The role of spiracles in the respiration of Polypterus, a freshwater air-breathing fish, was investigated in order to clarify the long-standing debate of their use in aerial inspiration in this ancient lineage. Observations of four species (Polypterus delhezi, Polypterus senegalus, Polypterus ornatipinnis, and Polypterus lapradei) under conditions of varying temperature, oxygen and disturbance level provide insight into the frequency of air breaths and the breath cycle, along with conclusive evidence that Polypterus utilizes its spiracles for air-breathing. The level of disturbance was shown to have a direct impact on spiracle use; under "unstressed" or "natural" conditions spiracles were used preferentially for air inhalation. Under "stressed" conditions the mouth was the primary inhalation method. However, air-breathing frequency was found to be independent of environmental factors including the temperature and dissolved oxygen content of the water, as well as disturbance levels. The breath cycle associated with Polypterus air-breathing was determined to have the following six key steps : 1) the head being brought parallel to the surface of the water, 2) the operculum expanding, 3) the floor of the mouth dropping with the release of the previous air breath, 4) the opening of the spiracular valve, 5) the floor of the mouth dropping a second time, and 6) the closing of the spiracular valve. Dissections performed as part of this research discovered a new muscle responsible for controlling the spiracular valve. The use of spiracles in Polypterus air-breathing may provide insight into the ecology and physiology of early swamp-dwelling tetrapodomorpha of the Devonian period

CRISPR-Cas9-based gene drive systems possess the inherent capacity to spread progressively throughout target populations. Here we describe two self-copying (or active) guide RNA-only genetic elements, called e-CHACRs and ERACRs. These elements use Cas9 produced in trans by a gene drive either to inactivate the cas9 transgene (e-CHACRs) or to delete and replace the gene drive (ERACRs). e-CHACRs can be inserted at various genomic locations and carry two or more gRNAs, the first copying the e-CHACR and the second mutating and inactivating the cas9 transgene. Alternatively, ERACRs are inserted at the same genomic location as a gene drive, carrying two gRNAs that cut on either side of the gene drive to excise it. e-CHACRs efficiently inactivate Cas9 and can drive to completion in cage experiments. Similarly, ERACRs, particularly those carrying a recoded cDNA-restoring endogenous gene activity, can drive reliably to fully replace a gene drive. We compare the strengths of these two systems.