An evaluation of potential reference genes for stability of expression in two salmonid cell lines after infection with either Piscirickettsia salmonis or IPNV

<p>Abstract</p> <p>Background</p> <p>Due to the limited number of species specific antibodies against fish proteins, differential gene expression analyses are vital for the study of host immune responses. Quantitative real-time reverse transcription PCR (qRT-PCR) is one of the most powerful tools for this purpose. Nevertheless, the accuracy of the method will depend on the careful selection of genes whose expression are stable and can be used as internal controls for a particular experimental setting.</p> <p>Findings</p> <p>The expression stability of five commonly used housekeeping genes [beta-actin (<it>ACTB</it>), elongation factor 1-alpha (<it>EF1A</it>), ubiquitin (<it>UBQ</it>), glyceraldehyd-3-phosphate dehydrogenase (<it>GAPDH</it>) and tubulin alpha (<it>TUBA</it>)] were monitored in salmonid cell lines CHSE-214 and RTS11 after infection with two of the most fastidious fish pathogens, the facultative bacterium <it>Piscirickettsia salmonis </it>and the aquabirnavirus IPNV (Infectious Pancreatic Necrosis Virus). After geNorm analysis, <it>UBQ </it>and <it>EF1A </it>appeared as the most stable, although <it>EF1A </it>was slightly upregulated at late stages of <it>P. salmonis </it>infection in RTS11. <it>ACTB </it>instead, showed a good performance in each case, being always considered within the three most stable genes of the panel. In contrast, infection-dependent differential regulation of <it>GAPDH </it>and <it>TUBA </it>was also demonstrated.</p> <p>Conclusion</p> <p>Based on the data presented here with the cell culture models CHSE-214 and RTS11, we suggest the initial choice of <it>UBQ</it>, <it>ACTB </it>and <it>EF1A </it>as reference genes in qRT-PCR assays for studying the effect of <it>P. salmonis </it>and IPNV on the host immune response.</p

Dealing with Food and Eggs in Mouthbrooding Cichlids: Structural and Functional Trade-Offs in Fitness Related Traits

As in any vertebrate, heads of fishes are densely packed with functions. These functions often impose conflicting mechanical demands resulting in trade-offs in the species-specific phenotype. When phenotypical traits are linked to gender-specific parental behavior, we expect sexual differences in these trade-offs. This study aims to use mouthbrooding cichlids as an example to test hypotheses on evolutionary trade-offs between intricately linked traits that affect different aspects of fitness. We focused on the oral apparatus, which is not only equipped with features used to feed and breathe, but is also used for the incubation of eggs. We used this approach to study mouthbrooding as part of an integrated functional system with diverging performance requirements and to explore gender-specific selective environments within a species.Because cichlids are morphologically very diverse, we hypothesize that the implications of the added constraint of mouthbrooding will primarily depend on the dominant mode of feeding of the studied species. To test this, we compared the trade-off for two maternal mouthbrooding cichlid species: a "suction feeder" (Haplochromis piceatus) and a "biter" (H. fischeri). The comparison of morphology and performance of both species revealed clear interspecific and intersex differences. Our observation that females have larger heads was interpreted as a possible consequence of the fact that in both the studied species mouthbrooding is done by females only. As hypothesized, the observed sexual dimorphism in head shape is inferred as being suboptimal for some aspects of the feeding performance in each of the studied species. Our comparison also demonstrated that the suction feeding species had smaller egg clutches and more elongated eggs.Our findings support the hypothesis that there is a trade-off between mouthbrooding and feeding performance in the two studied haplochromine cichlids, stressing the importance of including species-specific information at the gender level when addressing interspecific functional/morphological differences

Jaw Laterality and Related Handedness in the Hunting Behavior of a Scale-Eating Characin, Exodon paradoxus

