Detection of Viral Hemorrhagic Septicemia Virus (VHSV) from Diporeia spp. (Pontoporeiidae, Amphipoda) in the Laurentian Great Lakes, USA

The viral hemorrhagic septicemia virus (VHSV), genotype IVb, is a recent invader to the Laurentian Great Lakes basin and has been associated with mortalities in a number of resident freshwater fish species [1], [2], [3], [4]. While laboratory studies demonstrated that the virus can be transmitted to na？ve fish by both immersion and injection [5], [6], [7], the mode of VHSV transmission in the Great Lakes basin is largely unknown. In a previous study, it was concluded the pisocolid intermittent leech Myzobdella lugubris harbors VHSV [8]. Whether other macroinvertebrates can act as a vector or reservoir for VHSV remains to be elucidated.In the Great Lakes foodweb, amphipods of the genera Diporeia, Gammarus, and Hyalella occupy a central position as they transform energy from lower to higher trophic levels [9]. Unfortunately, Diporeia spp. have experienced a sharp decline sharp decline in abundance over the last two decades [10]; the cause(s) of which puzzle scientists. To tackle this enigma, a study was designed that involved comprehensive parasitological and microbiological analysis of Diporeia spp. collected from lakes Ontario, Huron, Michigan and Superior [Faisal and Winters: Pathogens impacting Diporeia spp. in the Great Lakes, submitted].Diporeia spp. were collected between August 2007 and April 2008 by taking Ponar grabs from seven locations in the Great Lakes basin at depths between 74-190 meters. The approximate locations of collections of Diporeia spp. are shown in Figure 1. Collected Diporeia spp. were pooled (five amphipods/pool), immersed briefly in absolute ethanol for surface disinfection, and then rinsed several times in sterile water. Samples (~100 μg) were homogenized with a sterile mortar and pestle and then diluted with 1 ml Earle's salt-based minimal essential medium (MEM, INVITROGEN). Homogenized Diporeia contents were removed with a sterile transfer pipette, dispensed into a sterile 1.5 ml centrifuge tube, and centrifuged at 5500 rcf for 20 min a