SC formulation. Simulated rainfall treatments did not alter fenamiphos concentrations in any soil layer (except for the 0- to 8-cm depth in 1992) or concentration of FSO and total fenamiphos residue in the 15- to 30-cm soil layer. Root-gall indices were greater from untreated than most fenamiphos-treated plots, but were not affected by formulations of fenamiphos or simulated rainfall treatments. Concentrations of total residue in squash ranged from 1 to 4 μg FSO₂/g.     

Interactions Between the Nematode Parasite of Pigs,Ascaris suum,and the Earthworm Aporrectodea longa

Pig faeces in which Ascaris suum eggs had been embryonating for 57 days were placed in buckets of soil containing either 30 or no earthworms (Aporrectodea longa). When present, earthworms consumed the faeces and transported the eggs down into the soil, without inflicting any visible damage on the eggs. In later experiments 10 earthworms from the above experiment were fed to each of ten pigs, and another 40 earthworms were dissected. None of the 10 pigs became infected with A. suum through consumption of earthworms, and none of the dissected earthworms were found to contain A. suum larvae. This experiment indicates that A. longa did not act as a paratenic host for A. suum but shows that earthworms are very efficient in transporting A. suum eggs from faeces deposited on the soil surface into the soil.     

Pentalysine Clusters Mediate Silica Targeting of Silaffins in Thalassiosira pseudonana

The biological formation of inorganic materials (biomineralization) often occurs in specialized intracellular vesicles. Prominent examples are diatoms, a group of single-celled eukaryotic microalgae that produce their SiO₂ (silica)-based cell walls within intracellular silica deposition vesicles (SDVs). SDVs contain protein-based organic matrices that control silica formation, resulting in species specifically nanopatterned biosilica, an organic-inorganic composite material. So far no information is available regarding the molecular mechanisms of SDV biogenesis. Here we have investigated by fluorescence microscopy and subcellular membrane fractionation the intracellular transport of silaffin Sil3. Silaffins are a group of phosphoproteins constituting the main components of the organic matrix of diatom biosilica. We demonstrate that the N-terminal signal peptide of Sil3 mediates import into a specific subregion of the endoplasmic reticulum. Additional segments from the mature part of Sil3 are required to reach post-endoplasmic reticulum compartments. Further transport of Sil3 and incorporation into the biosilica (silica targeting) require protein segments that contain a high density of modified lysine residues and phosphoserines. Silica targeting of Sil3 is not dependent on a particular peptide sequence, yet a lysine-rich 12–14-amino acid peptide motif (pentalysine cluster), which is conserved in all silaffins, strongly promotes silica targeting. The results of the present work provide the first insight into the molecular mechanisms for biogenesis of mineral-forming vesicles from an eukaryotic organism.