Obscure Puffer Takifugu Obscurus Research Articles

Identification and characterization of an L-type lectin from obscure puffer Takifugu obscurus in response to bacterial infection

L-type lectins (LTLs) contain a carbohydrate recognition domain homologous to leguminous lectins, and have functions in selective protein trafficking, sorting and targeting in the secretory pathway of animals. In this study, a novel LTL, designated as ToERGIC-53, was cloned and identified from obscure puffer Takifugu obscurus. The open reading frame of ToERGIC-53 contained 1554 nucleotides encoding 517 amino acid residues. The deduced ToERGIC-53 protein consisted of a signal peptide, a leguminous lectin domain (LTLD), a coiled-coil region, and a transmembrane region. Quantitative real-time PCR showed that ToERGIC-53 was expressed in all examined tissues, with the highest expression level in the liver. The expression of ToERGIC-53 was significantly upregulated after infection with Vibrio harveyi and Staphylococcus aureus. Recombinant ToERGIC-53-LTLD (rToERGIC-53-LTLD) protein could not only agglutinate and bind to one Gram-positive bacterium (S. aureus) and three Gram-negative bacteria (V. harveyi, V. parahaemolyticus and Aeromonas hydrophila), but also bind to glycoconjugates on the surface of bacteria such as lipopolysaccharide, peptidoglycan, mannose and galactose. In addition, rToERGIC-53-LTLD inhibited the growth of bacteria in vitro. All these results suggested that ToERGIC-53 might be a pattern recognition receptor involved in antibacterial immune response of T. obscurus.

Functional characterization of obscure puffer ToNK-lysin: A novel immunomodulator possessing anti-bacterial and anti-inflammatory properties

NK-lysins are one of the most abundant antimicrobial peptides produced by cytotoxic T lymphocytes (CTLs) and natural killer cells (NKs), and identified as a new class of intrinsically disordered proteins, playing critical roles in the cell-mediated cytotoxicity response, as well as immunomodulatory and antimicrobial activities upon a significant range of pathogens. In the present study, an NK-lysin was identified from Obscure puffer Takifugu obscurus (ToNK-lysin). The open reading frame of ToNK-lysin sequence spans 423 bp, encoding a peptide with 140 amino acids which shares a moderate residue identity (18%–60%) with NK-lysin of mammals and other teleost species. Phylogenetic analysis revealed that ToNK-lysin was most closely related to NK-lysins from the Pleuronectiformes (Bastard halibut Paralichthys olivaceus and Pacific halibut Hippoglossus stenolepis). Comprehensive computational analysis revealed that ToNK-lysin have substantial level of intrinsic disorder, which might be contribute to its multifunction. The transcripts of the ToNK-lysin were detected in multiple examined tissues and most abundant in gills. After bacterial and Poly I:C challenge, the transcriptional levels of ToNK-lysin were significantly up-regulated in the head kidney, liver and spleen at different time points. The recombinant ToNK-lysin showed significant antibacterial activity against Vibrio harveyi and Escherichia coli, and the ToNK-lysin treatment not only reduced the bacterial loads in liver and head kidney, but also alleviated the pathogen-mediated upregulation of immune-related genes. In addition, the co-incubation with rToNK-lysin protein remarkably degraded bacterial genomic DNA, suggesting the potential mechanism of ToNK-lysin against microbes. These results suggest that ToNK-lysin possess antibacterial and immunoregulatory function both in vivo and in vitro, which may allow it a potential applicability to the aquaculture industry.

Three novel L-type lectins from obscure puffer Takifugu obscurus promote antimicrobial immune response

