High water-temperature tolerance in photosynthetic activity of <Emphasis Type="Italic">Zostera marina</Emphasis> seedlings from Ise Bay,Mie Prefecture,central Japan

Photosynthetic activities of seedlings of Zostera marina were successively measured using a gas volumeter for 6 days at seven light (0–400 μmol photons/m² per s) and 11 water temperature conditions (5–35°C). The seedlings were collected from mature plants (Ise Bay, central Japan), and stored and cultured in incubators accurately controlled at each test temperature. The maximum gross photosynthesis (P _maxg) was recorded at an optimal water temperature of 29°C after 0 days. After 6 days, P _maxg appeared at 25°C and most plants cultured at 29–30°C bleached and withered after the drastic increase of light compensation point (I _c). On the contrary, at 5–28°C, the photosynthetic activities either changed little (5–25°C) or recovered after a temporal reduction (26–28°C); seedlings survived and looked healthy after being cultured for 6 days. The recovery was thought to be an acclimation to tolerate higher water temperature. As a result, the critical upper water temperature for Z. marina seedlings was proposed as 28°C. The temperature was consistent with the previously reported maximum water temperature in habitats around the southern boundary of Z. marina in the northern hemisphere.     

Cryptic occurrence of <Emphasis Type="Italic">Chattonella marina</Emphasis> var. <Emphasis Type="Italic">marina</Emphasis> in mangrove sediments in Probolinggo,East Java Province,Indonesia

Mangrove forests and adjacent creeks are known to be highly productive estuaries, which are partly supported by benthic microalgae that grow on the sediments. During surveys investigating the microalgal floras of mangrove swamps in the eastern part of Java Island, a mud sample unexpectedly included large numbers of a notorious fish killer, Chattonella-like motile cells, and its resting cysts. These motile cells were established as clonal cultures for further identification and physiological tests. The cysts were examined through palynological and molecular biological means. Identification based on light microscopy and ribosomal RNA gene sequences confirmed that these cells and cysts were Chattonella marina var. marina. While the strains were genetically identical to the temperate strains isolated from Japan and China, temperature experiments showed that the Indonesian strains possessed a high maximum quantum yield of photosystem II even after exposure to 34 °C, a temperature at which the Japanese strain could not survive. Salinity experiments showed adaptation of the strains to a salinity of 15. These findings, together with the discovery of populations of cysts in the mangrove sediment, highlight the tough and unique nature of the Indonesian strains, which are likely adapted to wide fluctuations of temperature and salinity in mangrove swamps, and pose a potential risk to fisheries in Indonesia.     

Relationship between light and diel vertical migration of <Emphasis Type="Italic">Diaphus theta</Emphasis> and <Emphasis Type="Italic">Euphausia pacifica</Emphasis> off eastern Hokkaido

Underwater irradiance in sound-scattering layers (SSLs) during diel vertical migration (DVM) of marine organisms was investigated by a drifting vessel at locations in the western North Pacific during the period August 24–27, 2008. The inhabitants of the layers were identified via sampling as Diaphus theta, a type of myctophid fish, and Euphausia pacifica, a species of zooplankton. Underwater irradiance was estimated by the Beer–Lambert law using irradiance measurements on the deck and the diffuse attenuation coefficients obtained in the water. During DVM, the mean irradiances at 490 nm at the lower and upper boundaries of the SSL composed of E. pacifica were ?54.0 and ?45.3 dB, respectively. In contrast, the values for the boundaries of the D. theta SSL were ?82.5 and ?59.3 dB, respectively. It was possible to distinguish between D. theta and E. pacifica based on these ranges and the differences between the mean volume-backscattering strength (ΔMVBS) at 120 and 38 kHz. It was also possible to distinguish between D. theta/E. pacifica and other causes of scattering more clearly using these two parameters together than when using the ΔMVBS parameters separately. Our results suggest that underwater irradiance is an important parameter for discriminating among organisms in DVM.     

The structural and histochemical analyses and chemical characters of the cuticle and epidermal walls of cotyledon in ungerminated seeds of <Emphasis Type="Italic">Zostera marina</Emphasis> L.