BACKGROUND: Asymmetry in animal bodies and behavior has evolved several times, but our knowledge of their linkage is limited. Tanganyikan scale-eating cichlids have well-known antisymmetry in their bodies and behavior; individuals open their mouths leftward (righty) or rightward (lefty), and righties always attack the right flank of the prey, whereas lefties attack the left. This study analyzed the morphological asymmetry in a scale-eating characiform, Exodon paradoxus, and its behavioral handedness. METHODOLOGY/PRINCIPAL FINDINGS: Each eight E. paradoxus was observed for 1-h with a prey goldfish in an aquarium to detect the behavioral handedness. Following the experiment, the lateral differences in the mandibles and head-inclination of these eight and ten additional specimens were analyzed. Both measurements on the morphology showed a bimodal distribution, and the laterality identified by these two methods was always consistent within a given individual, indicating that the characin has morphological antisymmetry. Furthermore, this laterality significantly corresponded to behavioral handedness; that is, lefties more often rasped scales from the right flank of the prey and vice versa. However, the correlation between laterality and handedness is the opposite of that in the cichlids. This is due to differences in the feeding apparatus and technique. The characin has cuspids pointing forward on the external side of the premaxilla, and it thrusts its dominant body side outward from its body axis on the flank of the prey to tear off scales. By contrast, the cichlids draw their dominant body side inward toward the axis or rotate it to scrape or wrench off scales with the teeth lined in the opened mouth. CONCLUSIONS/SIGNIFICANCE: This study demonstrated that the antisymmetry in external morphology and the corresponding behavioral handedness have evolved in two lineages of scale-eating fishes independently, and these fishes adopt different utilization of their body asymmetry to tear off scales

Mice Null for Calsequestrin 1 Exhibit Deficits in Functional Performance and Sarcoplasmic Reticulum Calcium Handling

In skeletal muscle, the release of calcium (Ca2+) by ryanodine sensitive sarcoplasmic reticulum (SR) Ca2+ release channels (i.e., ryanodine receptors; RyR1s) is the primary determinant of contractile filament activation. Much attention has been focused on calsequestrin (CASQ1) and its role in SR Ca2+ buffering as well as its potential for modulating RyR1, the L-type Ca2+ channel (dihydropyridine receptor, DHPR) and other sarcolemmal channels through sensing luminal [Ca2+]. The genetic ablation of CASQ1 expression results in significant alterations in SR Ca2+ content and SR Ca2+ release especially during prolonged activation. While these findings predict a significant loss-of-function phenotype in vivo, little information on functional status of CASQ1 null mice is available. We examined fast muscle in vivo and in vitro and identified significant deficits in functional performance that indicate an inability to sustain contractile activation. In single CASQ1 null skeletal myofibers we demonstrate a decrease in voltage dependent RyR Ca2+ release with single action potentials and a collapse of the Ca2+ release with repetitive trains. Under voltage clamp, SR Ca2+ release flux and total SR Ca2+ release are significantly reduced in CASQ1 null myofibers. The decrease in peak Ca2+ release flux appears to be solely due to elimination of the slowly decaying component of SR Ca2+ release, whereas the rapidly decaying component of SR Ca2+ release is not altered in either amplitude or time course in CASQ1 null fibers. Finally, intra-SR [Ca2+] during ligand and voltage activation of RyR1 revealed a significant decrease in the SR[Ca2+]free in intact CASQ1 null fibers and a increase in the release and uptake kinetics consistent with a depletion of intra-SR Ca2+ buffering capacity. Taken together we have revealed that the genetic ablation of CASQ1 expression results in significant functional deficits consistent with a decrease in the slowly decaying component of SR Ca2+ release

Effect of phosphate and temperature on force exerted by white muscle fibres from dogfish.

Effects of Pi (inorganic phosphate) are relevant to the in vivo function of muscle because Pi is one of the products of ATP hydrolysis by actomyosin and by the sarcoplasmic reticulum Ca pump. We have measured the Pi sensitivity of force produced by permeabilized muscle fibres from dogfish (Scyliorhinus canicula) and rabbit. The activation conditions for dogfish fibres were crucial: fibres activated from the relaxed state at 5, 12, and 20°C were sensitive to Pi, whereas fibres activated from rigor at 12°C were insensitive to Pi in the range 5-25 mmol l. Rabbit fibres activated from rigor were sensitive to Pi. Pi sensitivity of force produced by dogfish fibres activated from the relaxed state was greater below normal body temperature (12°C for dogfish) in agreement with what is known for other species. The force-temperature relationship for dogfish fibres (intact and permeabilized fibres activated from relaxed) showed that at 12°C, normal body temperature, the force was near to its maximum value