L-type lectins (LTLs) have leguminous lectin domains that bind to high-mannose-type oligosaccharides. LTLs are involved in glycoprotein secretory pathways and associated with many immune responses. In the present research, three LTL homologs from obscure puffer Takifugu obscurus, designated as ToVIP36-1, ToVIP36-2, and ToVIP36-3, were first cloned and identified. The open reading frames of ToVIP36-1, ToVIP36-2, and ToVIP36-3 were 1068, 1002, and 1086 bp in length, respectively, and encode polypeptides with 355, 333, and 361 amino acids, respectively. Key conserved residues and functional domains, including lectin_leg-like domain (LTLD), transmembrane region, and C-terminal trafficking signal KRFY, were identified in all ToVIP36s. Quantitative real-time PCR analysis showed that the three ToVIP36s were widely expressed in six examined tissues and had relatively high expression levels in the liver and intestine. The expression levels of ToVIP36s were remarkably altered in the liver and kidney after induction by Vibrio harveyi and Staphylococcus aureus. Subsequently, the recombinant LTLDs of ToVIP36s (rToVIP36-LTLDs) were prepared by prokaryotic expression. Three rToVIP36-LTLD proteins agglutinated with S. aureus, V. harveyi, Vibrio parahaemolyticus, and Aeromonas hydrophila in a calcium-dependent manner. In the absence of calcium, rToVIP36-LTLD proteins bound to the bacteria by binding to lipopolysaccharides, peptidoglycans, d-mannose, and d-galactose and inhibited the growth of S. aureus and V. harveyi. Our results indicated that ToVIP36s function as pattern-recognition receptors in T. obscurus immunity, providing insights into the role of LTLs in the antibacterial immunity of fishes.

Molecular cloning, characterization and gene expression analysis of twelve interleukins in obscure puffer Takifugu obscurus

Interleukins (ILs) are a subgroup of secreted cytokines, which are molecules involved in the intercellular regulation of the immune system. In this study, 12 IL homologs were cloned and functionally identified from obscure puffer Takifugu obscurus, and they were termed as ToIL-1β, ToIL-1, ToIL-6, ToIL-10, ToIL-11, ToIL-12, ToIL-17, ToIL-18, ToIL-20, ToIL-24, ToIL-27, and ToIL-34. Multiple alignment results showed that except for ToIL-24 and ToIL-27, other deduced ToIL proteins shared typical characteristics and structure with other known fish ILs. Phylogenetic analysis revealed that 12 ToILs were evolutionarily closely related to their counterparts in other selected vertebrates. Tissue distribution assay demonstrated that the mRNA transcripts of most ToIL genes were constitutively expressed in all tissues examined, with relatively high expression in immune tissues. Following Vibrio harveyi and Staphylococcus aureus infection, the expression levels of 12 ToILs in the spleen and liver were significantly upregulated, and their response over time varied. Taken together, these data were discussed accordingly with the ToIL expression and the immune response under the different situations tested. The results suggest that the 12 ToIL genes are involved in the antibacterial immune response in T. obscurus.

Characterization and functional analysis of a c-type lysozyme gene from obscure puffer Takifugu obscurus

Lysozyme (Lyz) is an alkaline enzyme that hydrolyzes mucopolysaccharides in bacteria and is highly conserved vertebrates and invertebrates. In this study, a c-type lysozyme gene (named ToLyzC) from the obscure puffer Takifugu obscurus was cloned and characterized. The full-length cDNA of ToLyzC was 432 bp, encoding 143 amino acids, with a predicted molecular mass of 16.2 kDa and a theoretical pI of 8.86. The depicted protein sequence contained a LYZ1 domain from 16 to 142 amino acids, seven conserved cysteine residues. Phylogenetic analysis indicated that ToLyzC clustered with Lyzs from other teleost fishes. Quantitative real-time PCR analysis revealed that ToLyzC mRNA was mainly expressed in the liver. The transcript level of ToLyzC gene was significantly upregulated after Staphylococcus aureus and Vibrio harveyi challenge. The optimal pH and temperature of recombinant ToLyzC protein (rToLyzC) lytic activity was detected to be 7.5 and 35 °C, respectively. rToLyzC exhibited significant antibacterial and bacterial binding activities against S. aureus, Aeromonas hydrophila, V. harveyi, and Edwardsiella tarda at different time points. In addition, the morphological changes of V. harveyi cells treated with rToLyzC were observed under scanning electron microscope, which further confirmed the antibacterial and bacteriolytic activity of rToLyzC. Taken together, our current study indicated that ToLyzC is involved in the immune response to bacterial infection in obscure puffers.

