Expression pattern of nanos, piwil, dnd, vasa and pum genes during ontogenic development in Nile tilapia Oreochromis niloticus

Primordial germ cells (PGCs) are specified by maternally provided determinants in fish. PGCs migrate then into prospective gonadal sites during early development and give rise to germ cell lineage. PGC disrupted animals do not sexually mature which has a range of commercial as well as environmental benefits. To find potential target genes for sterilisation of Nile tilapia, relative mRNA abundance patterns and tissue distribution of four nanos, two piwil, dnd1, vasa and three pum genes were investigated during ontogenic development from unfertilised eggs to newly hatched larvae and in adult tissues, respectively. The ontogenic pattern of RNA abundance revealed that all the investigated gene transcripts are maternally deposited to varying degrees, except for nanos2 which is not expressed in eggs. The ontogenic patterns of relative RNA abundance could be grouped into three categories. The first one, including nanos3, piwil1, piwil2, dnd1 and vasa, showed abundant transcript levels during early developmental stages which are then degraded during the period of maternal to zygotic transition between blastula and gastrula stages with a reduction in expression of four to five orders of magnitude by hatching stage. Another, including pum2 and pum3, showed similar patterns to the first group, but the transcript levels are reduced by only two orders of magnitude. The third group, including nanos1a, nanos1b and pum1, was characterised by a zygotic increase. nanos2 had no detectable transcripts until hatching stage. The tissue screening of nanos1a, nanos1b, pum1, pum2 and pum3 showed that they are expressed in various tissues, implying their potential pleiotropic effects in these tissues apart from gonads. In contrast, nanos3, piwil1, piwil2, dnd1 and vasa appeared to be exclusively expressed in gonads (both ovary and testis), and nanos2 showed testis-specific expression. Based on these results nanos3, piwil1, piwil2, dnd1 and vasa were prioritised among the 11 selected genes as potential target genes for sterilisation in Nile tilapia as they have no significant zygotic expression during embryogenesis, they are expressed exclusively in gonads and maternally deposited. These features suggest a potential role of these genes in the specification and maintenance of PGCs during the ontogenic development of Nile tilapia

The evolution of apolipoprotein B and its mRNA editing complex. Does the lack of editing contribute to hypertriglyceridemia?

The evolution of apolipoprotein B (Apob) has been intensely researched due to its importance during lipid transport. Mammalian full-length apob100 can be post-transcriptionally edited by the enzyme apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like complex-one (Apobec1) resulting in a truncated Apob, known as Apob48. Whilst both full-length and truncated forms of Apob are important for normal lipid homeostasis in mammals, there is no evidence for the presence of apob mRNA editing prior to the divergence of the mammals, yet, non-mammalian vertebrates appear to function normally with only Apob100. To date, the majority of the research carried out in non-mammalian vertebrates has focused on chickens with only a very limited number examining apob mRNA editing in fish. This study focused on the molecular evolution of Apobec1 and Apob in order to ascertain if apob mRNA editing occurs in eels, a basal teleost which represents an evolutionarily important animal group. No evidence for the presence of Apobec1 or the ability for eel apob to be edited was found. However, an important link between mutant mice and the evident hypertriglyceridemia in the plasma of non-mammalian vertebrates was made. This study has provided imperative evidence to help bridge the evolutionary gap between fish and mammals and provides further support for the lack of apob mRNA editing in non-mammalian vertebrates

Temperature-induced testicular germ cell loss and recovery in Nile tilapia Oreochromis niloticus

Water temperature is a critical external factor influencing gonadal development in fish. This research aimed to study the impact of elevated temperature on testicular germ cell survival and reproductive capacity of Nile tilapia. Male Nile tilapia were exposed to high temperatures of either 36 (HT1) or 37°C (HT2) for 3000 degree-days (DD) and thereafter returned to the control temperature of 27°C (CT) for 2200 DD. The deleterious effects on testicular germ and somatic cells were observed histologically, characterised by vacuolisation, atrophy and the loss of spermatogenic cells in testes with a more severe impact of HT2 compared to HT1. Interestingly, serum 11-ketotestosterone (11-KT) and testosterone (T) levels tended to be higher during the heat treatments than CT. Expression levels of germline-specific genes piwil1, piwil2 and nanos2 and Bcl-2 family genes, bcl-xLb and baxa were significantly reduced during the heat treatment compared to CT, more so in the HT2, while the levels of nanos3 and gfra1 transcripts were only significantly reduced in HT2, implying a significant loss of spermatogonial stem cell (SSC) and spermatogonia in HT2. The effect of HT2 is further evidenced by the significantly reduced sperm density and fertilisation rate compared to CT and HT1 at the end of the recovery period but complete sterility was not induced by HT2. Overall, the present study showed significant effects of HT2 on germ cell survival with histological changes in testes, reduced milt quality, increased 11-KT, and decreased expression of germline-specific genes, SSC marker genes and Bcl-2 family genes in testes which could therefore be potential target genes for sterilisation by genome editing