Structural, Metabolic, and Functional Brain Abnormalities as a Result of Prenatal Exposure to Drugs of Abuse: Evidence from Neuroimaging

Prenatal exposure to alcohol and stimulants negatively affects the developing trajectory of the central nervous system in many ways. Recent advances in neuroimaging methods have allowed researchers to study the structural, metabolic, and functional abnormalities resulting from prenatal exposure to drugs of abuse in living human subjects. Here we review the neuroimaging literature of prenatal exposure to alcohol, cocaine, and methamphetamine. Neuroimaging studies of prenatal alcohol exposure have reported differences in the structure and metabolism of many brain systems, including in frontal, parietal, and temporal regions, in the cerebellum and basal ganglia, as well as in the white matter tracts that connect these brain regions. Functional imaging studies have identified significant differences in brain activation related to various cognitive domains as a result of prenatal alcohol exposure. The published literature of prenatal exposure to cocaine and methamphetamine is much smaller, but evidence is beginning to emerge suggesting that exposure to stimulant drugs in utero may be particularly toxic to dopamine-rich basal ganglia regions. Although the interpretation of such findings is somewhat limited by the problem of polysubstance abuse and by the difficulty of obtaining precise exposure histories in retrospective studies, such investigations provide important insights into the effects of drugs of abuse on the structure, function, and metabolism of the developing human brain. These insights may ultimately help clinicians develop better diagnostic tools and devise appropriate therapeutic interventions to improve the condition of children with prenatal exposure to drugs of abuse

Loss of PTB or Negative Regulation of Notch mRNA Reveals Distinct Zones of Notch and Actin Protein Accumulation in Drosophila Embryo

Polypyrimidine Tract Binding (PTB) protein is a regulator of mRNA processing and translation. Genetic screens and studies of wing and bristle development during the post-embryonic stages of Drosophila suggest that it is a negative regulator of the Notch pathway. How PTB regulates the Notch pathway is unknown. Our studies of Drosophila embryogenesis indicate that (1) the Notch mRNA is a potential target of PTB, (2) PTB and Notch functions in the dorso-lateral regions of the Drosophila embryo are linked to actin regulation but not their functions in the ventral region, and (3) the actin-related Notch activity in the dorso-lateral regions might require a Notch activity at or near the cell surface that is different from the nuclear Notch activity involved in cell fate specification in the ventral region. These data raise the possibility that the Drosophila embryo is divided into zones of different PTB and Notch activities based on whether or not they are linked to actin regulation. They also provide clues to the almost forgotten role of Notch in cell adhesion and reveal a role for the Notch pathway in cell fusions

The Metabolic Consequences of Hepatic AMP-Kinase Phosphorylation in Rainbow Trout

AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, is proposed to function as a “fuel gauge” to monitor cellular energy status in response to nutritional environmental variations. However, in fish, few studies have addressed the metabolic consequences related to the activation of this kinase. This study demonstrates that the rainbow trout (Oncorhynchus mykiss) possesses paralogs of the three known AMPK subunits that co-diversified, that the AMPK protein is present in the liver and in isolated hepatocytes, and it does change in response to physiological (fasting-re-feeding cycle) and pharmacological (AICAR and metformin administration and incubations) manipulations. Moreover, the phosphorylation of AMPK results in the phosphorylation of acetyl-CoA carboxylase, a main downstream target of AMPK in mammals. Other findings include changes in hepatic glycogen levels and several molecular actors involved in hepatic glucose and lipid metabolism, including mRNA transcript levels for glucokinase, glucose-6-phosphatase and fatty acid synthase both in vivo and in vitro. The fact that most results presented in this study are consistent with the recognized role of AMPK as a master regulator of energy homeostasis in living organisms supports the idea that these functions are conserved in this piscine model