Changes in Transcriptome and Ultrastructure Reveal Salinity Tolerance of Obscure Puffer Takifugu obscurus

Fish can maintain a stable intracellular concentration of ions in environments of variable salinities through osmoregulation. In this study, we focused on obscure puffer Takifugu obscurus (T. obscurus), an anadromous fish with high commercial value and rapid depression of wide populations, to investigate changes at molecular and physiological levels underlying salinity tolerance through multifaceted analyses integrating transcriptomics and micrography. We found that with the increase of salinity, the structure of the three main osmoregulation organs, i.e., gill, intestine, and kidney had remarkable changes. The results of transcriptome demonstrated that the ATP-binding box transporter and cyclic adenosine monophosphate (cAMP) signaling pathway in the gill and kidney were significantly reduced. Growth hormone, prolactin, and cortisol regulated more transporters of body composition and stimulated chloride cell proliferation and differentiation, which change the capacity for membrane transport between ion and water molecules. Adenosine-activating protein kinase and thyroid hormone signaling pathway were also significantly upregulated. These transcriptional levels changes of T. obscurus combined with ultrastructure in response to salinity increase indicated that osmoregulation is a complex process involving multiple organs and signaling pathways. Overall, this study can deepen the understanding of osmotic regulation during fish migration.

Calreticulin functions in antimicrobial immunity of obscure puffer Takifugu obscurus

Calreticulin (Crt) is a highly conserved and multi-functional protein with lectin-like properties and important immunological activities. In this study, a Crt homolog, namely, ToCrt, was cloned and characterized from the obscure puffer Takifugu obscurus with an open reading frame of 1278 bp encoding a putative protein of 425 amino acids. The deduced amino acid sequence of ToCrt consisted of three conserved structural domains: N-domain, P-domain, and C-terminal domain. In the phylogenetic tree, ToCrt formed a separate cluster with three Crts from other pufferfish species (Takifugu rubripes, Takifugu flavidus, and Takifugu bimaculatus). The mRNA transcript of ToCrt was ubiquitously expressed in all the examined tissues in a decreasing order: liver, spleen, kidney, gills, intestine, and heart. After Vibrio harveyi, Edwardsiella tarda, and Aeromonas hydrophila stimulations, the levels of ToCrt mRNA in the kidney and spleen were significantly upregulated compared with that in the control group. The recombinant calreticulin domain of ToCrt (rToCrt) could bind three Gram-negative bacteria (V. harveyi, E. tarda, and A. hydrophila) and polysaccharides from bacterial cell walls such as lipopolysaccharide and peptidoglycan. Meanwhile, rToCrt could agglutinate different kinds of microorganisms and exhibit antimicrobial activity. These results suggested that T. obscurus ToCrt could serve as an antimicrobial effector in the host immune response against invading microorganisms.

A new calnexin modulates antibacterial immune response in obscure puffer Takifugu obscurus

Calnexin (Cnx) is a membrane-bound lectin chaperone of the endoplasmic reticulum. In this study, a novel Cnx homologue from the obscure puffer Takifugu obscurus was characterized, tentatively named ToCnx. The cDNA of ToCnx was 1803 bp, and it contained an open reading frame encoding a polypeptide of 600 amino acid residues with a calculated molecular weight of 67.5 kDa. Multiple alignment of the deduced amino acid sequences of ToCnx and other related fish Cnxs revealed that ToCnx had typical characteristics of fish Cnxs. Sequence comparison and phylogenetic tree analysis showed that ToCnx had the closest relationship with Cnxs from Takifugu flavidus and Takifugu rubripes. ToCnx transcripts were detected in all the tissues examined, and they were mainly expressed in the liver, kidney, and intestine. Upon Vibrio harveyi, Edwardsiella tarda, and Aeromonas hydrophila infection, ToCnx transcripts were all significantly upregulated in the kidneys. The recombinant calreticulin domain of ToCnx (rToCnx) was prepared by prokaryotic expression. In the absence of calcium, rToCnx was able to bind three Gram-negative bacteria (V. harveyi, E. tarda, and A. hydrophila) and two bacterial saccharides, such as lipopolysaccharide and peptidoglycan. In the presence of calcium, rToCnx could agglutinate all the detected microorganisms. In addition, rToCnx possessed the effect of inhibiting the growth of three microbe strains. These observations suggested that ToCnx is an important participant in host immune defense against bacteria.

