Food effect on the physiological condition of the mussel Perna viridis (Bivalvia: Mytilidae), using the RNA/DNA ratio

The green mussel, Perna viridis, became widespread in the northern coast of Sucre State since its arrival to Venezuela in 1993. RNA/DNA and Protein/DNA ratios were used to study the effect of starvation on its instantaneous growth. The mussels were collected in La Esmeralda and Chacopata, acclimatized in the laboratory for four weeks and maintained for another six weeks in two groups: one fed ad libitum and another without food (this later group was later fed for two additional weeks). Protein (colorimetric method), and nucleic acid concentrations (RNA and DNA, fluorometric method with ethidium bromide) were measured in adductor muscle, digestive gland and gills. The instantaneous growth was assessed using RNA/DNA and Protein/DNA rations. These indexes were always higher in the fed organisms. Animals from Chacopata were in better physiological condition that those from La Esmeralda during the abstinence time (six weeks). Muscle was the best tissue to determine instantaneous growth. The RNA/DNA ratio is a reliable index to determine the physiological condition and instantaneous growth of this species.     

The effect of starvation on RNA: DNA ratios and growth of larval striped bass, Morone saxatalis

Hatchery reared larval striped bass, Morone saxatilis , 8-days-post-hatching were subjected to various feeding/starvation regimes over a period of 14 days.
Batches of larvae from each treatment were sampled over the 14-day period and subdivided for determination of notochord length and RNA:DNA ratio. The best growth was found in fully fed F1000 larvae (exposed to 1000 Artermia nauplii l⁻¹), which reached 8.2 mm after 11 days and 9.6 mm after 14 days. Starved animals after 11 days had notochord lengths of 4.9 mm. Growth curves from feeding-delayed larvae indicated that animals fed after up to 5 days starvation were capable of complete recovery. F100 larvae (exposed to 100 Anemia nauplii 1^−l) had a slower growth rate than F1000 larvae, reaching a notochord length of 7.3 mm after 14 days. RNA:DNA ratios over time closely followed notochord growth curves, with clear differences between starved, F100 and F1000 larvae being established after only 2 days. Equilibrium RNA:DNA ratios of 3.0 and 2.25 were established in F1000 and F100 larvae, respectively, 6.8 days after the beginning of the experiment. The average lag time between a change from the starved to the fed condition and a change in RNA:DNA ratio as determined by the divergence of the nucleic acid curve from the starved condition was 0.66 days.
In treatments where starvation followed various periods of feeding, larvae regressed in notochord length such that the final length at 14 days reflected the degree of feeding. RNA:DNA ratios in these animals again closely followed growth curves with a lag time of 0.81 days.
It was concluded that RNA:DNA ratios provided very accurate indices of growth in striped bass larvae which were highly sensitive to feeding status.     

Assessment of the nutritional status of field-caught larval Pacific bluefin tuna by RNA/DNA ratio based on a starvation experiment of hatchery-reared fish

RNA/DNA ratio is a useful and reliable indicator of the nutritional status of fish larvae and juveniles. In order to assess the nutritional status of field-caught larval Pacific bluefin tuna Thunnus orientalis (Temminck et Schlegel), starvation experiments of hatchery-reared larvae were conducted and changes in the RNA/DNA ratio of fed and starved larvae were analyzed. Starvation experiments were conducted every 3 days after first feeding. The survival rate of Pacific bluefin tuna larvae ranged 10-50% after 1 day of starved conditions and growth retardation was observed immediately. These results suggest that Pacific bluefin tuna larvae have a very low tolerance to starvation. The RNA/DNA ratios of fed larvae were approximately 2.0-4.0. On the other hand, the value of starved larvae significantly decreased to 1.0-3.0. The nutritional status of 3 cohorts of field-caught tuna larvae collected in the northwestern Pacific Ocean was examined based on the value of the RNA/DNA ratio of the 1 day starved larvae. 4.35-25.77% of the cohorts were regarded as the “starving condition”, which was negatively correlated to the ambient prey densities. These findings suggest that the nutritional condition of larval Pacific bluefin tuna was influenced by the ambient prey density, and starvation itself and starvation-induced predation could greatly contribute to mortality in the larval period of Pacific bluefin tuna.     

