Liquid-chromatographic hybrid triple–quadrupole linear-ion-trap MS/MS analysis of fatty-acid esters of dinophysistoxin-1 in bivalves and toxic dinoflagellates in Japan

Fatty-acid esters of dinophysistoxin-1 (DTX1) in scallops Patinopecten yessoensis, mussels Mytilus coruscus, and toxic dinoflagellate Dinophysis species, collected from Japanese seawater, were analyzed by liquid-chromatography mass spectrometry (LC–MS). Precursor ion monitoring, multiple reaction monitoring for 18 fatty-acid esters of DTX1, and full-scan MS/MS spectra obtained with a hybrid triple–quadrupole linear-ion-trap mass spectrometer showed that 14:0, 16:0, and 16:1 esters were the most abundant 7-O-acyl-DTX1 analogues in bivalves. Fatty-acids esters formed by conjugations at hydroxyl positions other than the 7-position of DTX1 were not detected in the bivalves. DTX1 and okadaic acid-16:0 fatty-acid esters have been reported as the most abundant ester in bivalves in several previous studies; however, we found that 7-O-16:1-DTX1 was the most abundant ester in some mussels in which 16:1 was more dominant than 16:0 in the free fatty-acid profile. Comparison between 7-O-acyl-DTX1 and free fatty-acid profiles in the same bivalve samples suggests that polyunsaturated fatty acids are selectively excluded in enzymatic acylation of DTX1. No 7-O-acyl-DTX1 was detected in any single-cell isolates of D. fortii, D. acuminata, D. mitra, D. norvegica, D. tripos, D. infundibulus, and D. rotundata.     

Annual variation of potential predation impacts on larval and juvenile marbled sole <Emphasis Type="Italic">Pseudopleuronectes yokohamae</Emphasis> by sand shrimp <Emphasis Type="Italic">Crangon uritai</Emphasis> in Hakodate Bay,Hokkaido

An investigation was conducted to evaluate the annual variation in potential predation impact (PPI) on larval and juvenile Pseudopleuronectes yokohamae by Crangon uritai in Hakodate Bay using predator-prey size relationships. Laboratory experiments were designed to estimate the favorable prey size of C. uritai through back-calculation of body length (BL) of P. yokohamae from sagittal otolith diameter observed in the stomachs of C. uritai. The most favorable prey-predator size ratio (BL of P. yokohamae-total length (TL) of C. uritai) class was 0.15–0.19, and ranged from 0.12–0.31. There was a significant positive correlation between the BL increase of P. yokohamae and the bottom water temperature in the field, such that BLs stagnated during the cold year of 1999 from April to June, and increased during the warm year of 2002. In contrast, no significant correlation was found between the TL increase of C. uritai and the bottom water temperature. Moreover, there were no significant differences in the mean TL of C. uritai between months (April–June) or years (1998–2002). Variation of PPI depended on the relationships between the growth rates of P. yokohamae and bottom water temperature. Therefore, the warm year of 2002 resulted in higher potential predation impact (PPI≥20), and it was at least 20 days shorter than that of the cold year of 1999. These results suggest that increased bottom water temperature in the nursery area was one of the most important factors for cumulative predation loss.     

Effect of ration size on growth,body composition,and energy and protein maintenance requirement of fingerling Indian major carp, <Emphasis Type="Italic">Labeo rohita</Emphasis> (Hamilton)

An eight-week feeding trial has been conducted to determine the optimum ration for Indian major carp, Labeo rohita, fingerling (4.10 ± 0.30 cm, 0.55 ± 0.16 g) by feeding a purified diet (40% CP; 3.61 kcal g⁻¹ GE) at six levels, 2, 4, 6, 8, 10, and 12% of body weight per day, at 0800 and 1600 h, in triplicate, to 20 fish per trough fitted with a water flow-through system. Highest weight gain, best feed conversion ratio (FCR), best specific growth rate (SGR%), and highest protein efficiency ratio (PER) were evident for rations of 6–8% body weight. Second-degree polynomial regression analysis for FCR, PER, protein, and energy retention data indicated the break-points occurred at 6.55, 6.75, 6.80, and 6.95% bw per day, respectively. Significant (P < 0.05) differences between body composition were observed for fish fed different rations. Maximum body protein content was recorded for 6% and 8% rations. A linear increase in body fat content was evident with increasing ration. Body moisture and ash content remained non-significantly (P > 0.05) low for higher rations, however. On the basis of these results it is recommended that feeding in the range 6.5–7.0% bw per day corresponding to 2.6–2.8 g protein and 23.49–25.31 kcal energy per 100 g of the diet per day is optimum for growth and efficient feed utilization of Labeo rohita. Results for 2–4% rations (0.8–1.6 g protein and 7.23–14.46 kcal energy) suggest these amounts approximate to the maintenance requirement of this fish.     