In silico characterization and expression analysis of eight C-type lectins in obscure puffer Takifugu obscurus

C-type lectins (CTLs) are a group of carbohydrate-binding proteins that play crucial roles in innate immune defense against invading pathogens. CTLs have been extensively studied in lower vertebrates, such as fish, for their roles in eliminating pathogens; however, their homologs in pufferfish are not well known. In the present study, eight CTLs from obscure puffer Takifugu obscurus (designated as ToCTL3–10 according to the order they were discovered) were obtained. All predicted ToCTL proteins contained a single carbohydrate recognition domain (CRD). ToCTL7 also contained one calcium-binding epidermal growth factor (EGF)-like domain (EGF_CA) and a transmembrane region. ToCTL9 also contained an SCP domain, an EGF domain, and an EGF-like domain. Bioinformatics analysis revealed that ToCTL3–10 mainly clustered with the corresponding CTL homologs of other pufferfish species. Tissue distribution analysis detected ToCTL3–10 in all tissues examined, including kidneys, liver, gills, spleen, intestines, and heart. Moreover, the expressions of ToCTL3–10 were significantly induced in the kidneys of obscure puffer following challenges with three Gram-negative bacterial pathogens, namely, Vibrio harveyi, Aeromonas hydrophila, and Edwardsiella tarda, and a synthetic analog of double-stranded RNA poly(I:C). The expression patterns of ToCTL3–10 in response to different immune stimulants were different. Our results indicated that the eight ToCTLs obtained herein might be involved in host defense against bacterial and poly(I:C) infections in T. obscurus.

Immunohistochemical characterization and change in location of branchial ionocytes after transfer from freshwater to seawater in the euryhaline obscure puffer, Takifugu obscurus.

The obscurepuffer Takifugu obscurus is a euryhaline fish species suitable for studying the molecular mechanism of osmoregulation. The distributional changes of branchial ionocytes were detected following the transfer from freshwater (FW) to seawater (SW) based on two main ion transporters, Na+/K+-ATPase (NKA) and Na+/K+/ 2Cl- cotransporter 1 (NKCC1). The mRNA and protein expression levels of NKA and NKCC1 in the gills all increased rapidly in the first four days after transfer to SW. Double immunofluorescence staining showed that NKCC1 and NKA were colocalized in the branchial ionocytes and the immunoreaction of NKCC1 was stronger after transfer. Moreover, following transfer to SW, the number of lamellar ionocytes in the gills is reduced and the number of filament ionocytes is increased significantly. Taken together, these findings indicated that SW transfer of obscure puffer promotes the changes of distribution, function and size of branchial ionocytes.

Characterization and Functional Analysis of Two Transmembrane C-Type Lectins in Obscure Puffer (Takifugu obscurus).

C-type lectins (CTLs) have received widespread attention in animal immune responses. In the present study, two CTLs (ToCTL1 and ToCTL2) were identified from obscure puffer Takifugu obscurus. The open reading frames of ToCTL1 and ToCTL2 were 687 and 1,380 bp, respectively. The predicted ToCTL1 and ToCTL2 proteins contained a single transmembrane region and one typical carbohydrate recognition domain (CRD). Quantitative real-time polymerase chain reaction detected ToCTL1 and ToCTL2 transcripts in all examined tissues, with high levels in the intestine and kidney, and their expression levels were remarkably altered upon Vibrio harveyi and Aeromonas hydrophila infection. The recombinant proteins ToCTL1-CRD and ToCTL2-CRD agglutinated the Gram-negative and Gram-positive bacteria in a Ca2+-dependent manner. rToCTL1-CRD and rToCTL2-CRD exhibited evident binding activities against seven kinds of bacteria and polysaccharides (lipopolysaccharide and peptidoglycan) in a Ca2+-independent manner. Moreover, rToCTL1-CRD and rToCTL2-CRD could inhibit the growth of four types of bacteria in vitro. These findings collectively demonstrated that ToCTL1 and ToCTL2 could be involved in host defense against bacterial infection in T. obscurus.

