A novel application of motion analysis for detecting stress responses in embryos at different stages of development.

Motion analysis is one of the tools available to biologists to extract biologically relevant information from image datasets and has been applied to a diverse range of organisms. The application of motion analysis during early development presents a challenge, as embryos often exhibit complex, subtle and diverse movement patterns. A method of motion analysis able to holistically quantify complex embryonic movements could be a powerful tool for fields such as toxicology and developmental biology to investigate whole organism stress responses. Here we assessed whether motion analysis could be used to distinguish the effects of stressors on three early developmental stages of each of three species: (i) the zebrafish Danio rerio (stages 19 h, 21.5 h and 33 h exposed to 1.5% ethanol and a salinity of 5); (ii) the African clawed toad Xenopus laevis (stages 24, 32 and 34 exposed to a salinity of 20); and iii) the pond snail Radix balthica (stages E3, E4, E6, E9 and E11 exposed to salinities of 5, 10 and 15). Image sequences were analysed using Sparse Optic Flow and the resultant frame-to-frame motion parameters were analysed using Discrete Fourier Transform to quantify the distribution of energy at different frequencies. This spectral frequency dataset was then used to construct a Bray-Curtis similarity matrix and differences in movement patterns between embryos in this matrix were tested for using ANOSIM

Mechanisms of Na+ uptake, ammonia excretion, and their potential linkage in native Rio Negro tetras (Paracheirodon axelrodi, Hemigrammus rhodostomus, and Moenkhausia diktyota)

Mechanisms of Na+ uptake, ammonia excretion, and their potential linkage were investigated in three characids (cardinal, hemigrammus, moenkhausia tetras), using radiotracer flux techniques to study the unidirectional influx (Jin), efflux (Jout), and net flux rates (Jnet) of Na+ and Cl−, and the net excretion rate of ammonia (JAmm). The fish were collected directly from the Rio Negro, and studied in their native “blackwater” which is acidic (pH 4.5), ion-poor (Na+, Cl− ~20 µM), and rich in dissolved organic matter (DOM 11.5 mg C l−1). Jin Na, Jin Cl, and JAmm were higher than in previous reports on tetras obtained from the North America aquarium trade and/or studied in low DOM water. In all three species, Jin Na was unaffected by amiloride (10−4 M, NHE and Na+ channel blocker), but both Jin Na and Jin Cl were virtually eliminated (85–99 % blockade) by AgNO3 (10−7 M). A time course study on cardinal tetras demonstrated that Jin Na blockade by AgNO3 was very rapid (<5 min), suggesting inhibition of branchial carbonic anhydrase (CA), and exposure to the CA-blocker acetazolamide (10−4 M) caused a 50 % reduction in Jin Na .. Additionally, Jin Na was unaffected by phenamil (10−5 M, Na+ channel blocker), bumetanide (10−4 M, NKCC blocker), hydrochlorothiazide (5 × 10−3 M, NCC blocker), and exposure to an acute 3 unit increase in water pH. None of these treatments, including partial or complete elimination of Jin Na (by acetazolamide and AgNO3 respectively), had any inhibitory effect on JAmm. Therefore, Na+ uptake in Rio Negro tetras depends on an internal supply of H+ from CA, but does not fit any of the currently accepted H+-dependent models (NHE, Na+ channel/V-type H+-ATPase), or co-transport schemes (NCC, NKCC), and ammonia excretion does not fit the current “Na+/NH4 + exchange metabolon” paradigm. Na+, K+-ATPase and V-type H+-ATPase activities were present at similar levels in gill homogenates, Acute exposure to high environmental ammonia (NH4Cl, 10−3 M) significantly increased Jin Na, and NH4 + was equally or more effective than K+ in activating branchial Na+,(K+) ATPase activity in vitro. We propose that ammonia excretion does not depend on Na+ uptake, but that Na+ uptake (by an as yet unknown H+-dependent apical mechanism) depends on ammonia excretion, driven by active NH4 + entry via basolateral Na+,(K+)-ATPase. © 2014, Springer-Verlag Berlin Heidelberg