Juvenile Flounder Research Articles

A combined measurement of metal bioaccumulation and condition indices in juvenile European flounder, Platichthys flesus, from European estuaries

Condition indices and metal bioaccumulation of early life stages of juvenile flounder (5–10cm) were determined in three anthropogenic estuaries (the Scheldt, Seine and Loire) and compared to a reference site (the Canche). Significant correlations were found between metal concentrations in sediment and (i) fish liver for Cd, Cu, Ni, Pb, V and Zn and (ii) fish gills for Cd and Mn. Metal accumulation in juvenile flounder from the three anthropogenic estuaries coincided with significantly lower Fulton’s K indices (from 0.99±0.03 to 1.06±0.01mgmm−3) compared to those from the Canche estuary (from 1.02±0.01 to 1.13±0.01mgmm−3). This discrepancy in fish condition index increased with fish size and therefore, strongly depends on the time juvenile spend in estuary. Muscle lipid contents and Triacylglycerol to Sterol ratios were significantly lower in fish collected in the Scheldt (lipid content: 21.3±3.6%), Seine (17.9±19.8%) and Loire (19.5±2.4%) estuaries compared to those originating from the Canche (38.3±4.6%). This study highlights that combined measures of both fish metal contents and condition indices gives a relevant assessment of juvenile fish health growing in anthropogenic estuaries.

Cloning of the cDNA encoding adenosine 5′-monophosphate deaminase 1 and its mRNA expression in Japanese flounder Paralichthys olivaceus

AMP deaminase catalyzes the conversion of AMP into IMP and ammonia. In the present study, a full-length cDNA of AMPD1 from skeletal muscle of Japanese flounder Paralichthys olivaceus was cloned and characterized. The 2 526 bp cDNA contains a 5′-UTR of 78 bp, a 3′-UTR of 237 bp and an open reading frame (ORF) of 2 211 bp, which encodes a protein of 736 amino acids. The predicted protein contains a highly conserved AMP deaminase motif (SLSTDDP) and an ATP-binding site sequence (EPLMEEYAIAAQVFK). Phylogenetic analysis showed that the AMPD1 and AMPD3 genes originate from the same branch, but are evolutionarily distant from the AMPD2 gene. RT-PCR showed that the flounder AMPD1 gene was expressed only in skeletal muscle. QRT-PCR analysis revealed a statistically significant 2.54 fold higher level of AMPD1 mRNA in adult muscle (750±40 g) compared with juvenile muscle (7.5±2 g) (P<0.05). HPLC analysis showed that the IMP content in adult muscle (3.35±0.21 mg/g) was also statistically significantly higher than in juvenile muscle (1.08±0.04 mg/g) (P<0.05). There is a direct relationship between the AMPD1 gene expression level and IMP content in the skeletal muscle of juvenile and adult flounders. These results may provide useful information for quality improvement and molecular breeding of aquatic animals.

김(Porphyra yezoensis)에서 추출한 당단백질의 사료내 첨가가 넙치(Paralichthys olivaceus) 치어 성장 및 Edwardsiella tarda 저항성에 미치는 영향

This study examined the effects of dietary glycoprotein extracted from Porphyra yezoensi on growth performance and resistance against the pathogenic bacteria Edwardsiella tarda in olive flounder. A porphyra-originated glycoprotein (P) was extracted using sequential processes of water and ethanol treatment. P extracts were added to a fish-meal-based diet at concentrations of 0.0, 0.5, and 1.0% (designated as Con, , and , respectively). Fish were fed one of the three experimental diets for 10 weeks. All fish groups exhibited over 96.7% survival during the experimental period. Results indicated that the fish fed diets containing P showed an increase in growth performance, including enhanced weight gain, specific growth rate, and feed efficiency. An increase in insulin-like growth factor (IGF-1) was observed in the fish fed the diet, as compared to those fed Con. At the end of the 10-week feeding trial, all fish were infected with E. tarda, and accumulated mortality was monitored for 8 days. Fish fed the Con diet exhibited increasing mortality from day 3 to the end of the challenge test, whereas the mortality of P-fed fish ceased at day 5. We suggest that supplementation with P-originated glycoprotein in aquafeed may increase growth performance and resistance against pathogenic bacteria in olive flounder juveniles.