Identification of overwintering vegetative cells of the bivalve-killing dinoflagellate <Emphasis Type="Italic">Heterocapsa circularisquama</Emphasis> in Uranouchi Inlet,Kochi Prefecture,Japan

ABSTRACT:   Red tides of Heterocapsa circularisquama have led to serious damage of bivalve aquacultures in western coastal areas of Japan. To understand the whole picture regarding the ecology of this species, it is essential to clarify its overwintering mechanisms. In this study, the population dynamics of H. circularisquama were investigated from February 2004 to November 2005, and overwintering cells were identified for the first time in water columns of Uranouchi Inlet, Kochi Prefecture, Japan. Heterocapsa circularisquama cells were detected by the indirect fluorescent antibody technique using monoclonal antibodies that specifically recognize and react to this species. Vegetative cells were almost always detected from the first observation in February 2004 to November 2005 with temperatures of 10.5–30.6°C. During the period from winter to spring, this species survived in areas with a temperature higher than 10°C. The overwintering cells of H. circularisquama were isolated in March 2004, and identification was made via observation of the morphology and body scales of the cultured cells. These overwintering cells were identified as H. circularisquama and reacted to the monoclonal antibody. These results indicate that H. circularisquama can overwinter and survive throughout the year in a vegetative cell state in Uranouchi Inlet.     

Diet and fish choice of Eurasian otters (<Emphasis Type="Italic">Lutra lutra</Emphasis> L.) in fish wintering ponds in Hungary

The diet composition and fish preference of piscivorous Eurasian otters (Lutra lutra) were studied in two fish farm systems in Hungary using spraint (otter faeces) analysis during two wintering periods. The primary food source of otters in both fish farms was fish (97–99% of biomass). The main fish prey was small-sized, below 100 g in weight (96% in both areas), while fish prey above 500 g comprised only 0.1–0.4% of the diet. The bulk of the otters’ diet consisted of less-valued species, especially non-native Prussian carp (Carassius auratus gibelio). Consumption of commercial fish species ranged between 15 and 31% of the total diet. Otters preferred fish below 100 g in weight (Ivlev’s electivity index, E _i = 0.65–0.70), and showed a lesser preference for (or avoided) fish above 100 g in weight (E _i = −0.37–1.00). With regard to species distribution, otters preferred small (below 100 g) grass carp (Ctenopharyngodon idella), zander (Sander lucioperca), pike (Esox lucius), Prussian carp, topmouth gudgeon (Pseudorasbora parva), while they consumed common carp (Cyprinus carpio), the most important commercial species, proportionally to its abundance in the environment (E _i = −0.18–0.29).     

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.     

Age and growth of Gerres sp. (Perciformes: Gerreidae) in Okinawa Island of Southern Japan

A total of 518 Gerres sp. were collected around Okinawa Island, Japan, from November 2002 to July 2005, with monthly sampling where the standard length of females (n=218) were 56.2–147.1 mm, and males (n=149) were 62.2–139.4 mm. The maximum ages observed for females were 5⁺ years and males were 4⁺ years, estimated by transverse sectioned sagittal otoliths. Mean marginal increment indicated that opaque rings were formed once a year during April to July. The standard length (SL; mm) — body wet weight (BW; g) relationships were described as BW=(3.26×10⁻⁵) SL^2.97 and BW=(3.13×10⁻⁵) SL ^2.98 for females and males, respectively, and the standard length at age described by von Bertalanffy growth function for females, L _t=137.1(1−e^{−0.80[t+0.80]}) and males, L _t=127.3(1−e^{−0.82[t+0.93]}).     