Estimating recent growth in the cuttlefish Sepia officinalis: are nucleic acid-based indicators for growth and condition the method of choice?

A laboratory calibration study was undertaken with juvenile Sepia officinalis (80-85 g initial wet weight) to investigate the effects of different food rations and different starving intervals on RNA/dry weight (DW) ratios and RNA/DNA ratios in cephalopod mantle muscle at two different temperatures. The digestive gland index was also used as an additional indicator of recent growth. High food rations and low temperature went along with high RNA/DW ratios and high RNA/DNA ratios. Starving resulted in a linear decline in growth performance and a concomitant decrease in RNA/DW and RNA/DNA ratio, with RNA/DNA ratios representing the growth data better. RNA/DNA ratios decreased faster at higher temperatures. A fluorimetric assay for nucleic acid analysis was optimized for cephalopod mantle tissues and yielded reproducible RNA/DNA ratios with a relative variance below 10%. Thus, it may be possible to use this estimator of recently encountered feeding regime for the evaluation of mortality rates of early teuthid paralarvae to eventually support stock management. Also, log relative digestive gland weight showed a strong relationship with starving time, but, surprisingly, not with temperature. Data from the two temperatures analyzed could be combined to form a common regression line of relative digestive gland index with starving time. This indicator for recent growth might be especially suitable for large specimens with a well-developed digestive gland.     

Biochemical Indices and Life Traits of Loggerhead Turtles (Caretta caretta) from Cape Verde Islands

The loggerhead turtle (Caretta caretta) is an endangered marine reptile for whom assessing population health requires knowledge of demographic parameters such as individual growth rate. In Cape Verde, as within several populations, adult female loggerhead sea turtles show a size-related behavioral and trophic dichotomy. While smaller females are associated with oceanic habitats, larger females tend to feed in neritic habitats, which is reflected in their physiological condition and in their offspring. The ratio of RNA/DNA provides a measure of cellular protein synthesis capacity, which varies depending on changes in environmental conditions such as temperature and food availability. The purpose of this study was to evaluate the combined use of morphometric data and biochemical indices as predictors of the physiological condition of the females of distinct sizes and hatchlings during their nesting season and how temperature may influence the physiological condition on the offspring. Here we employed biochemical indices based on nucleic acid derived indices (standardized RNA/DNA ratio-sRD, RNA concentration and DNA concentration) in skin tissue as a potential predictor of recent growth rate in nesting females and hatchling loggerhead turtles. Our major findings were that the physiological condition of all nesting females (sRD) decreased during the nesting season, but that females associated with neritic habitats had a higher physiological condition than females associated with oceanic habitats. In addition, the amount of time required for a hatchling to right itself was negatively correlated with its physiological condition (sRD) and shaded nests produced hatchlings with lower sRD. Overall, our results showed that nucleic acid concentrations and ratios of RNA to DNA are an important tool as potential biomarkers of recent growth in marine turtles. Hence, as biochemical indices of instantaneous growth are likely temperature-, size- and age-dependent, the utility and validation of these indices on marine turtles stocks deserves further study.     

Ontogenetic changes in RNA,DNA and protein contents of Chinese loach,Paramisgurnus dabryanus (Dabry de Thiersant, 1872), larvae and juveniles

The changes in nucleic acid‐based indices and protein variables of Chinese loach, Paramisgurnus dabryanus, larvae and juveniles from hatching to 60 days after hatching (DAH) were conducted to assess its growth potential. The nucleic acid contents were analysed using a UV‐based method (n = 3, rearing temperature 24.4 ± 0.4°C, dissolve oxygen 7.1 ± 0.5 mg L^?1, pH 7.9 ± 0.4). Ribonucleic acid (RNA) concentration significantly decreased from 2 to 5 DAH, then increased rapidly until 10 DAH, declining slightly thereafter. Deoxyribonucleic acid (DNA) concentration increased 2–5 DAH, decreased until 9 DAH, slightly increased again around 26 DAH, and then declined to a relatively stable level. Both RNA‐DNA and protein‐DNA ratios showed a statistically obvious relationship with growth rates. A significantly positive relationship was found between RNA‐DNA ratio and growth rates during the early life stage of Chinese loach. According to the results, growth of Chinese loach is characterized by rapid hyperplasia from hatching through completion of the yolk‐sac stage followed by continued rapid hyperplasia combined with increasing hypertrophy after feeding commences. The stage preceding 17 DAH of Chinese loach P. dabryanus is presumed to be critical for its survival and growth at 24°C.     