How Zostera marina L. adapts to environmental stresses is of major interest to researchers wanting to obtain a better understanding of how to preserve this ecologically important seagrass. We have examined the structure and chemical properties of the cuticle and epidermal walls of Z. marina cotyledons at the pre-germination stage by microscopy, histochemistry, and chemical analyses. The epidermal cells, which have a smooth plasma membrane and slight wall-ingrowth, are surrounded by thickened outer tangential walls consisting of cellulose and pectic substances. The thick cuticle layer covers the outer surface of the outer tangential walls; the former were observed here to be much thicker than has been reported earlier for the leaves and sheaths of Z. marina. Chemical analysis of the isolated cuticle by attenuated total reflectance–infrared Fourier transform spectrometry and gas chromatography–mass spectrometry detected C₁₆ and C₁₈ fatty acids, ω-hydroxy fatty acids and β-sitosterol as components of the wax and cutin. The outermost cuticle layer was also observed to be covered with a slimy layer consisting of some polysaccharides. These results suggest that the extremely thick cuticle and slimy layer could play a significant role in protecting the cotyledons from environmental stresses at the pre-emergence stage and just after emergence.     

Genetic difference between Ezo-awabi <Emphasis Type="Italic">Haliotis discus hannai</Emphasis> and Kuro-awabi <Emphasis Type="Italic">H. discus discus</Emphasis> populations: Microsatellite-based population analysis in Japanese abalone

ABSTRACT:   This study documents the genetic population relationships between Ezo-awabi Haliotis discus hannai and Kuro-awabi H. discus discus by means of the microsatellite technique with an emphasis on the extent of genetic difference between Ezo- and Kuro-awabi populations. Eight markers were employed to screen five populations each collected from Ezo- and Kuro-awabi habitats. All eight loci showed polymorphisms in all populations (number of alleles per locus, 11.1–14.1; averaged expected heterozygosity, 0.64–0.70). The number of loci that yielded significant genetic heterogeneities (allele frequency distribution and/or pairwise θ ) between Ezo- and Kuro-awabi populations was larger than that generated in comparisons between populations within Ezo- and Kuro-awabi habitats. According to the bootstrap neighbor-joining trees constructed on the basis of two genetic distance measures ( D _A and D _ST), the 10 populations fell into two clusters of populations (Ezo-awabi and Kuro-awabi habitat groups), and the results of hierarchical amova testing also supported the neighbor-joining clustering. The outcomes presented here suggest that the microsatellite markers used in this study are potentially an efficient means to differentiate Ezo- and Kuro-awabi populations.     

The carbohydrate catabolism of <Emphasis Type="Italic">Zostera marina</Emphasis> influenced by lower salinity during the pre-germination stage

The distribution of starch granules in ungerminated seeds of Zostera marina was examined by electron microscopy and histochemistry. Quantitative changes during germination in starch and sugars [glucose/maltosaccharides (MS), sucrose, and fructose] were examined in the seeds using biochemical methods. At natural seawater salinity (30.2 psu), the number of starch granules and the amount of starch markedly decreased, resulting in no increase in the sugar content in the embryos during the early germination stage. A substantial increase in sugars in the shoots occurred after the development of true leaves, probably due to photosynthetic activity in the green true leaves. At natural seawater salinity, the amount of starch did not significantly change during the initial 14 days of germination. In contrast, at lower salinities, starch levels decreased significantly from day 0 to day 6, resulting in higher levels of glucose/MS and fructose in the basal hypocotyls. These results suggest that compared with the salinity of natural seawater, lower salinity may interfere with starch catabolism in seeds, thereby causing the seed coat to break open earlier.     

Differences in longitudinal distribution patterns along a Honshu stream of brown trout <Emphasis Type="Italic">Salmo trutta</Emphasis>, white-spotted charr <Emphasis Type="Italic">Salvelinus leucomaenis</Emphasis> and masu salmon <Emphasis Type="Italic">Oncorhynchus masou</Emphasis>

Brown trout Salmo trutta were first introduced into Japan in 1892, and they currently naturally reproduce in several rivers in Honshu and Hokkaido, Japan. Although negative impacts of brown trout introductions on native salmonid fishes have been documented in some Hokkaido rivers, studies of ecological interactions between brown trout and native salmonid fishes on Honshu are limited. In this study, we describe the longitudinal distribution patterns of introduced brown trout, white-spotted charr Salvelinus leucomaenis and masu salmon Oncorhynchus masou in a 4 km stretch of a stream in central Honshu. Underwater observations were conducted in all pools within upstream, middle and downstream sections (190–400 m in length) of this stretch in order to estimate the densities of these species. Only white-spotted charr was observed in the upstream section, while brown trout and masu salmon were observed in the middle and downstream sections. Masu salmon densities, however, were much lower than brown trout densities. In the downstream section, white-spotted charr was absent. These results are consistent with results from previous studies of Hokkaido rivers, where it was found that white-spotted charr in low-gradient areas tend to be displaced by brown trout.     