The relationship between paralarval feeding and morphological changes in the proboscis and beaks of the neon flying squid <Emphasis Type="Italic">Ommastrephes bartramii</Emphasis>

We compared the diet of Ommastrephes bartramii paralarvae with morphological changes in their beaks and proboscis (fused tentacles). The paralarvae were collected north of the Hawaiian Islands during 2001 and 2002 and ranged in mantle length (ML) from 1.1 to 13.2 mm. They fed on crustaceans, including copepods (copepodite stage) and amphipods. The rostral tips of upper and lower beaks began to protrude anteriorly at around 3–4 mm ML, and the smallest paralarva with identifiable prey in its digestive tract was 4.2 mm ML, which suggests that the paralarvae can masticate prey soon after the beaks protrude. The proboscis separated into two tentacles at 9.3–13.2 mm ML, but the newly formed tentacles were weakly developed even in the largest specimen, suggesting that tentacles do not operate functionally and that the arms are used to capture prey.     

Age and growth of the lizardfish <Emphasis Type="Italic">Saurida elongata</Emphasis> from the Tsushima/Korea Strait

We studied the age and growth of the lizardfish Saurida elongata using ground thin sections of otoliths (sagittae) from specimens collected in the Tsushima/Korea Strait between May 1999 and June 2001. A total of 695 individuals with fork lengths (FL) ranging from 189 to 478 mm were examined. The frequency of translucent zone occurrence at the outer margin of the otoliths indicated that translucent zones formed once a year between November and February. Most of the males examined were estimated to be 2–7 years old and the females, 3–9 years old. The maximum estimated age of a male specimen was 10 years and that of a female, 11 years. The estimated von Bertalanffy growth curves were FL _t  = 451{1 − exp[−0.172(t + 2.50)]} and FL _t  = 515{1 − exp[−0.151(t + 2.47)]} for males and females, respectively. At all ages, the FL at a specific age for females was greater than that for males, suggesting that females of this species grow faster than males.     

Substrate specificity of brain acetylcholinesterase and its sensitivity to carbamate insecticides in <Emphasis Type="Italic">Carassius auratus</Emphasis>

The substrate specificity of brain acetylcholinesterase (AChE) in adult Carassius auratus fish and its sensitivity to carbamate insecticides were investigated in vitro. The results showed that the order of four substrates hydrolyzed by brain AChE in C. auratus was acetylthiocholine iodide > β-methylthiocholine iodide > propionylthiocholine iodide > butyrylthiocholine iodide, and the maximum velocity (V _max) of AChE hydrolyzing acetylthiocholine iodide (ATCh) was the highest among the four substrates, and the V _max values were 0.067 and 0.082 mmol min⁻¹ mg⁻¹ for male and female fish respectively. But their Michaelis–Menten constants (K _m) were the lowest, only 0.071 and 0.072 mmol/l respectively. Compared with other carbamate insecticides, the sensitivity of brain AChE to carbofuran was the highest and the IC₅₀ values were 1.04 × 10⁻⁶ mol/l for females and 1.17 × 10⁻⁶ mol/l for males. The inhibitory tendencies of eserine, methomyl, and aldicarb to brain AChE were very similar, and the percentage inhibition increased with time at the concentration of 1 × 10⁻⁶ mol/l. The order of inhibition potential of the three inhibitors from the highest to the lowest was eserine, aldicarb, and methomyl.     

A new species of freshwater eel <Emphasis Type="Italic">Anguilla luzonensis</Emphasis> (Teleostei: Anguillidae) from Luzon Island of the Philippines

Anguilla luzonensis, a new species of freshwater eel, family Anguillidae, is described on the basis of 29 specimens collected from the Pinacanauan River system, which is a tributary of the Cagayan River on northern Luzon Island of the Philippines. The new species and A. celebesensis, which is distributed in the same region, both have variegated color marking and broad maxillary bands of teeth, but show statistically significant differences in the following characters of the new species: predorsal-fin length 28.6–33.1% of total length (TL); preanal length 39.6–44.8% of TL; trunk length 26.4–31.3% of TL; distance between the verticals through the anus and origin of the dorsal fin 9.3–13.9% of TL; length of gape 35.5–46.9% of head length; the total number of vertebrae 103–107; and number of abdominal vertebrae 40–42. These morphological differences, together with the present knowledge about anguillid eel taxonomy, genetics, and ecology, strongly support the presence of the new species A. luzonensis in the area of northern Luzon Island of the Philippines.     