Development Of A Nucleic Acid Lateral Flow Strip For Detection Of Hepatitis C Virus (Hcv) Core Antigen

The object of this study was to develop a simple, rapid, specific, and highly sensitive method to detect HCV core antigen. A nucleic acid aptamer was designed with the high specificity and sensitivity in a nucleic acid lateral flow strip to compete with HCV core antigen and DNA probes. The lower detection limit of the test strip was calculated to be 10 pg/mL with the scanner and 100 pg/mL with naked eyes. Results showed that there were no cross-interactions with other proteins such as HCV NS3, E1/E2 antigens, HIV p24 antigens, or BSA proteins (HCV unrelated protein). When the viral load exceeded 10⁴ copies/mL, the positive coincidence rates of ELISA and strip detection, when compared with the HCV RNA assay, were 98.44% and 97.28%, respectively. The results indicated that the ELISA detection and strip assay were in good agreement with the measured value. The results indicated that a nucleic acid lateral flow strip was a simple, rapid, specific, highly sensitive, and cost-effective field-based method for detecting HCV core antigen. The strip assay is an acceptable alternative to diagnose HCV core antigen and to investigate its epidemiology in clinical laboratories lacking specialized equipment and skills.     

Effect of denervation on the synthesis of ribonucleic acid and deoxyribonucleic acid in rat diaphragm muscle

1. The effect of unilateral denervation of rat diaphragm muscle on its content of nucleic acids and their incorporation of precursors was investigated. 2. After denervation the paralysed hemidiaphragm hypertrophies and within 3 days its content of RNA increases considerably. The concentration of DNA/unit mass remains fairly constant. 3. During this period there is some increase in the rate of incorporation of [(14)C]adenine into RNA, whereas there is some diminution in the rate of incorporation of [(14)C]orotic acid. 4. Incorporation of [(14)C]adenine and [(3)H]thymidine into DNA is much increased in the paralysed tissue, reaching its maximum by about the third day, but returning to normal by the tenth. 5. The significance of these results in relation to the hypertrophy after denervation is discussed.     

Identification of nucleic acid binding sites on translin-associated factor X (TRAX) protein

Translin and TRAX proteins play roles in very important cellular processes such as DNA recombination, spatial and temporal expression of mRNA, and in siRNA processing. Translin forms a homomeric nucleic acid binding complex and binds to ssDNA and RNA. However, a mutant translin construct that forms homomeric complex lacking nucleic acid binding activity is able to form fully active heteromeric translin-TRAX complex when co-expressed with TRAX. A substantial progress has been made in identifying translin sites that mediate its binding activity, while TRAX was thought not to bind DNA or RNA on its own. We here for the first time demonstrate nucleic acid binding to TRAX by crosslinking radiolabeled ssDNA to heteromeric translin-TRAX complex using UV-laser. The TRAX and translin, photochemically crosslinked with ssDNA, were individually detected on SDS-PAGE. We mutated two motifs in TRAX and translin, designated B2 and B3, to help define the nucleic acid binding sites in the TRAX sequence. The most pronounced effect was observed in the mutants of B3 motif that impaired nucleic acid binding activity of the heteromeric complexes. We suggest that both translin and TRAX are binding competent and contribute to the nucleic acid binding activity.     