Spatial learning-induced <Emphasis Type="Italic">egr</Emphasis>-<Emphasis Type="Italic">1</Emphasis> expression in telencephalon of gold fish <Emphasis Type="Italic">Carassius auratus</Emphasis>

The immediate-early gene (egr-1) expression was used to examine the neuron’s response in telencephalon of goldfish during spatial learning in small space. Fishes were pre-exposed in the experimental apparatus and trained to pick food from the tray in a rectangular-shaped arena. The apparatus was divided into identical compartments comprising three gates to provide different spatial tasks. After the fish learned to pass through the gate one, two more gates were introduced one by one. Fish made more number of attempts and took longer time (P < 0.05) to pass through the first gate than the gate two or three. This active learning induces the expression of egr-1 in telencephalon as established by western blot analysis. Subsequently, the fish learn quickly to cross the similar type of second and third gate and make fewer errors with a corresponding decline in the level of egr-1 expression. As the fish learned to pass through all the three gates, third gate was replaced by modified gate three. Interestingly, the level of egr-1 expression increased again, when the fish exhibit a high exploratory behavior to cross the modified gate three. The present study shows that egr-1 expression is induced in the telencephalon of goldfish while intensively acquiring geometric spatial information to pass through the gates.     

Genetic population structure of <Emphasis Type="Italic">Nibea albiflora</Emphasis> in Yellow Sea and East China Sea

ABSTRACT:   The population genetic structure and level of gene flow of Nibea albiflora from the Yellow Sea and the East China Sea were examined with a 479-bp segment of a mtDNA control region. In total, 65 samples were collected from three locations and 37 haplotypes were obtained. Mean haplotype diversity and nucleotide diversity for the three populations ranged from 0.9130 ± 0.0308 (Zhoushan) to 0.9926 ± 0.0230 (Xiamen), and from 0.0073 ± 0.0043 (Qingdao) to 0.0099 ± 0.0057 (Xiamen). Analysis of molecular variance and pairwise F _ST revealed little genetic structure between the Yellow Sea and the East China Sea in N. albiflora . But based on the exact test of differentiation, the null hypothesis that N. albiflora within the Yellow Sea and the East China Sea constitutes a panmictic mtDNA gene pool was rejected. This might be caused by the broad spawning areas but not by the Yangtze River outflow. Mismatch distribution revealed that N. albiflora has undergone population expansion, possibly before the last 85 000–170 000 years. The existence of high gene flow between stocks in the studied area was supported by our results. Annual migrations, larval drift in the ocean currents, and recent range expansion could be the reasons for little genetic structure in the studied area.     

Characterization of <Emphasis Type="Italic">kiss2</Emphasis> and <Emphasis Type="Italic">kissr2</Emphasis> genes and the regulation of kisspeptin on the HPG axis in <Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>

Reproduction allows organisms to produce offspring. Animals shift from immature juveniles into mature adults and become capable of sexual reproduction during puberty, which culminates in the first spermiation and sperm hydration or ovulation. Reproduction is closely related to the precise control of the hypothalamic–pituitary–gonadal (HPG) axis. Kisspeptin peptides are considered as the important regulator of HPG axis in mammalian. However, the current understanding of kisspeptin in flatfish is not comprehensive. In this study, we cloned and analyzed the kiss2 and kissr2 genes in Cynoglossus semilaevis. Interesting alternative splicing in the 5′-untranslated regions (UTR) of the Cskissr2 gene was found. The expression profiles of Cskiss2 and Cskissr2 showed relative high messenger RNA (mRNA) levels at the late gastrula stage during embryonic development, at total length = 40 mm during early gonadal differentiation, and in the brains and gonads of all investigated tissues. These results suggested that the kisspeptin system participated in embryogenesis and in the regulation of gonadal differentiation and development. Considering that the control and regulatory mechanisms of kisspeptin in the central reproductive axis are still unclear, we documented that the intramuscular injection of kisspeptin caused different sGnRH and cGnRH mRNA levels in a dose- and tissue-dependent manner. The mRNA expressions of FSH and LH were stimulated in the ovary and were inhibited in the testis under the kisspeptin treatments. These results provided foundations for understanding the roles of kisspeptin in the neuroendocrine system in fish. The manipulation of the kisspeptin system may provide new opportunities to control the gonadal development and even reproduction in fish.     