Genetically similar VHSV isolates are differentially virulent in olive flounder Paralichthys olivaceus

Two viral hemorrhagic septicemia virus (VHSV) isolates, VHSV-KR-CJA and VHSV-KR-YGH, were isolated from viral hemorrhagic septicemia disease outbreaks in flounder farms in South Korea. The VHSV-KR-CJA isolate was isolated from a flounder farm with high mortality (80%), while the VHSV-KR-YGH isolate was isolated from a flounder farm with low mortality (15%), suggesting that these isolates differ in virulence. The virulence of these isolates was evaluated in juvenile flounder via intraperitoneal injection. Consistent with their virulence in the field, mortality data revealed that the VHSV-KR-CJA isolate was highly pathogenic (cumulative mortality of 80%), while the VHSV-KR-YGH isolate was less pathogenic in flounder (cumulative mortality of 20%). To characterize the genotypes of these viruses, the full open reading frames (ORFs) encoding nucleoprotein N, phosphoprotein P, matrix protein M, glycoprotein G, nonstructural viral protein NV, and polymerase L of these viruses were sequenced and analyzed. Sequence analysis revealed that both isolates are genetically very similar (identical amino acid sequences for P, M, NV, and L and >99.7 and 99.8% amino acid sequence identity for N and G, respectively). Phylogenetic analysis indicated that both of these viruses belong to the Genotype IVa group, suggesting that they originated from a common ancestral virus. The low pathogenicity VHSV strain may potentially evolve to become a more pathogenic strain through only a few nucleotide substitutions. Further functional analyses of mutations in VHSV genes are necessary to identify factors that determine VHSV pathogenicity in flounder.

Responses of juvenile European flounder (Platichthys flesus) to multistress in the Vilaine estuary, during a 6-month survey

Physiological and genetic responses of age 0+ Platichthys flesus were investigated in the eutrophicated and moderately contaminated Vilaine estuary, during a 6-month survey. The main objective of this study was to explore the biological responses of fishes during their juvenile period in an estuarine system in order to detect a possible selective pressure induced by the environmental stress. Our results showed a general convergence in physiological responses along the survey: an increase in genotoxicity was associated with an increase in mRNA expression of ATPase and betaine homocysteine methyltransferase. These results could suggest an increase of cellular damage, energetic request, and detoxification rate related to the growing exposure time to stress. Considering the aging of the cohort, the genetic characteristics of the Vilaine flounder cohort came closer to the one observed in a highly stressed system, the Seine estuary, suggesting a potential selective pressure mainly induced by the chemical stress.

Tissue-specific bioaccumulation and oxidative stress responses in juvenile Japanese flounder (Paralichthys olivaceus) exposed to mercury

To understand mercury (Hg) toxicity in marine fish, we measured Hg accumulation in juvenile Japanese flounder (Paralichthys olivaceus) and assessed the effects on growth and antioxidant responses. After Hg exposure (control, 5, 40, and 160 μg/L Hg) for 28 d, fish growth was significantly reduced. The accumulation of Hg in fish was dose-dependent and tissue-specific, with the maximum accumulation in kidney and liver, followed by gills, bone, and muscle. Different antioxidants responded differently to Hg exposure to cope with the induction of lipid peroxidation (LPO), which was also tissue-specific and dosedependent. As Hg concentration increased, superoxide dismutase (SOD) and catalase (CAT) activities increased significantly, whereas glutathione S -transferase (GST) activity and glutathione (GSH) levels decreased significantly in the gills. SOD and glutathione peroxidase (GPx) activities and the GSH level increased significantly in the liver. SOD activity and GSH levels increased significantly, but CAT activity decreased significantly with an increase in Hg concentration in the kidney. LPO was induced significantly by elevated Hg in the gills and kidney but was least affected in the liver. Therefore, oxidative stress biomarkers in gills were more sensitive than those in the liver and kidney to Hg exposure. Thus, the gills have potential as bioindicators for evaluating Hg toxicity in juvenile flounder.