The uptake of [3H]uridine into the nucleic acids of rat uterus during early pregnancy

1. Changes in content and uptake of [(3)H]uridine into the nucleic acids of rat uterus during the first 9 days of pregnancy were studied. 2. From day 6 implantation sites were separated from the rest of the uterine tissue for independent analysis. 3. Up to day 5 of pregnancy no changes were found in the total dry matter or in RNA and DNA content/unit dry matter nor in the RNA/DNA ratios. 4. From day 6, when implantation sites are visible, the water content of the implantation sites increased by 2-3%, and the RNA content/unit dry wt. and the RNA/DNA ratios increased. The DNA content/unit dry wt. did not increase in the implantation sites until day 8. 5. Uptake of [(3)H]uridine into the acid-soluble fraction of the tissues was markedly higher in implantation sites than in non-implantation sites. 6. Uptake of [(3)H]uridine into RNA was significantly increased on day 3 of pregnancy and again on day 5. 7. On days 6 and 7, the incorporation into RNA of implantation sites was significantly higher than in the remainder of the uterine tissue but decreased on days 8 and 9 to the same value as that of the normal tissue. 8. No change occurred in uptake into DNA until day 6, when there was an increase in uptake by the implantation sites. 9. It is suggested that the increase in RNA synthesis on day 3 is a preparation of the uterus for the onset of implantation on day 5, and that increased synthesis in implantation sites on days 6 and 7 is the elaboration of new RNA necessary for this early stage of pregnancy to commence.     

Comparison of Day and Night Fyke Netting, Electrofishing and Snorkelling for Monitoring a Population of the Threatened Golden Galaxias (Galaxias auratus)

The littoral zone of small off-stream water storage containing a translocated population of Galaxias auratus was sampled fortnightly at day and night with fyke nets, electrofishing and snorkelling over 3 months. Variation in population data provided by each method, including relative abundance indices, size structure, and habitat preferences, were examined. Aspects of behaviour and activity patterns were also investigated. Night sampling using all methods consistently yielded larger catches than day sampling. The size structure of catches varied, with electrofishing at night and fyke netting during the day having higher proportions of juveniles, whilst snorkelling at night and electrofishing during the day had higher proportions of adults. Fyke netting at night yielded by far the largest catches (~3-fold more than other methods) and also captured balanced proportions of juveniles and adults. Galaxias auratus had a strong diel activity pattern and were most active at night. The majority of the population migrated into the littoral zone during the night and back into deeper water during the day. A small number of juveniles remained in the littoral zone and some adults sheltered in the dense cover of species-rich littoral vegetation during the day. Shores with shallow depth profiles appeared to be preferred due to higher catches in these areas using all methods. Based on the results of this study, fyke netting at night in littoral habitats is recommended for monitoring populations of G. auratus. Fyke netting is likely to be an effective method for monitoring other lacustrine galaxiid species; however, further work is required to investigate the effects of habitat variables and fish community structure on activity patterns of galaxiids, and hence their catchability with various methods, in more extensive lentic environments.     

Effects of feeding regime on food consumption, growth rates and tissue nucleic acids in juvenile Arctic charr, Salvelinm alpinus, with particular respect to compensatory growth

Juvenile Arctic charr responded to a change from restricted to satiation feeding by showing a growth spurt (compensatory growth). During this period of rapid growth the fish became hyper-phagic and in the days immediately following transfer from restricted to satiation feeding showed improved food conversion efficiency compared to their counterparts raised on a liberal feeding regime. Tissue (liver and muscle) nucleic acid concentrations were influenced by feeding regime, and RNA : DNA ratios were low in both starved fish and those fed restricted rations. Following transfer from restricted to satiation feeding, tissue RNA : DNA ratios were rapidly restored to initial levels. The uses of tissue RNA: DNA ratios both in evaluating nutritional status and as growth indices are discussed.     

Nutritional condition, starvation status and growth of early juvenile Japanese sea bass (Lateolabrax japonicus) related to prey distribution and feeding in the nursery ground