Production and characterization of monoclonal antibodies to leukocytes and serum immunoglobulin in Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

Two monoclonal antibodies (MAbs: JFW1 and JFW10) were produced against peripheral blood leukocytes (PBL) in Japanese flounder. Additionally, MAbs against flounder immunoglobulin (Ig; JFW20 and JFW21) were generated for the surface marker of Ig⁺ leukocytes using purified serum Ig as an antigen. MAb JFW1 recognized the surface marker of granulocytes and monocytes and MAb JFW10 specifically bound to the surface antigen of thrombocytes. Flow cytometric analysis of PBL incubated with JFW1, JFW10, JFW20 and JFW21 revealed that 2.5–7.4, 23.7–50.1, 25.2–26.1 and 5.2–8.3% of all leukocytes were positive for these markers. Analysis of head kidney leukocytes (HKL) showed that JFW1, JFW10, JFW20 and JFW21 bound to 30.5–36.3, 1.9–2.8, 6.4–8.3 and 1.9–3.0% of all leukocytes, respectively. Western blot analysis after SDS-PAGE showed that JFW10 recognizes a protein of 115 kDa from lysed PBL. JFW20 recognized the 70 and 74 kDa proteins of the heavy chain of Ig from serum. No band was observed for either JFW1 or JFW21. These antibodies will be useful for the identification and isolation of Japanese flounder leukocyte subpopulations and will facilitate immunological studies of flounder.     

Growth and feed utilization of gilthead sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis>, L.) fed to satiation and restrictively at increasing dietary energy levels

Three isoproteic (47% protein) diets were formulated to contain graded levels of crude fat (diet D16:16%, diet D24: 24% and diet D32: 32%). Each diet was fed to satiation in three and to 80% satiation in two replicate groups of gilthead sea bream (Sparus aurata), having an initial body weight of 72–74 g. The trial lasted 81 days. Groups fed to satiation showed higher final body weight (FBW; 238.8–252.3 g vs. 218.0–229.3 g) and daily growth index (DGI; 2.49–2.65%/day vs. 2.27–2.34%/day) than those fed to 80% satiation. Feed intake was significantly different both for feeding level and for diet composition. Fish fed to satiation had higher feed conversion rate (FCR) compared to the 80% satiation groups (1.33–1.44 vs. 1.13–1.17; P ≤ 0.001). Within satiation groups, FCR was significantly lower in fish fed D16 compared to fish fed D32 (1.33 vs. 1.44, P ≤ 0.05), whereas no statistical differences were found within the 80% satiation groups. The increase in dietary lipid level did not improve growth performance, feed efficiency and protein utilization but decreased gross lipid efficiency. Conversely, a reduction in ration from satiation to 80% satiation decreased DGI, thus improving FCR. Feed costs were influenced by dietary energy level and feeding ratio, the lowest energy diet at 80% satiation being the most profitable combination among the variables.     

Henneguya pseudorhinogobii n. sp. (Myxozoa: Myxosporea) parasitizing the gills of the freshwater goby Rhinogobius sp. OR from the Nagara River and redescription of Henneguyarhinogobii

Two types of infection with myxosporean parasites, which were different in cyst size, were found from the gill of Rhinogobius sp. OR collected from the Nagara River, Gifu Prefecture, Japan. A myxosporean forming large-type cysts in the gill filament was morphologically identified as Henneguya rhinogobii. Another myxosporean forming small-type cysts was found to be parasitic in the gill lamella. Average spore sizes of the latter myxosporean were 15.8 (range 14.2–17.8) μm in length, 5.3 (4.7–5.8) μm in width, 6.5 (5.9–7.6) μm in polar capsule length and 34.9 (25.3–42.9) μm in caudal appendage length, which were almost identical to those of H. rhinogobii. However, the two were distinguishable by the location in the gill (intrafilamental or intralamellar) and by the difference in the sequence of SSU rDNA (about 96% similarity). We propose the myxosporean forming intralamellar small-type cysts as Henneguya pseudorhinogobii n. sp. and redescribe H. rhinogobii by morphological and molecular studies.     