Comparisons of liver proteomes in the European flounder Platichthys flesus from three contrasted estuaries

Estuaries are important areas highly susceptible to anthropogenic degradations like pollution. Estuarine species have thus to cope with many types of constraints depending on the estuaries' characteristics. The European flounder Platichthys flesus is considered as a sentinel species for the monitoring of estuarine water quality. In this study, juvenile flounders (0+ group) were sampled from three contrasted Channel estuaries, i.e. the Seine, the Canche and the Tamar, and we characterized their liver proteomes by using a two-dimensional electrophoresis based proteomic approach.We showed that 27 protein spots differentially accumulated between the 3 populations. Six of these proteins were identified by MALDI TOF–TOF mass spectrometry. Flounders from the Seine and from the Tamar, two highly polluted estuaries, displayed common differences, i.e. an increase of the energetic- and the glutathione-metabolism. The most accumulated protein in the Seine's samples (6.7-fold) was a Vitelline Membrane Outer layer protein 1 homolog, suggesting oogenesis deregulation in these juvenile (sexually immature) flounders. Future works applying this kind of proteomic approach on flounders experimentally exposed to conditions that mimic environmental constraints will help to better understand the significance of these environmental proteomic signatures.

The Effects of Composite Enzymes in Diets on Growth, Carcass Composition and Specific Enzyme Activities of Juvenile &lt;i&gt;Paralichthys lethostigma&lt;/i&gt;

The study was conducted to investigate the effects of commercial composite enzymes (CE) on growth, carcass composition and specific enzyme activities of juvenile flounder Paralichthys lethostigma. Four diets were formulated: one diet contained no CE (the control), the three others provided graded levels of CE (0.5%, 1% and 1.5%). Each diet was fed to pentaplicate of flounder juveniles for 28 days in aquaria (50×40×30 cm, water volume 50 L). The growth parameters (final wet weight, specific growth rate: SGR, feed conversion rate: FCR and apparent digestibility: AD) were significantly affected (P0.05). The CE had significant effects (P&lt;0.05) on the specific activities of neural protease, alkine protease and amylase, but not on acid protease, amimopeptidase and alkine phosphatase. The pattern of the specific activities of neural protease, alkine protease and amylase was similar to that of SGR with fish fed diets containing 1.5% of CE exhibited the significantly higher activities (P&lt;0.05). The results demonstrated that the supplement of CE in diets could significantly improve growth performance and digestive enzyme activities of flounder juveniles.

Response to “Comment on ‘Otolith Microchemistry Reveals Substantial Use of Freshwater by Southern Flounder in the Northern Gulf of Mexico’” by Pedro Morais

Lowe et al. (Estuar Coast 34:630–639, 2011) used Sr/Ca in the otoliths of juvenile southern flounder (Paralichthys lethostigma) to demonstrate the importance of low-salinity habitats in the northern Gulf of Mexico during early life. While otolith cores indicated that most southern flounder originated in high-salinity environments (elevated Sr/Ca in otolith core) and moved into lower salinity habitats (decreased Sr/Ca outside of the otolith core), approximately one third of the sampled individuals had otolith core chemistry that indicated spawning or hatching in or near freshwater habitats (low Sr/Ca throughout the otolith). Morais (Estuar Coast 34:630–639, 2012) proposes a different interpretation: he suggests that the high Sr/Ca found in 68 % of juvenile otolith cores (and low Sr/Ca just outside of the core region) may be due to females hydrating their eggs in offshore (high-salinity) waters, with spawning, hatching, and egg/larval development all actually occurring in low-salinity environments (his “sea-run mother” hypothesis). While there is ample literature supporting the importance of maternal contributions to otolith microchemistry during vitellogenesis, it is our contention that such contributions ultimately depend on the ecology and reproductive physiology of each species. Further, a mass inshore migration of adult southern flounder during their spawning season is counter to the well-documented life history patterns for this species throughout its range. While we appreciate Morais' perspective, we continue to stand by the conclusion of our original paper.

Influence of temperature on clove oil anaesthesia in flounder (Platichthys flesus Linnaeus, 1758)

Summary The efficacy of clove oil as an anaesthetic in juvenile flounder was evaluated at two temperatures (10–12 and 16–18 °C) and the effective concentration determined. Flounder juveniles with a body length of 9.0–10.2 cm and a weight of 6–14 g were individually exposed to clove oil at concentrations ranging from 200 to 1000 mg L−1 in a 5-L bath with three replicates per treatment with 10 fish. The induction and recovery times were recorded in seconds for all doses and temperatures. Temperature had no effect on induction, but higher temperature shortened the recovery time. The effective concentration (EC50) was estimated as 753 (692–820) mg L−1 for ≤3 min with 95% confidence limits. The study demonstrated that clove oil can be used as an effective anaesthetic in juvenile flounder and that temperature has an effect on recovery time from anaesthesia with clove oil.