Aspirin inhibits the acute venodilator response to furosemide in patients with chronic heart failure

OBJECTIVES: We sought to determine the effect of aspirin on the venodilator effect of furosemide in patients with chronic heart failure (CHF) BACKGROUND: Furosemide has an acute venodilator effect preceding its diuretic action, which is blocked by nonsteroidal anti-inflammatory drugs. The ability of therapeutic doses of aspirin to block this effect of furosemide in patients with CHF has not been studied. For comparison, the venodilator response to nitroglycerin (NTG) was also studied. METHODS: Eleven patients with CHF were randomized to receive placebo, aspirin at 75 mg/day or aspirin at 300 mg/day for 14 days in a double-blind, crossover study. The effect of these pretreatments on the change in forearm venous capacitance (FVC) after 20 mg of intravenous furosemide was measured over 20 min by using venous occlusion plethysmography. In a second study, the effect of 400 μg of sublingual NTG on FVC was documented in 11 similar patients (nine participated in the first study). RESULTS: Mean arterial pressure, heart rate and forearm blood flow did not change in response to furosemide. After placebo pretreatment, furosemide caused an increase in FVC of 2.2% (95% confidence interval [CI] −0.9% to 5.2%; mean response over 20 min). By comparison, FVC fell by −1.1% (95% CI −4.2% to 1.9%) after pretreatment with aspirin at 75 mg/day, and by −3.7% (95% CI −6.8% to −0.7%) after aspirin at 300 mg/day (p = 0.020). In the second study, NTG increased FVC by 2.1% (95% CI −1.6% to 5.8%) (p = 0.95 vs. furosemide). CONCLUSIONS: In patients with CHF, venodilation occurs within minutes of the administration of intravenous dose of furosemide. Our observation that aspirin inhibits this effect further questions the use of aspirin in patients with CHF

Physiological impact and comparison of mutant screening methods in piwil2 KO founder Nile tilapia produced by CRISPR/Cas9 system

The application of genome engineering techniques to understand the mechanisms that regulate germ cell development opens promising new avenues to develop methods to control sexual maturation and mitigate associated detrimental effects in fish. In this study, the functional role of piwil2 in primordial germ cells (PGCs) was investigated in Nile tilapia using CRISPR/Cas9 and the resultant genotypes were further explored. piwil2 is a gonad-specific and maternally deposited gene in Nile tilapia eggs which is known to play a role in repression of transposon elements and is therefore thought to be important for maintaining germline cell fate. A functional domain of piwil2, PIWI domain, was targeted by injecting Cas9 mRNA and sgRNAs into Nile tilapia embryos at 1 cell stage. Results showed 54% of injected mutant larvae had no or less putative PGCs compared to control fish, suggesting an essential role of piwil2 in survival of PGCs. The genotypic features of the different phenotypic groups were explored by next generation sequencing (NGS) and other mutant screening methods including T7 endonuclease 1 (T7E1), CRISPR/Cas-derived RNA-guided engineered nuclease (RGEN), high resolution melt curve analysis (HRMA) and fragment analysis. Linking phenotypes to genotypes in F0 was hindered by the complex mosacism and wide indel spectrum revealed by NGS and fragment analysis. This study strongly suggests the functional importance of piwil2 in PGCs survival. Further studies should focus on reducing mosaicism when using CRISPR/Cas9 system to facilitate direct functional analysis in F0

Reproductive performance and offspring quality of non-ablated Pacific white shrimp (Litopenaeus vannamei) under intensive commercial scale conditions

This study evaluated reproductive performance of non-ablated Litopenaeus vannamei and the quality of their offspring under commercial conditions. Five tanks were stocked with non-ablated female and other five with ablated individuals as control. Two different larval rearing trials (Larviculture I and II) have been conducted. Six larviculture tanks (n = 3) were used on the first trial (LI) and ten for the second one (LII) (n = 3). Postlarvae from LII were used for nursery and grow-out. Spawning event and hatching rate per day were similar between both treatments. Mating success, mortality of female and number of eggs and nauplii per tank per day of non-ablated group were significantly lower than ablated female. Non-ablated female fecundity (number of eggs and nauplii per spawned female per day) was significantly higher than control. There was no significant difference between daily larval stage index of larvae in LI and LII. The response to the salinity stress test, and final survival and weight in LI was similar. However in LII, postlarvae derived from non-ablated had significantly higher survival to salinity stress test. Identical survival, final weight, weekly growth, feed conversion rate and yield were observed in nursery. The same was observed in grow-out, including weight gain and specific growth rate. Overall this study demonstrates that non-ablated females can have comparable level of productivity to ablated females in intensive commercial hatchery conditions. Their offspring perform comparably in all culture stages with evidence of enhanced resistance to stress in larvae derived from non-ablated female broodstock

