A method for CRISPR/Cas9 mutation of genes in fathead minnow (Pimephales promelas).

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing allows for the disruption or modification of genes in a multitude of model organisms. In the present study, we describe and employ the method for use in the fathead minnow (Pimephales promelas), in part, to assist in the development and validation of adverse outcome pathways (AOPs). The gene coding for an enzyme responsible for melanin production, tyrosinase (tyr), was the initial target chosen for development and assessment of the method since its disruption results in abnormal pigmentation, a phenotype obvious within 3-4 d after injection of fathead minnow embryos. Three tyrosinase-targeting guide strands were generated using the fathead minnow sequence in tandem with the CRISPOR guide strand selection tool. The strands targeted two areas: one stretch of sequence in a conserved region that demonstrated homology to EGF-like or laminin-like domains as determined by Protein Basic Local Alignment Search Tool in concert with the Conserved Domain Database, and a second area in the N-terminal region of the tyrosinase domain. To generate one cell embryos, in vitro fertilization was performed, allowing for microinjection of hundreds of developmentally-synchronized embryos with Cas9 proteins complexed to each of the three guide strands. Altered retinal pigmentation was observed in a portion of the tyr guide strand injected population within 3 d post fertilization (dpf). By 14 dpf, fish without skin and swim bladder pigmentation were observed. Among the three guide strands injected, the guide targeting the EGF/laminin-like domain was most effective in generating mutants. CRISPR greatly advances our ability to directly investigate gene function in fathead minnow, allowing for advanced approaches to AOP validation and development.

Use of the oligochaete, Lumbricuilus variegatus, as a prey organism for toxicant exposure of fish through the diet.

The oligochaete, Lumbriculus variegatus, has several characteristics that make it desirable as a prey organism for conducting dietary exposure studies with fish. We conducted 21- and 30-d experiments with young fathead minnows (Pimephales promelas) and rainbow trout (Oncorhynchus mykiss), respectively, to determine whether a diet consisting solely of L. variegatus would support normal growth and to compare performance with standard diets (Artemia nauplii, frozen brine shrimp, or trout chow). All diets were readily accepted, and fish survived and grew well. Food conversion in both fathead minnows and rainbow trout was as high as or higher for the oligochaete diet compared with others, although this comparison is influenced by differences in ration, ingestion rate, or both. The oligochaete diet had gross nutritional analysis similar to the other diets, and meets fish nutrition guidelines for protein and essential amino acids. Methodologies and practical considerations for successfully using oligochaetes as an experimental diet are discussed. Considering their ready acceptance by fish, their apparent nutritional sufficiency, the ease of culturing large numbers, and the ease with which they can be loaded with exogenous chemicals, we believe that L. variegatus represents an excellent choice of exposure vector for exposing fish to toxicants via the diet.

Time course of metal loss in Lumbriculus variegatus following sediment exposure.

After exposure for 21 d to sediment spiked with Cd, Pb, Cu, or Zn, oligochaetes (Lumbriculus variegatus) held in clean water depurated metal rapidly over the first few hours but much more slowly from 8 h up to 32 h. Results are consistent with previous work suggesting a 6-h depuration period as generally appropriate for sediment bioaccumulation studies with L. variegatus.