Comments on Lowe et al. “Otolith Microchemistry Reveals Substantial Use of Freshwater by Southern Flounder in the Northern Gulf of Mexico”

Lowe et al. (Estuaries and Coasts, 34:630–639, 2011) hypothesized that juvenile southern flounder Paralichthys lethostigma (Jordan and Gilbert 1884) would migrate from the Gulf of Mexico into the Mobile-Tensaw River Delta (AL, USA) and use low-salinity (oligohaline/freshwater) habitats during, at least, a portion of their first year of life. Thus, they analyzed the Sr/Ca ratio profiles along the sagittal otoliths of southern flounder collected in the Mobile-Tensaw River Delta and observed that one third of the flounders had low Sr/Ca levels in the otoliths’ core and throughout the otolith, suggesting that these fishes hatched in freshwater or low-salinity habitats where they spend the majority of their life. The other two thirds of southern flounder showed high levels of Sr/Ca ratio in the otoliths’ core following a marked decline of Sr/Ca ratio, which then maintained along the remainder of the otolith. This pattern was interpreted as larvae hatched in higher salinity waters before entering the Mobile-Tensaw River Delta; however, in this paper, I list several arguments to support an alternative interpretation for this pattern. I suggest that the high levels of Sr/Ca ratios in the otoliths’ core of southern flounder does not reflect the saline conditions where larvae hatched, instead it reflects the location where the female progenitor hydrated the eggs. Thus, adding my interpretation on the data of Lowe et al. (Estuaries and Coasts, 34:630–639, 2011), it seems that southern flounder might hatch in or near freshwater habitats and the migration of southern flounder into an estuarine ecosystem to spawn might exist.

Metal concentrations, growth and condition indices in European juvenile flounder (Platichthys flesus) relative to sediment contamination levels in four Eastern English Channel estuaries

The aim of the present study was to evaluate the effects of metal contamination on the biological responses of 0-group juvenile European flounder and to assess and compare the quality of four estuarine habitats located in the Eastern English Channel. Fish otolith growth and condition indices (RNA : DNA ratio, Fulton's K condition index) were measured and found to be significantly lower in individuals from the Seine estuary compared to those of the Canche, Authie and Somme estuaries. No obvious effects of hydrological condition or food availability on the flounder biological responses were observed. Sediments from the Seine showed the highest metal concentrations, bioavailable proportion and enrichment factors. Higher metal concentrations were observed in fish from the Seine compared to the other ones caught in less polluted estuaries. These results suggest that contaminants may have a negative impact on the early life history stage of flounder.

SCALES OF VARIABILITY IN FIN RAY COUNTS OF FLOUNDER, PLATICHTHYS FLESUS L. ON IRISH AND WELSH COASTS

Meristic variation within a fish species can arise due to environmental factors and can aid investigation between fish stocks and closely related juvenile fish species. The aim of this study was to investigate the spatial variation in meristic characteristics for juvenile flounder Platichthys flesus from Irish and Welsh coasts. Five meristic fin ray counts were taken from 120 fish from nursery grounds in three regions (west Irish coast, east Irish coast and Welsh coast). Statistical analysis revealed regional variability for two of the meristic counts (dorsal and anal fin rays). A discriminant function analysis revealed a weak separation of the three regions. This separation improved when samples from the east Irish region were eliminated, with 65% of the fish from the Irish west coast and 73% of the fish from Wales correctly classified.

Effect of Dietary Starch Level and Kind on the Growth and Body Composition of Juvenile Olive Flounder Paralichthys olivaceus

