Abstract

Among microbes, viruses are known as being the most abundant members of microbial communities, yet only a modest amount of information on viriobenthos abundance and diversity is available. Spatial and temporal dynamics of total sediment viral and bacterial abundances were examined in the polyhaline, mesohaline and oligohaline zones of the Chesapeake Bay. Storage conditions for three sediment fractions [pore water (PW), whole sediment (WS), and sediment after pore water removal (AP)] were examined to determine freezing effects on viral and bacterial abundance. No significant difference in viral abundance was apparent, but bacterial abundances were significantly lower in frozen samples. The effect of formalin fixed/refrigerated storage of viral extracts was also examined. Within the first two hours, viral abundance decreased ∼30% in fixed extracts and 66% in unfixed extracts while stored at 4°C.

The efficacy of viral extraction with sodium pyrophosphate and vortexing was investigated and confirmed to be an adept means of removing ∼75% of viruses in one step. DNase experiments were also performed to determine the validity of epifluorescence enumerations of viruses, showing no significant differences in the numbers of viruses in DNase treated or untreated extracts. Viral abundances from surface sediments ranged from 2.2 × 10 ¹⁰ viruses ml^-1 (February 2004) to 1.7 × 10 ¹¹ viruses ml^-1 (April 2003) and direct bacterial counts ranged from 3.8 × 10⁸ cells ml^-1 (February 2004) to 3.7 × 10⁹ cells ml^-1 (April 2003) and were positively correlated to one another. Vertical depth profiles of viral and bacterial abundances showed an overall decrease in abundances with depth in the sediments.

Viral diversity of sediments was measured via both transmission electron microscopy (TEM) and random amplified polymorphic DNA-PCR (RAPD-PCR). TEM showed that viruses with small capsids (20-50 nm) from the Myoviridae and Podoviridae families dominated the sediment samples. Seasonally, the most diverse viral communities for autumn samples were from the lower bay, sandy, polyhaline station but the mesohaline station showed the greatest viral diversity in spring samples. Filamentous phages were rare but mostly observed in sandier sediments while elongated capsid phages were observed in more silty-clay sediments. Given that viral morphological diversity was determined via TEM analysis, the likelihood exists that extraction may alter observed viral morphologies. Six known phage representing three tailed phage families were added to sediments and subjected to equivalent extraction procedures. These investigations showed that Siphoviridae exhibited the greatest loss of tails, 32%-55%. Thus Myoviridae and Podoviridae frequencies often may be over-represented in TEM analyses.

RAPD-PCR was utilized to examine genotypic diversity of Chesapeake Bay sediment viruses. Using a single 10-mer primer for all stations, unique banding patterns were found. In contrast to previously reported water column data, benthic viral banding patterns claded according to station rather than season and showed the highest similarity at 75% for the mesohaline station between August 2003 and February 2004. Autumn, polyhaline station samples contained the greatest genetic diversity, whereas the highest genetic diversity was in the oligohaline zone. Temporal changes in viral abundance and morphology suggest that the viriobenthos community is more dynamic than previously considered.