The effects of dietary lipid sources and lecithin on the production of giant freshwater prawn <Emphasis Type="Italic">Macrobrachium rosenbergii</Emphasis> larvae in the Mekong Delta region of Vietnam

The giant freshwater prawn, Macrobrachium rosenbergii, is cultured widely in the Mekong Delta region of Vietnam but it is often difficult or expensive for hatchery operators to purchase commercial diets used as a feeding supplement to Artemia nauplii. Therefore, in the present study, the effects of lipid sources and lecithin on the growth and survival rate of M. rosenbergii larvae were examined in order to develop suitable hand-prepared larval diets for seed production of M. rosenbergii in this area. Six egg custard diets consisting of various ratios of lipid (originating from soybean oil and squid oil) and lecithin were used for rearing Macrobrachium rosenbergii larvae. Treatments in which larvae were fed diets containing squid oil exhibited the highest body length and survival rates (7.14–7.43 mm and 51.1–68.1%, respectively), and differed significantly from other treatments (P<0.05). Use of dietary soybean oil yielded the lowest body length and survival rates (6.29–6.75 mm and 22.0–48.7%), respectively). The supplementation of dietary lecithin did not increase final body weight but did improve larval survival rates. The n-3 HUFA content of prawns fed dietary squid oil was higher than those of animals provided with other diets. These results indicated that the most appropriate diet for rearing M. rosenbergii larvae is the diet containing 3% squid oil and 1.5% lecithin.     

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.     

Changes in growth of marbled sole <Emphasis Type="Italic">Pseudopleuronectes yokohamae</Emphasis> between high and low stock-size periods in Tokyo Bay,Japan

We examined the age and growth of the marbled sole Pseudopleuronectes yokohamae collected from Tokyo Bay, Japan, during two study periods between 2002 and 2008, when the stock size was consistently low. The von Bertalanffy growth equations were found to differ significantly between sexes, and females attained a larger standard length than males of the same age. These results were then compared with those reported for 1974–1983 and 1986–1988, two study periods when the stock size was high. The growth of both sexes of marbled sole in Tokyo Bay during the 2000s was found to be significantly higher than that in the 1970s and 1980s. To explore possible factors causing these changes in the growth, we examined bottom water temperature and population density. We found that the population density decreased and the bottom water temperature increased between the late 1970s and the late 2000s. However, despite evidence of changes in population densities and water temperatures, further study is needed to determine the main factors causing the observed changes in growth.     

Age and growth of two gizzard shads, <Emphasis Type="Italic">Nematalosa come</Emphasis> and <Emphasis Type="Italic">N. japonica</Emphasis>, in coastal waters around Okinawa Island,southwestern Japan

The age and growth of two Nematalosa species around Okinawa Island were examined using sectioned otoliths collected from September 2003 to April 2006. Monthly changes in the frequency of the appearance of a translucent band on the outer margin of the otoliths indicated that ring formation occurred once a year from January to July for Nematalosa come and from January to March for Nematalosa japonica. The von Bertalanffy growth equations for both species were as follows: N. come: L _t  = 365.5{1 − exp[−0.111 × (t + 0.288)]} for females and L _t  = 214.7{1 − exp[−0.700 × (t – 1.110)]} for males; N. japonica: L _t  = 205.1{1 − exp[−1.068 × (t − 1.180)]} for females and L _t  = 195.5 {1 − exp[−1.293 × (t − 1.269)]} for males. The maximum ages observed for N. come and N. japonica were 11 and 6 years old, respectively. The growth of these species was characterized by the slow growth of N. come over many years, resulting in a larger size than N. japonica.     

Life-history traits of eight <Emphasis Type="Italic">Lethrinus</Emphasis> species from two local populations in waters off the Ryukyu Islands

The number of growth rings was counted on broken and burnt otoliths of eight Lethrinus spp. from two local populations: Lethrinus atkinsoni, L. harak, L. miniatus, L. nebulosus, L. obsoletus, L. ornatus, L. ravus, and L. rubrioperculatus, collected from 1985 to 1996 in waters off the Ryukyu Islands. Growth rings were revealed to be formed annually from about October to June by marginal analysis. In addition to obtaining the three parameters in von Bertalanffy growth equation and the maximum age in specimens, the relationship between age and both ovarian maturity rate and sex ratio (percentage female) was analyzed. The age at 50% of ovarian maturity was the lowest at 1–2 years old in L. harak, L. ravus, and L. rubrioperculatus and was the highest at approximately 4 years old in L. nebulosus. The age at which the sex ratio decreased to 50% due to sexual transition from female in protogynous hermaphrodite species was the lowest at 3–4 years old in the L. atkinsoni Okinawa population and was the highest at 7–8 years old in L. miniatus. The oldest maximum age for specimens was 26 years in L. nebulosus and the youngest maximum age was 12 years in L. ornatus.