A 7-week feeding trial was conducted to investigate the effect of dietary starch level and kind on the growth and body composition of juvenile olive flounder. Triplicate groups of fish (average weight: 1.5 g) were fed iso-nitrogenous (48% crude protein) and isocaloric (4.8 kcal/g diet) diets containing 15-25% <TEX>${\alpha}$</TEX>-potato starch and 15% <TEX>${\beta}$</TEX>-potato starch. Survival was not affected by dietary starch level and kind. The weight gain of fish fed the diet containing 20% <TEX>${\alpha}$</TEX>-potato starch was significantly higher than that of fish fed the diets containing 15% and 25% <TEX>${\alpha}$</TEX>-potato starch levels. The feed efficiency and protein efficiency ratios of fish fed the diets containing 15% <TEX>${\beta}$</TEX>-potato starch were significantly lower than those of the other groups (P < 0.05). The protein efficiency ratio tended to increase with increasing <TEX>${\alpha}$</TEX>-potato starch. The daily feed intake of fish fed the diet containing 15% <TEX>${\beta}$</TEX>-potato starch was significantly higher than that of the other groups (P < 0.05). The hepatosomatic index, condition factor, and proximate composition of the whole body were not affected by the dietary starch level and kind. These results indicate that up to 20% <TEX>${\alpha}$</TEX>-potato starch could be incorporated into the juvenile flounder diet for optimum growth.

Tissue-specific accumulation of cadmium and its effects on antioxidative responses in Japanese flounder juveniles

This study investigated the accumulation of cadmium (0–8 mg Cd L −1) and its toxicological effects on oxidative stress biomarkers in different tissues of Japanese flounder juveniles. Following Cd exposure for 28 d, accumulation of Cd in fish was dose-dependent and tissue-specific, with the greatest accumulation in the liver, followed by the kidney, gill, and muscle. Although the gill and liver mounted active antioxidant responses at ≥4 mg L −1 Cd including a decrease in glutathione level and GST and GPx activities, the antioxidant response failed to prevent lipid peroxidation induction in these organs. In the kidney, increased GPx and GST activities and decreased SOD activity were observed in fish exposed to high Cd concentrations, but LPO levels did not significantly differ among the exposure concentrations. The gill was most sensitive to oxidative damage, followed by the liver; the kidney was the least affected tissue.

넙치 치어 Paralichthys olivaceus의 혈액학적 성상에 미치는 나프탈렌의 급성독성영향

Naphthalene was composed of a substantial fraction of polycyclic aromatic ydrocarbons(PAHs) in crude oil and causes acute toxicity. In this study, we examined the toxicity of different kinds concentrations 0, 1000, 1800, 3200, 5600, of naphthalene to juvenile flounder, Paralichthys olivaceus for 24h to determine 24-median lethal concentration() and acute effect on the hematological properties. 24h- value of this species was . Hematocrit value significantly increased at 5600 and naphthalene exposed group by 24h compared to control fish. Plasma. Glucose was significantly higher in the (P. Plasma [] and [] significantly increased in the 5600 and , however [] was not affected by acute naphthalene exposure. The results of this study suggest the acute exposure to naphthalene affects both ionoregulation and osmoregulation in juvenile flounder.

Feeding habits of juvenile slime flounder Microstomus achne in the coastal area of southern Hokkaido

A total of 45 juvenile [30.0–57.4 mm total length (TL)] slime flounder Microstomus achne were collected in the coastal area of southern Hokkaido from April to July in 2001 and April to June in 2002. Their diets were analyzed. Slime flounder juveniles of 30.0–39.9 mm TL fed predominantly on small crustaceans (gammarid amphipods, harpacticoids and cumaceans) and those of 40.0–57.4 mm TL on gammarid amphipods, cumaceans and polychaetes. The major prey items changed with growth from small crustaceans (e.g., harpacticoids) to polychaetes, although gammarid amphipods were the major prey items throughout the juvenile period (30.0–57.4 mm TL).

배합사료의 다양한 첨가제가 넙치(Paralichthys olivaceus) 치어의 성장, 혈액 성상, 지방산 조성, 유전자 발현 및 조직 성상에 미치는 영향

