Practical Euthanasia Method for Common Sea Stars (Asterias rubens) That Allows for High-Quality RNA Sampling

Sea stars in research are often lethally sampled without available methodology to render them insensible prior to sampling due to concerns over sufficient sample quality for applied molecular techniques. The objectives of this study were to describe an inexpensive and effective two-step euthanasia method for adult common sea stars (Asterias rubens) and to demonstrate that high-quality RNA samples for further use in downstream molecular analyses can be obtained from pyloric ceca of MgCl2-immersed sea stars. Adult common sea stars (n = 15) were immersed in a 75 g/L magnesium chloride solution until they were no longer reactive to having their oral surface tapped with forceps (mean: 4 min, range 2–7 min), left immersed for an additional minute, and then sampled with sharp scissors. RNA from pyloric ceca (n = 10) was isolated using a liquid–liquid method, then samples were treated with DNase and analyzed for evaluation of RNA integrity number (RIN) for assessment of the quantity and purity of intact RNA. Aversive reactions to magnesium chloride solution were not observed and no sea stars regained spontaneous movement or reacted to sampling. The calculated RIN ranged from 7.3–9.8, demonstrating that the combination of animal welfare via the use of anesthesia and sampling for advanced molecular techniques is possible using this low-cost technique.

Effects of Short-Term Fasting on mRNA Expression of Ghrelin and the Peptide Transporters PepT1 and 2 in Atlantic Salmon (Salmo salar)

Food intake is a vital process that supplies necessary energy and essential nutrients to the body. Information regarding luminal composition in the gastrointestinal tract (GIT) collected through mechanical and nutrient sensing mechanisms are generally conveyed, in both mammals and fish, to the hypothalamic neurocircuits. In this context, ghrelin, the only known hormone with an orexigenic action, and the intestinal peptide transporters 1 and 2, involved in absorption of dietary di- and tripeptides, exert important and also integrated roles for the nutrient uptake. Together, both are potentially involved in signaling pathways that control food intake originating from different segments of the GIT. However, little is known about the role of different paralogs and their response to fasting. Therefore, after 3 weeks of acclimatization, 12 Atlantic salmon (Salmo salar) post-smolt were fasted for 4 days to explore the gastrointestinal response in comparison with fed control (n = 12). The analysis covered morphometric (weight, length, condition factor, and wet content/weight fish %), molecular (gene expression variations), and correlation analyses. Such short-term fasting is a common and recommended practice used prior to any handling in commercial culture of the species. There were no statistical differences in length and weight but a significant lower condition factor in the fasted group. Transcriptional analysis along the gastrointestinal segments revealed a tendency of downregulation for both paralogous genes slc15a1a and slc15a1b and with significant lowered levels in the pyloric ceca for slc15a1a and in the pyloric ceca and midgut for slc15a1b. No differences were found for slc15a2a and slc15a2b (except a higher expression of the fasted group in the anterior midgut), supporting different roles for slc15 paralogs. This represents the first report on the effects of fasting on slc15a2 expressed in GIT in teleosts. Transcriptional analysis of ghrelin splicing variants (ghrl-1 and ghrl-2) showed no difference between treatments. However, correlation analysis showed that the mRNA expression for all genes (restricted to segment with the highest levels) were affected by the residual luminal content. Overall, the results show minimal effects of 4 days of induced fasting in Atlantic salmon, suggesting that more time is needed to initiate a large GIT response.

Intestinal electrogenic sodium-dependent glucose absorption in tilapia and trout reveal species differences inSLC5A-associated kinetic segmental segregation

Electrogenic sodium-dependent glucose transport along the length of the intestine was compared between the omnivorous Nile tilapia ( Oreochromis niloticus) and the carnivorous rainbow trout ( Oncorhynchus mykiss) in Ussing chambers. In tilapia, a high-affinity, high-capacity kinetic system accounted for the transport throughout the proximal intestine, midintestine, and hindgut segments. Similar dapagliflozin and phloridzin dihydrate inhibition across all segments support this homogenous high-affinity, high-capacity system throughout the tilapia intestine. Genomic and gene expression analysis supported findings by identifying 10 of the known 12 SLC5A family members, with homogeneous expression throughout the segments with dominant expression of sodium-glucose cotransporter 1 (SGLT1; SLC5A1) and sodium-myoinositol cotransporter 2 (SMIT2; SLC5A11). In contrast, trout’s electrogenic sodium-dependent glucose absorption was 20–35 times lower and segregated into three significantly different kinetic systems found in different anatomical segments: a high-affinity, low-capacity system in the pyloric ceca; a super-high-affinity, low-capacity system in the midgut; and a low-affinity, low-capacity system in the hindgut. Genomic and gene expression analysis found 5 of the known 12 SLC5A family members with dominant expression of SGLT1 ( SLC5A1), sodium-glucose cotransporter 2 (SGLT2; SLC5A2), and SMIT2 ( SLC5A11) in the pyloric ceca, and only SGLT1 ( SLC5A1) in the midgut, accounting for differences in kinetics between the two. The hindgut presented a low-affinity, low-capacity system partially attributed to a decrease in SGLT1 ( SLC5A1). Overall, the omnivorous tilapia had a higher electrogenic glucose absorption than the carnivorous trout, represented with different kinetic systems and a greater expression and number of SLC5A orthologs. Fish differ from mammals, having hindgut electrogenic glucose absorption and segment specific transport kinetics.