Unionid mussels are ecologically important and are globally imperiled. Toxicants contribute to mussel declines, and toxicity tests using juvenile mussels-a sensitive life stage-are valuable in determining thresholds used to set water quality criteria. In vitro culture methods provide an efficient way to propagate juveniles for toxicity testing, but their relative chemical sensitivity compared with in vivo propagated juveniles is unknown. Current testing guidelines caution against using in vitro cultured juveniles until this sensitivity is described. Our objective was to evaluate the relative sensitivity of juvenile mussels produced from both in vitro and in vivo propagation methods to selected chemicals. We conducted 96-h acute toxicity tests according to ASTM International guidelines with 3 mussel species and 6 toxicants: chloride, nickel, ammonia, and 3 copper-based compounds. Statistically significant differences between in vitro and in vivo juvenile 96-h median effect concentrations were observed in 8 of 17 tests, and in vitro juveniles were more sensitive in 6 of the 8 significant differences. At 96 h, 4 of the 8 statistically different tests for a given chemical were within a factor of 2, which is the intralaboratory variation demonstrated in a recent evaluation of mussel toxicity tests. We found that although differences in chemical sensitivity exist between in vitro and in vivo propagated juvenile mussels, they are within normal toxicity test variation. Therefore, in vitro propagated juvenile mussels may be appropriate for use in ASTM International-based toxicity testing. Environ Toxicol Chem 2018;37:3077-3085. © 2018 SETAC.
The Trispot Darter (Etheostoma trisella) is an imperiled small-bodied freshwater fish that inhabits headwaters of the upper Coosa River watershed in Alabama, Georgia, and Tennessee. We sequenced the complete mitochondrial genome of this species to develop non-invasive environmental DNA (eDNA) surveillance protocols. A mitochondrial phylogenomic analysis reveals the Trispot Darter to have diverged from soon after the common ancestor of genus Etheostoma. The mitochondrial genome sequence of E. trisella is similar to other darter species in terms of GC content and gene order, but the sequence is sufficiently divergent to permit the design of species-specific eDNA primers.
Environmental DNA (eDNA) has increasingly been used in the surveillance of imperiled aquatic species. Despite recent efforts in drawing genetic material from the environment, there are still pitfalls surrounding this field. We created a novel protocol which implements loop-mediated isothermal amplification (LAMP) to detect target DNA. Our methods are applied here in the surveillance of Etheostoma trisella, the Trispot Darter, a freshwater fish that recently received protection under the U.S. Endangered Species Act. Water samples (n = 256) were collected at sites in Alabama and Georgia to determine whether E. trisella still occupies historic sites and whether it inhabits previously unknown areas. We found evidence of E. trisella presence in 69 water samples while 187 were negative. Our LAMP protocol is capable of amplifying low quantities of DNA in the water, and is a robust technique for freshwater species surveillance. Verification of positive results from eDNA experiments is essential to confirm reaction reliability. Application of methods such as ours are necessary for recognizing species under threat that require conservation.Surveillance of a federally protected freshwater fish using loop-mediated isothermal amplification (LAMP) and eDNA
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