【This feeding experiment was conducted to investigate the effects of dietary inclusion of various additives on growth performance, hematological parameters, fatty acid composition, gene expression and histopathological changes in juvenile olive flounder (Paralichthys olivaceus). Eleven isonitrogenous (49% crude protein) and isolipidic (10% crude lipid) experimental diets were formulated: no additives (Con); 5% kelp meal (Ke); 10% krill meal (Kr); 1% garlic powder (Ga); 1% citrus meal (Ci); 3% onion powder (On); 1% ginger powder (Gi); 1% mugwort powder (Mu); 1% licorice powder (Li); 1% wasabi powder (Wa); and a mixture (Mix) of these additives. Three replicate groups of juvenile flounder (average weight of 8.5 g) were fed one of the experimental diets to visual satiety twice a day for 15 weeks. The dietary inclusion of additives did not affect survival, weight gain, specific growth rate feed efficiency, daily feed intake, daily protein intake, protein efficiency ratio, hepatosomatic index and visceralsomatic index of the fish. Plasma triglyceride levels were significantly lower in fish fed the Ke, Ga, On, Gi, Mu, Li, and Mix diets than in fish fed the control diet. Plasma glucose, glutamic oxaloacetic transaminase and total cholesterol did not differ among dietary treatments. No significant difference was observed in fatty acid composition and lipid content of the dorsal muscle in fish fed the experimental diets. Myosin gene expression did not differ significantly among treatments after 5 weeks but was significantly lower in fish fed the Kr, Ci, Li, and Mix diets than in control group after 15 weeks. Histopathological analysis showed mild gill hyperplasia and mild necrosis of liver parenchymal cells in several individuals of each experimental group. These conditions were also observed in the control group and were not thought to be related to the inclusion of feed additives. The present findings indicate that the dietary inclusion of additives did not affect growth performance, fatty acid composition, gene expression, and histopathological changes in juvenile flounder. However, plasma triglyceride content may be reduced by supplementation with 5% kelp meal, 3% onion powder, 1% garlic powder, 1% ginger powder, 1% mugwort powder, and the additive mixture.】

Growth and Lipid Composition of Winter Flounder Juveniles Reared under Natural and Fixed Photoperiod and Temperature Conditions

AbstractIn age‐0 winter flounder Pseudopleuronectes americanus, a fixed long photoperiod (14.5 h light: 9.5 h dark) applied during the first 45 d postsettlement did not improve growth compared with fish held under natural photoperiod conditions. After being reared under these two regimes until 29 October, juveniles exposed to the long photoperiod after settlement were maintained under the same conditions during the winter, but temperature was not allowed to decrease below 4°C. Juveniles previously exposed to the natural photoperiod were maintained under one of the following conditions: (1) natural photoperiod and natural temperature (1–9°C), (2) natural photoperiod and a minimum winter temperature of 4°C, or (3) long photoperiod and a minimum temperature of 4°C. Most mortality (80%) occurred within the first 2 months of the experiment. Juveniles that experienced the transition from natural to long photoperiod conditions displayed higher growth through the winter than did juveniles exposed to natural conditions; at the end of winter, fish that were transitioned to the long photoperiod were 25% longer (19 mm), were twice as heavy (125 mg), and contained twice as much total lipids (803 μg/mg) and five times more triacylglycerols (24% of total lipids) than juveniles subjected to the natural photoperiod. These results indicate that we may be able to eliminate the winter fasting that occurs under natural conditions and to elicit winter growth in this species by using photoperiod manipulation. In addition, it appears that a decrease in photoperiod is needed for fish to respond to a subsequent increase in photoperiod.Received November 3, 2009; accepted May 27, 2010

First description and collection records of juvenile slime flounder Microstomus achne from the eastern and western coastal waters off Honshu Island, Japan

Morphological characteristics and body pigmentations of juvenile slime flounder Microstomus achne were described from one specimen [27.8 mm total length (TL)] collected off Niigata Prefecture from the Sea of Japan (35 m depth) in May 1993 and from three specimens (30.1, 36.2, and 40.9 mm TL) collected off Fukushima Prefecture on the Pacific coast of northern Japan (15 and 50 m depths) during April–May 2008 and 2009. These age-0 juveniles had a characteristically slenderly oval-shaped body and slightly protruded front nostrils. Sparsely distributed melanophores and xanthophores were clearly visible throughout the ocular side of one specimen, which was judged as a newly settled juvenile, whereas several spots aggregated by small melanophores were found in the others with elongated pectoral fins and small cycloid scales. Melanophores were only slightly detectable on the blind side. Comparisons of the depth distributions of slime flounder juveniles and sympatrically collected age-0 pleuronectids (Pseudopleuronectes herzensteini, Pseudopleuronectes yokohamae, Platichthys bicoloratus, and Dexistes rikuzenius) suggested that slime flounder can settle on broad nursery areas of coastal and offshore pleuronectid flatfishes, probably depending on local oceanic conditions during the protracted pelagic phase.