Monitoring of <Emphasis Type="Italic">Francisella noatunensis</Emphasis> subsp. <Emphasis Type="Italic">orientalis</Emphasis> in farmed Nile tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>) in Brazil

Francisella noatunensis orientalis is a bacterium that causes emerging bacteriosis in Nile tilapia (Oreochromis niloticus) in many parts of the world, including Brazil. It is a non-motile, Gram-negative, strictly aerobic, facultative intracellular coccobacillus. This species of bacteria is responsible for low to high mortality in fish farms, causing economic losses for fish farmers. This study aimed to detect the presence of F. noatunensis orientalis using qPCR (real-time polymerase chain reaction) and to describe lesions caused by the bacterium in O. niloticus in Brazilian aquaculture. For this purpose, 360 fish from six fish farms (30 per farm) were sampled at two time points (n = 180 per sampling). Necropsies and histopathology were performed for lesion observation, in addition to qPCR and sequencing for detection and identification of Francisella species. Environmental data were collected using a multiparameter sonde YSI EXO2. All measured limnological variables were within the optimum range for cultivation of Nile tilapia. The major lesions present were melanization of the skin, splenomegaly, granulomas, and inflammatory cell responses. The prevalence of francisellosis varied from 0 to 86.66% between time periods and fish farms analyzed, and an outbreak was observed during the second sampling period. This study describes the prevalence of francisellosis in O. niloticus and reports that the lesions found are not exclusively associated with this bacterial disease.     

Comparison of larval thermal maxima between <Emphasis Type="Italic">Fundulus heteroclitus</Emphasis> and <Emphasis Type="Italic">F. grandis</Emphasis>

Fundulus heteroclitus and F. grandis are resident salt marsh fishes that overlap in distribution over a narrow range in northeastern Florida. The objective of the present study was to examine whether the limits of the species’ ranges could be explained by differences in thermal tolerance. Two populations of each species were collected and then spawned in the laboratory, and 9-day-old larvae were used for critical thermal maxima trials. Mean LOE temperatures of larvae ranged from 43.04 to 43.65°C and showed little difference between species. Therefore, differences in high temperatures experienced cannot account for the differences of the distributions of the two species. Condition-specific competition may play a greater role in determining the observed range of the two species.     

Effect of <Emphasis Type="Italic">Cratoxylum formosum</Emphasis> on innate immune response and disease resistance against <Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> in tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis>

The effect of aqueous extract of Cratoxylum formosum on innate immune response and disease resistance in tilapia Oreochromis niloticus was investigated. The fish were fed diets containing 0% (control), 0.5% (diet 1), 1% (diet 2), and 1.5% (diet 3) of C. formosum aqueous extract for 30 days. At the end of the experimental period, parameters of innate immune response including phagocytosis of blood leukocytes, lysozyme activity in plasma, and respiratory bust activity were examined. Feeding the fish with diet 2 and diet 3 for 20 days enhanced phagocytic activity and for 30 days stimulated lysozyme and respiratory burst activities. Diet 1 increased the phagocytic activity at 30 days, but did not affect the other measured parameters. All parameters were not significantly changed (P > 0.05) in the control group throughout the experiment. Following 30 days of feeding, fish were infected with S. agalactiae. The cumulative mortalities of bacterial-infected tilapia that were fed diet 1, diet 2, and diet 3 were 56, 12, and 10%, respectively, compared with 85% in the control group. These results indicate that the aqueous extract of C. formosum may elevate the innate immune response and enhance disease resistance in tilapia.     

Demographic and competition studies on <Emphasis Type="Italic">Brachionus ibericus</Emphasis> and <Emphasis Type="Italic">Proales similis</Emphasis> in relation to salinity and algal (<Emphasis Type="Italic">Nannochloropsis oculata</Emphasis>) density

We isolated the rotifers Brachionus ibericus and Proales similis from the sediment of shrimp tanks and studied their individual demographic characters and competition between them at two food levels (0.25?×?10⁶, 1.00?×?10⁶ cells ml^?1 of Nannochloropsis oculata at 25 °C) and salinities ranging from 10 to 30‰. Our hypothesis was that growth rates would be higher with increasing food levels and salinities. Observations were taken twice a day for life table studies and daily once for population growth experiments. Using survivorship and fecundity data, we derived various life history variables. Although the average life span (7.6?±?0.4 days) and gross reproductive rate (33.8?±?2.9 neonate female^?1 day^?1) of B. ibericus were higher than those of P. similis (average life span 5.4?±?0.6 days and gross reproductive rate 13.0?±?0.6 neonate female^?1 day^?1), the population growth experiments showed that P. similis had higher r values (0.32?±?0.005 day^?1) than B. ibericus (0.23?±?0.002 day^?1) at 1.0?×?10⁶ cells ml^?1 of N. oculata. The rotifer P. similis was more adversely affected due to the presence of B. ibericus than vice versa. The data are important for developing techniques for a large-scale culture of these rotifers as food in aquaculture.     