The nutritional condition and protein growth rates of Japanese temperate bass larvae and juveniles were studied in relation to prey distribution and feeding habits in the nursery grounds in Chikugo estuary, Ariake Sea, Japan. Samples were collected from a wide spatial area covering the nursery grounds of the fish in March and April 2003. Food habits of the fish were analyzed by examining the gut contents. Fish condition was evaluated by using RNA/DNA ratio and other nucleic acid-based indices and protein growth rates. The nucleic acid contents in individually frozen larvae and juveniles were quantified by fluorometric method. Two distinguished feeding patterns, determined by the distribution of prey copepods, were identified along the nursery ground. The first pattern showed the dependency of the fish on the calanoid copepod Sinocalanus sinensis, which was the single dominant prey in low saline upper river areas and the second pattern involved a multi-species dietary habit mainly dominated by Acartia omorii, Oithona davisae and Paracalanus parvus. Values of RNA, DNA, total protein, growth rates and for all the nucleic acid-based indices were higher in upstream areas than in the downstream areas. The proportion of the starving fish was higher in the downstream areas than in the upstream areas. Condition of juvenile sea bass was not equal throughout the nursery grounds; fish in the upper river were in better condition than those in the lower estuary. We speculated that utilization of S. sinensis, which appears a suitable prey item and provide a better foraging environment in the upstream nursery ground, is one of the key factors for early survival and growth of Japanese temperate bass larvae and juveniles in the Chikugo estuary.     

Alpha-L-RNA (alpha-L-ribo configured RNA): synthesis and RNA-selective hybridization of alpha-L-RNA/alpha-L-LNA chimera

Synthesis of the novel alpha-L-ribofuranosyl phosphoramidite derivative was accomplished via the alpha-L-ribofuranosyl thymine nucleoside. Amidite was used in automated syntheses of chimeric oligonucleotides composed of mixtures of the novel alpha-L-RNA nucleotide monomer ((alphaL)T, alpha-L-ribo configured RNA), and DNA, LNA (T(L), locked nucleic acid) or alpha-L-LNA ((alphaL)T(L), alpha-L-ribo configured locked nucleic acid) nucleotide monomers. For alpha-L-RNA/DNA and alpha-L-RNA/alpha-L-LNA chimeras, RNA-selective hybridization was obtained, for alpha-L-RNA/alpha-L-LNA chimera we found increased binding affinity compared to the corresponding DNA:RNA reference duplex. In addition, alpha-L-RNA/alpha-L-LNA chimera displayed significant stabilization towards 3'-exonucleolytic degradation. These results indicate that alpha-L-RNA/alpha-L-LNA chimeras deserve further evaluation as antisense molecules.     

Nucleic acids and protein content in relation to growth and regression of buffalo (Bubalus bubalis) corpora lutea

Information on nucleic acids and protein content of buffalo corpus luteum (CL) in relation to growth, development and regression is not available. An experiment was thus conducted to investigate the variation and relationship between nucleic acids and protein content in CL during different developmental stages and to determine the qualitative differences in protein constituents in any of these stages. Buffalo corpora lutea of different developmental stages viz., developing (day 5-10, n = 16), developed (day 11-17, n = 12) and regressed (day 18-21, n = 10) stages were collected from non-pregnant and -pathological genitalia (n = 38). The DNA, RNA and protein content in tissue extracts were determined and the proteins in pooled samples were analyzed by polyacrylamide gel electrophoresis. Developing stage CL had more total and per gram tissue level of DNA and RNA with significant positive relationship with total and per gram RNA and protein contents. Although there was no significant difference in total weight, a significant decrease in total DNA as well as per gram level of DNA and RNA was observed in developed stage compared to developing stage CL. The total protein content in developed stage CL was compared to developing and regressed stage CL. Non-denaturing PAGE analysis of CL proteins of different stages showed five protein bands of 210, 190, 82, 68 and 66 kDa and one that migrated with the dye front in all the stages however, not shown any differences in banding pattern. Denaturing PAGE showed 15 bands viz., 205, 66, 53, 42, 35, 27, 24, 22, 20, 18, 17, 14, 9, 7.5 and 6.5 kDa. Out of these 66 and 53 kDa bands appeared with maximum intensity in all the three stages of CL. Comparison of bands between the three stages revealed five 57, 31, 27, 19 and 16 kDa stage-specific bands in regressed stage CL. The present study indicated that the DNA, RNA and protein content of buffalo CL varied with the stages of development and regressed stage CL contained some unique protein bands which were not observed either in developed or developing stage CL.     

