Rapid CRISPR-Cas13a genetic identification enables new opportunities for listed Chinook salmon management.

Accurate taxonomic identification is foundational for effective species monitoring and management. When visual identifications are infeasible or inaccurate, genetic approaches provide a reliable alternative. However, these approaches are sometimes less viable (e.g., need for near real-time results, remote locations, funding concerns, molecular inexperience). In these situations, CRISPR-based genetic tools can fill an unoccupied niche between real-time, inexpensive, but error-prone visual identification and more expensive or time-consuming, but accurate genetic identification for taxonomic units that are difficult or impossible to visually identify. Herein, we use genomic data to develop CRISPR-based SHERLOCK assays capable of rapidly (<1 h), accurately (94%-98% concordance between phenotypic and genotypic assignments), and sensitively (detects 1-10 DNA copies/reaction) distinguishing ESA-listed Chinook salmon runs (winter- and spring-run) from each other and from unlisted runs (fall- and late fall-run) in California's Central Valley. The assays can be field deployable with minimally invasive mucus swabbing negating the need for DNA extraction (decreasing costs and labour), minimal and inexpensive equipment needs, and minimal training to conduct following assay development. This study provides a powerful genetic approach for a species of conservation concern that benefits from near real-time management decision-making but also serves as a precedent for transforming how conservation scientists and managers view genetic identification going forward. Once developed, CRISPR-based tools can provide accurate, sensitive, and rapid results, potentially without the prohibitive need for expensive specialty equipment or extensive molecular training. Further adoption of this technology will have widespread value for the monitoring and protection of our natural resources.

Does the bowfin gas bladder represent an intermediate stage during the lung-to-gas bladder evolutionary transition?

Whether phenotypic evolution occurs gradually through time has prompted the search for intermediate forms between the ancestral and derived states of morphological features, especially when there appears to be a discontinuous origin. The gas bladder, a derived character of the Actinopteri, is a modification of lungs, which characterize the common ancestor of bony vertebrates. While gas bladders and lungs are similar in many ways, the key morphological difference between these organs is the direction of budding from the foregut during development; essentially, the gas bladder buds dorsally and the lungs bud ventrally from the foregut. Did the shift from ventral lungs to dorsal gas bladder transition through a lateral-budding stage? To answer this question, the precise location of budding during gas bladder development in bowfin, representing the sister lineage to teleosts, has been debated. In the early 20th-century, it was suggested that the bowfin gas bladder buds laterally from the right wall of the foregut. We used nano-CT scanning to visualize the early development of the bowfin gas bladder to verify the historical studies of gas bladder developmental morphology and determine whether the direction of gas bladder budding in bowfin could be intermediate between ventrally budding lungs and dorsally budding gas bladders. We found that the bowfin gas bladder buds dorsally from the anterior foregut; however, during early development, the posterior gas bladder twists right. As development progresses, the posterior, right-hand twist becomes shallower, and the gas bladder itself shifts toward a mid-dorsal position. The budding site is definitively dorsal, despite the temporary lateral twist of the posterior gas bladder.

Changes in Nkx2.1, Sox2, Bmp4, and Bmp16 expression underlying the lung-to-gas bladder evolutionary transition in ray-finned fishes.

The key to understanding the evolutionary origin and modification of phenotypic traits is revealing the responsible underlying developmental genetic mechanisms. An important organismal trait of ray-finned fishes is the gas bladder, an air-filled organ that, in most fishes, functions for buoyancy control, and is homologous to the lungs of lobe-finned fishes. The critical morphological difference between lungs and gas bladders, which otherwise share many characteristics, is the general direction of budding during development. Lungs bud ventrally and the gas bladder buds dorsally from the anterior foregut. We investigated the genetic underpinnings of this ventral-to-dorsal shift in budding direction by studying the expression patterns of known lung genes (Nkx2.1, Sox2, and Bmp4) during the development of lungs or gas bladder in three fishes: bichir, bowfin, and zebrafish. Nkx2.1 and Sox2 show reciprocal dorsoventral expression patterns during tetrapod lung development and are important regulators of lung budding; their expression during bichir lung development is conserved. Surprisingly, we find during gas bladder development, Nkx2.1 and Sox2 expression are inconsistent with the hypothesis that they regulate the direction of gas bladder budding. Bmp4 is expressed ventrally during lung development in bichir, akin to the pattern during mouse lung development. During gas bladder development, Bmp4 is not expressed. However, Bmp16, a paralogue of Bmp4, is expressed dorsally in the developing gas bladder of bowfin. Bmp16 is present in the known genomes of Actinopteri (ray-finned fishes excluding bichir) but absent from mammalian genomes. We hypothesize that Bmp16 was recruited to regulate gas bladder development in the Actinopteri in place of Bmp4.