Dietary supplementation of curcumin augments heat stress tolerance through upregulation of <Emphasis Type="Italic">nrf-2</Emphasis>-mediated antioxidative enzymes and <Emphasis Type="Italic">hsps</Emphasis> in <Emphasis Type="Italic">Puntius sophore</Emphasis>

Heat stress is one of the major environmental concerns in global warming regime and rising temperature has resulted in mass mortalities of animals including fishes. Therefore, strategies for high temperature stress tolerance and ameliorating the effects of heat stress are being looked for. In an earlier study, we reported that Nrf-2 (nuclear factor E2-related factor 2) mediated upregulation of antioxidative enzymes and heat shock proteins (Hsps) provide survivability to fish under heat stress. In this study, we have evaluated the ameliorative potential of dietary curcumin, a potential Nrf-2 inducer in heat stressed cyprinid Puntius sophore. Fishes were fed with diet supplemented with 0.5, 1.0, and 1.5% curcumin at the rate 2% of body weight daily in three separate groups (n = 40 in each group) for 60 days. Fishes fed with basal diet (without curcumin) served as the control (n = 40). Critical thermal maxima (CTmax) was determined for all the groups (n = 10, in duplicates) after the feeding trial. Significant increase in the CTmax was observed in the group fed with 1.5% curcumin- supplemented fishes whereas it remained similar in groups fed with 0.5%, and 1% curcumin-supplemented diet, as compared to control. To understand the molecular mechanism of elevated thermotolerance in the 1.5% curcumin supplemented group, fishes were given a sub-lethal heat shock treatment (36 °C) for 6 h and expression analysis of nrf-2, keap-1, sod, catalase, gpx, and hsp27, hsp60, hsp70, hsp90, and hsp110 was carried out using RT-PCR. In the gill, expression of nrf-2, sod, catalase, gpx, and hsp60, hsp70, hsp90, and hsp110 was found to be elevated in the 1.5% curcumin-fed heat-shocked group compared to control and the basal diet-fed, heat-shocked fishes. Similarly, in the liver, upregulation in expression of nrf-2, sod, catalase, and hsp70 and hsp110 was observed in 1.5% curcumin supplemented and heat shocked group. Thus, this study showed that supplementation of curcumin augments tolerance to high temperature stress in P. sophore that could be attributed to nrf-2-induced upregulation of antioxidative enzymes sod, catalase, gpx, and the hsps.     

Effect of combination feeding of <Emphasis Type="Italic">Nannochloropsis</Emphasis> and freshwater <Emphasis Type="Italic">Chlorella</Emphasis> on the fatty acid composition of rotifer <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> in a continuous culture

ABSTRACT:   A continuous culture of rotifer was conducted to investigate the effect of combination feeding of both a high density of Nannochloropsis oculata (N) and condensed freshwater Chlorella (FC) on the fatty acid composition of L-type rotifer Brachionus plicatilis in a continuous culture system. The algal feeding of the rotifers was carried out in three successive steps: N-feeding → N+FC-feeding → FC-feeding. The culture was conducted at 24°C and 25–27 psu in a 2000 mL bottle with 50% of water exchanged daily. The combination N+FC-feeding was effective in increasing rotifer density. The rotifers fed on N+FC (N+FC-R) had more non-polar lipids than polar ones, similar to those on N (N-R), opposite to the rotifers fed on FC (FC-R). N+FC-R contained higher levels of 16:2, 18:2n-6 (linoleic acid [LA]) and 20:2n-6, but lower levels of 18:1, 20:4n-6 (arachidonic acid), 20:5n-3 (eicosapentaenoic acid [EPA]) and 22:5n-3 (docosapentaenoic acid [DPA]) compared with N-R. Whereas N+FC-R contained higher levels of 16:1n-7, EPA and DPA, but lower levels of 16:2 and LA compared with FC-R. N+FC-R had more DPA in polar lipids than in non-polar ones. The Σn-6/Σn-3 ratio in N+FC-R was 0.9–1.0, significantly different from those in N-R (0.4) and FC-R (6.6–8.4). Therefore, it is inferred that the fatty acid profile of the N+FC-R cultured in a continuous culture system was affected by both N and FC. Also, the combination N+FC-feeding may be effective in manipulating the Σn-6/Σn-3 ratio in continuously cultured rotifers.