Insertion of YFP at P5CS1 and AFL1 shows the potential, and potential complications, of gene tagging for functional analyses of stress-related proteins.

Crispr/CAS9-enabled homologous recombination to insert a tag in frame with an endogenous gene can circumvent difficulties such as context-dependent promoter activity that complicate analysis of gene expression and protein accumulation patterns. However, there have been few reports examining whether such gene targeting/gene tagging (GT) can alter expression of the target gene. The enzyme encoded by Δ1-pyrroline-5-carboxylate synthetase 1 (P5CS1) is key for stress-induced proline synthesis and drought resistance, yet its expression pattern and protein localisation have been difficult to assay. We used GT to insert YFP in frame with the 5' or 3' ends of the endogenous P5CS1 and At14a-Like 1 (AFL1) coding regions. Insertion at the 3' end of either gene generated homozygous lines with expression of the gene-YFP fusion indistinguishable from the wild type allele. However, for P5CS1 this occurred only after selfing and advancement to the T5 generation allowed initial homozygous lethality of the insertion to be overcome. Once this was done, the GT-generated P5CS1-YFP plants revealed new information about P5CS1 localisation and tissue-specific expression. In contrast, insertion of YFP at the 5' end of either gene blocked expression. The results demonstrate that GT can be useful for functional analyses of genes that are problematic to properly express by other means but also show that, in some cases, GT can disrupt expression of the target gene.

Largescale mullet (Planiliza macrolepis) can recover from thermal pollution-induced malformations.

It is well known in aquaculture that hyperthermic perturbations may cause skeleton malformations in fish, but this phenomenon has rarely been documented in wild species. One rare location where thermal pollution has increased the proportion of malformed fish in wild population is in the waters near the Kuosheng Nuclear Power Plant in Taiwan. At this site, the threshold temperature and critical exposure time for inducing deformations have not been previously determined. In addition, it was unclear whether juvenile fish with thermal-induced malformations are able to recover when the temperature returns below the threshold. In the present study, juvenile largescale mullet (Planiliza macrolepis) were kept at temperatures ranging from 26°C and 36°C for 1-4 weeks, after which malformed fish were maintained at a preferred temperature of 26°C for another 8 weeks. The vertebrae bending index (VBI) of fish was increased after 2 weeks at 36°C, and deformed vertebral columns were detected by radiography after 4 weeks. However, malformations were not observed in groups kept at or below 34°C. Moreover, at the end of the recovery period, both the VBI and the vertebrae malformations had returned to normal. The results of this study may help to more precisely determine potential environmental impacts of thermal pollution and raise the possibility that the capacity for fish vertebrae to recover from the impacts of chronic thermal exposures may be an important consideration in marine fish conservation.

The Effects of Continuous Acoustic Stress on ROS Levels and Antioxidant-related Gene Expression in the Black Porgy (Acanthopagrus schlegelii).

Hao-Yi Chang, Tzu-Hao Lin, Kazuhiko Anraku, and Yi Ta Shao (2018) Short-term exposure to strong underwater noise is known to seriously impact fish. However, the chronic physiological effects of continuous exposure to weak noise, i.e. the operation noise from offshore wind farms (OWF), remain unclear. Since more and more OWF will be built in the near future, their operation noise is an emerging ecological issue. To investigate the long-term physiological effects of such underwater noise on fish, black porgies (Acanthopagrus schlegelii) were exposed to two types of simulated wind farm noise-quiet (QC: 109 dB re 1 µPa / 125.4 Hz; approx. 100 m away from the wind turbine) and noisy (NC: 138 dB re 1 µPa / 125.4 Hz; near the turbine)-for up to 2 weeks. Measurement of auditory-evoked potentials showed that black porgies can hear sound stimuli under both NC and QC scenarios. Although no significant difference was found in plasma cortisol levels, the fish under NC conditions exhibited higher plasma reactive oxygen species (ROS) levels than the control group at week 2. Moreover, alterations were found in mRNA levels of hepatic antioxidant-related genes (sod1, cat and gpx), with cat downregulated and gpx upregulated after one week of QC exposure. Our results suggest that the black porgy may adapt to QC levels of noise by modulating the antioxidant system to keep ROS levels low. However, such antioxidant response was not observed under NC conditions; instead, ROS accumulated to measurably higher levels. This study suggests that continuous OWF operation noise represents a potential stressor to fish. Furthermore, this is the first study to demonstrate that chronic exposure to noise could induce ROS accumulation in fish plasma.