Nucleic acid indices of egg production in the tropical copepod Acartia sinjiensis

Two experiments were conducted to evaluate the effects of food and temperature on the nucleic acid content and egg production (and their relationship) of the tropical copepod Acartia sinjiensis. Experiment 1 evaluated the effect of food quality (as different algae species) on the relationship between nucleic acid content and egg production. In Experiment 2, the main and interaction effects of food type, food concentration and temperature on the total, Carbon and Nitrogen specific egg production and nucleic acid indices were evaluated in a factorial experimental design. Food quality, concentration and temperature significantly affected the nucleic acid content, egg production rate and the nucleic acid-egg production relationship of A. sinjiensis. RNA indices were correlated with egg production in females fed Pavlova salina, Tetraselmis chuii and Chaetoceros muelleri, but not in females fed Isochrysis aff. galbana. The slopes of the linear regressions of RNA indices as predictors of egg production were similar in females fed different algae species, suggesting that the slope of these relationships might be independent of food quality. The DNA content of females was significantly affected by food and temperature, suggesting that it is not a good index of cell number in this species. Nevertheless, the RNA:DNA ratio was as good a predictor of egg production as total RNA content. Egg production showed a weakly positive correlation with temperature. On the other hand, total, C- and N-specific nucleic acid indices had a strong negative correlation with temperature. In addition, temperature had a non linear effect on the slopes of the regression lines of RNA content and RNA:DNA ratio as predictors of egg production—slopes were similar at 25 °C and 30 °C, but significantly lower at 20 °C. Furthermore, the predictability of egg production was improved when the interaction term of nucleic acid indices with temperature was used instead of the nucleic acid indices alone in linear regression models. Our results suggest food quality has a limited influence on the nucleic acid-egg production relationship, and that temperature should be accounted for in models using nucleic acid indices as predictors of egg production in A. sinjiensis.     

Use of diphenylamine as a colorimetric reagent for ribonucleic acid

RNA has been demonstrated to react with diphenylamine when acid hydrolysis is performed for 1 hour or more at 100°C. This reaction can be used for quantitative analysis of RNA, since there is a linear relationship between RNA concentration and absorbance. The reaction of RNA with diphenylamine can be quanlitatively distinguished from the reaction of DNA: the absorption spectrum of the RNA-diphenylamine reaction product has a maximum at 650 mμ, and a second, smaller peak at 490 mμ, while the DNA-diphenylamine reaction product has a single maximum at 605 mμ. It was found that, when mixtures of DNA and RNA are reacted with diphenylamine, the spectra reflect both the DNA:RNA ratios and the total amounts of nucleic acids. When the two-wavelength method of spectrum analysis was applied to such spectra, good agreement was found between actual and calculated values of nucleic acid concentrations. In this way, diphenylamine can be used for the simultaneous determination of the concentrations of DNA and RNA in mixtures. As is the case for the reaction of DNA with diphenylamine, it was found that the reaction of RNA is not altered by the presence of protein and that it involves primarily the purine nucleotides. The reaction of RNA with diphenylamine is discussed in relation to its possible analytical applications.     

Action of inhibition by fluorodeoxyuridine and its reversal by thymidine on the nucleic acid fractions of the overground parts of photoperiodically inducedChenopodium rubrum plants

The nucleic acid fractions obtained by chromatography on MAK columns were compared in 4 variants ofChenopodium rubrum plants treated in different ways during floral induction. The first variant was normally induced to flowering. The second one was inhibited by application of FUDR to the apical bud on the third day of induction. In the third variant the inhibitory effect of FUDR was reversed by application of THY 24 h after FUDR treatment. In the fourth variant THY was applied 24 h after the termination of induction at a time when it was no longer able to reverse the inhibition of flowering. In plants treated with FUDR, a decrease in DNA and RNA synthesis was observed. After reversal of the inhibitory effect of FUDR by THY, DNA synthesis remained somewhat lower than in the control variant but RNA synthesis reached the same level as in the controls or even surpassed it. In plants to which THY was applied at a time when it was no longer possible to reverse flowering, the results obtained from different experiments were not identical. In some experiments nucleic acid synthesis remained lower than in the variant in which THY restituted flowering. In other experiments nucleic acid synthesis was fully restituted and reached the same level as in the control variant. This phenomenon is explained by the different size of the experimental plants at the beginning of the different experiments. The results are discussed with respect to the relation of the dynamics of nucleic acids to growth and development of the plants.