Determination of the Distribution of the Resident Inshore and Offshore Migratory Cod Populations Around Shetland (IVa) and Westwards into VIa

The current genetic analysis alludes to finer scale structuring of Atlantic cod stocks in the IVa and VIa stock units than had previously been reported by Heath et al. (2014). Consistent with previous studies of maturation, cod from Viking sampled in 2014 matured at a later age and larger size than other areas, providing a phenotypic population marker.  During spawning time there was no indication that the Viking group extended beyond the > 100 m waters of the northern North Sea. Indeed, the new genetic and maturity evidence suggests that Shetland coastal cod (ShIE) appear to extend into waters > 100 m east of Shetland.  The possible separation of cod from Scottish inshore waters from those offshore is also reminiscent of the inshore-offshore division seen in the northern North Sea.  There is some indication of mixing of populations outside the breeding season in the genetic analysis as well as the observation of large immature cod present in west coast samples.  The present study has considerably expanded our understanding of the Viking cod from northern IVa and when combined with the studies by Poulsen et al. (2011) and Heath et al. (2014), provides a good indication of population extent at spawning time and suggests a split around 0030 W

Removal of the adhesive gum layer surrounding naturally fertilised ballan wrasse (Labrus bergylta) eggs

Commercial production of ballan wrasse (Labrus bergylta) as a cleaner fish for the removal of sea lice (Lepeophtheirus salmonis) from farmed salmonids (Salmo salar) has increased due to its proven efficiency. One bottleneck in commercial hatchery production is working with the benthic adhesive eggs, which makes disinfection and incubation of eggs challenging; therefore, this study aimed to find a chemical or enzymatic treatment and process to remove the adhesive gum layer. Naturally spawned eggs were collected from artificial spawning substrates up to 24h post spawning from wild caught broodstock kept in captivity at the Marine Harvest, Machrihanish facility. Four treatments were tested: tannic acid (0.2, 0.1, and 0.05%), sodium sulfite (2, 1, and 0.5%),l-cysteine (2, and 1%), and enzyme alcalase® (4.0, 3.0, 2.0, 1.0, and 0.5%)in vitro. Eggs were exposed for 25min while being continually agitated, and the proportion of “degummed” eggs was counted at the end of each time period. Enzyme alcalase® was the only treatment that proved successful in degumming eggs, with the time to complete degumming (≥96%) inversely related to enzyme concentration. Complete degumming occurred between 15 and 30min for all enzyme alcalase® dose rates. Mean hatch rates for eggs treated with enzyme alcalase ® were not compromised by the treatment and in the highest dose tested were actually found to be higher in treated eggs (78.9±2.4%) than controls (71.3±3.3%). The use of enzyme alcalase® has proven effective in degumming ballan wrasse eggs without affecting hatch rates. However, translation of this method toin situdegumming and thus removal of eggs from spawning substrate on farm remains to be standardised

Bacterial Communities of Ballan Wrasse (Labrus bergylta) Eggs at a Commercial Marine Hatchery

Ballan wrasse (Labrus bergylta, Ascanius 1767) are cleaner fish cultured in northern Europe to remove sea lice from farmed Atlantic salmon (Salmo salar, Linnaeus 1758). Despite increasing appreciation for the importance of the microbiota on the phenotypes of vertebrates including teleosts, the microbiota of wrasse eggs has yet to be described. Therefore, the aim of this present study was to describe the bacterial component of the microbiota of ballan wrasse eggs shortly after spawning and at 5 days, once the eggs had undergone a routine incubation protocol that included surface disinfection steps in a common holding tank. Triplicate egg samples were collected from each of three spawning tanks and analysis of 16S rRNA gene sequences revealed that 88.6% of reads could be identified to 186 taxonomic families. At Day 0, reads corresponding to members of the Vibrionaceae, Colwelliaceae and Rubritaleaceae families were detected at greatest relative abundances. Bacterial communities of eggs varied more greatly between tanks than between samples deriving from the same tank. At Day 5, there was a consistent reduction in 16S rRNA gene sequence richness across the tanks. Even though the eggs from the different tanks were incubated in a common holding tank, the bacterial communities of the eggs from the different tanks had diverged to become increasingly dissimilar. This suggests that the disinfection and incubation exerted differential effects of the microbiota of the eggs from each tank and that the influence of the tank water on the composition of the egg microbiota was lower than expected. This first comprehensive description of the ballan wrasse egg bacterial community is an initial step to understand the role and function of the microbiota on the phenotype of this fish. In future, mass DNA sequencing methods may be applied in hatcheries to screen for pathogens and as a tool to assess the health status of eggs