Much of the microbiome research done in oysters, including the Crassostrea virginica or the eastern oyster, has utilized hemolymph samples. Hemolymph, the invertebrate analog to blood in that it transports waste and nutrients to and from various tissues, is a useful medium for microbial community analysis as its collection is straightforward, it hosts a complex microbiota, and many bacterial members can reflect or even be predictive of diseased states. However, this requires hemolymph not contaminated with external fluids or other matter. The current gold-standard of hemolymph extraction, the notch method, involves cutting a notch in the oyster shell followed by insertion of a needle into the adductor muscle. Due to the needle’s movement through the soft tissue exposed to seawater, and potentially other tissues surrounding the adductor muscle, concerns about potential contamination exist. An alternative hemolymph sampling method, hereby referred to as the hole method, minimizes potential sources of contamination by drilling a hole through decontaminated shell surface above the adductor muscle allowing needle insertion directly into the internal tissue.
To evaluate the potential for sampling method-dependent hemolymph contamination, and to compare the two described methods, hemolymph was sampled from oysters using both the notch and hole methods. Half of the oysters had the notch sample taken first whereas the other half had the hole sample taken first. Plates inoculated with notch sample hemolymph showed bacterial colony growth whereas those inoculated with hemolymph from hole samples did not. Analysis of 16S amplicon data revealed significant differences between the microbial communities of each sample type. Notably, hemolymph samples collected from a hole before notch sampling showed the highest rates of alpha diversity. Overall, these results clearly demonstrate that hemolymph sampling method impacts microbiome composition. Moving forward, researchers should carefully consider hemolymph sampling methods when designing microbiome studies and critically evaluate the potential impact of this method on their results.