Responses of Takifugu obscurus fertilized eggs and larvae to increased ammonia exposure.

Ammonia is a common toxicant in aquatic systems; this substance has become a critical threat to fish, especially in early life stages. This study aimed to evaluate the effects of unionized ammonia (NH3-N: 0, 0.068, 0.138, 0.206, 0.275, 0.343, 0.412, and 0.481 mg L(-1)) on fertilized eggs and larvae of obscure puffer Takifugu obscurus, a fish species with potential economic value. Results showed that hatch time was significantly retarded and hatch rate was significantly decreased as NH3-N concentrations increased; newly hatched larvae exhibited high rate of abnormalities and low viability. The survival rate of larvae also decreased significantly as NH3-N concentrations increased; larvae could tolerate NH3-N to a less extent than embryos. NH3-N also caused a significant decrease in superoxide dismutase (SOD) and Na(+)/K(+) ATPase activities but not in malondialdehyde (MDA) levels of larvae. Two-way ANOVA indicated that there was a statistically significant interaction between NH3-N concentrations and exposure times on SOD activity but not on Na(+)/K(+) ATPase activity. Such responses indicated that an increase in ammonia concentration in surface water may negatively affect the early development of T. obscurus and thus likely impair population recruitment and persistence of this fish species.

The combined effect of temperature and pH on embryonic development of obscure puffer Takifugu obscurus and its ecological implications

Rising temperature and decreasing pH caused by climate change may affect the development of fish. The objective of this study was to determine the combined effects of temperature (19, 22, 25, 28 °C) and pH (5, 6, 7, 8) on incubation time, total hatch rate, abnormal rate, and viability of newly hatched larvae 24 h post-hatch of obscure puffer Takifugu obscurus fertilized eggs. The results showed that incubation time was significantly shortened with increase in temperature and delayed at low pH. Significant interaction was detected between temperature and pH on incubation time. Both temperature and pH significantly affected the total hatch rate, abnormal rate, and the viability of newly hatched larvae, but no significant interaction was detected. Such results indicated that fluctuations in temperature and decreasing pH in waters during the spawning season of this anadromous species may have substantially negative impacts on its population recruitment and persistence.

Changes in plasma osmolality and Na+/K+ ATPase activity of juvenile obscure puffer Takifugu obscurus following salinity challenge

The osmoregulatory capabilities of 6-month-old juvenile obscure puffer Takifugu obscurus, transferred directly from fresh water to different salinities (0‰, freshwater control; 10‰; 20‰ and 30‰), were studied over an 8-day period. After transfer, plasma osmolality of the fish at 30‰ was significantly higher than those at all other salinities throughout the experiment. The Na+/K+ ATPase activity in the gills of the fish treated with various salinities increased significantly, peaking at 48 h, then decreased gradually to the control level at 192 h. Similar fluctuation trends of the Na+/K+ ATPase activity were observed in the kidneys. Modified Gaussian model provided accurate fits for the time-course changes in the Na+/K+ ATPase activities after abrupt salinity challenge. The results demonstrated that obscure puffer has strong capacity to tolerate abrupt salinity changes and can osmoregulate well over a wide range of salinities even in juvenile stage.

Effects of dietary amylose/amylopectin ratio on growth performance, feed utilization, digestive enzymes, and postprandial metabolic responses in juvenile obscure puffer Takifugu obscurus

The effect of dietary amylose/amylopectin (AM/AP) ratio on growth, feed utilization, digestive enzyme activities, plasma parameters, and postprandial blood glucose responses was evaluated in juvenile obscure puffer, Takifugu obscurus. Five isonitrogenous (430 g kg(-1) crude protein) and isolipidic (90 g kg(-1) crude lipid) diets containing an equal starch level (250 g kg(-1) starch) with different AM/AP ratio diets of 0/25, 3/22, 6/19, 9/16 and 12/13 were formulated. Each experimental diet was fed to triplicate groups (25 fish per tank), twice daily during a period of 60 days. After the growth trial, a postprandial blood response test was carried out. Fish fed diet 6/19 showed best growth, feed efficiency and protein efficiency ratio. Hepatosomatic index, plasma total cholesterol concentration, liver glycogen and lipid content, and gluconokinase, pyruvate kinase and fructose-1,6-bisphosphatase activities were lower in fish fed highest AM/AP diet (12/13) than in fish fed the low-amylose diets. Activities of liver and intestinal trypsin in fish fed diet 3/22 and diet 6/19 were higher than in fish fed diet 9/16 and diet 12/13. Activities of liver and intestinal amylase and intestinal lipase, and starch digestibility were negatively correlated with dietary AM/AP ratio. Fish fed diet 3/22 and diet 6/19 showed higher plasma total amino acid concentration than fish fed the other diets, while plasma urea nitrogen concentration and activities of alanine aminotransferase and aspartate aminotransferase showed the opposite trend. Equal values were found for viscerosomatic index and condition factor, whole body and muscle composition, plasma high-density and low-density lipoprotein cholesterol concentrations, and activities of lipase and hexokinase and glucose-6-phosphatase in liver. Postprandial plasma glucose and triglyceride peak value of fish fed diet 12/13 were lower than in fish fed the low-amylose diets, and the peak time of plasma glucose was later than in fish fed the other diets. Plasma glucose and triglyceride concentrations showed a significant difference at 2 and 4 h after a meal and varied between dietary treatments. According to regression analysis of weight gain against dietary AM/AP ratio, the optimum dietary AM/AP ratio for maximum growth of obscure puffer was 0.25. The present result indicates that dietary AM/AP ratio could affect growth performance and feed utilization, some plasma parameters, digestive enzyme as well as hepatic glucose metabolic enzyme activities in juvenile obscure puffer.

Effects of photoperiod, temperature, and salinity on growth and survival of obscure puffer Takifugu obscurus larvae

The effects of photoperiod, temperature, and salinity on growth and survival of obscure puffer Takifugu obscurus larvae were determined from 3 to 19 days after hatching (dah). The results showed that the optimal photoperiod for obscure puffer larvae was under continuous light conditions (24 h light:0 h dark). Increasing daylength resulted in improved larval growth and survival. Larvae cultured under total darkness grew very slowly and even negatively, and all died within 12 days (15 dah). The optimal and critical high temperatures for obscure puffer larvae were at 22–23 and 32 °C, respectively. The specific growth rates (SGR) of obscure puffer larvae increased with an increase in temperature from 20 to 29 °C, while no corresponding increase in SGR was observed for temperatures from 29 to 32 °C. Larval survival rate significantly decreased when the temperature increased from 23 to 32 °C, and there was a tendency for a low survival rate at 20 °C. A high salinity (25‰) resulted in reduced larval growth and survival probabilities. The highest survival and specific growth rates were both predicted to occur at 8‰ to 10‰ based on quadratic equations. High SGR (5.71–6.14% d − 1 ) and survival rates (> 90%) were found within a wide salinity range (0–20‰). The optimal and suitable salinity ranges of larviculture were 8–10‰ and 0–20‰, respectively. Therefore, obscure puffer larvae can feasibly be cultured in brackish water (0–20‰) existing in majority of coastal areas.

Effects of salinity on survival and Na+/K+ ATPase activity of obscure puffer Takifugu obscurus embryos

Effects of salinity on survival and Na+/K+ ATPase activity of obscure puffer Takifugu obscurus embryos

Differences in reproductive strategies between obscure pufferTakifugu obscurusand ocellated pufferTakifugu ocellatusduring their spawning migration

Summary Reproductive strategies were compared between obscure puffer Takifugu obscurus and ocellated puffer Takifugu ocellatus captured in waters near Yangzhong Island in the lower reaches of the Yangtze River during the spawning migration season from February to June in two consecutive years (1995 and 1996). Results showed that obscure puffer and ocellated puffer have adopted different reproductive strategies, including different spawning times, different size at maturity, and different number and size of oocytes, resulting in two different larval sizes. In detail, the timing of the spawning migration and status of gonad development of obscure puffer was about 1 month earlier than that of ocellated puffer; the obscure puffer was obviously longer and heavier than ocellated puffer in both mature male and female fish; mean GSI of obscure puffer females (15.8%) was significantly higher than that of ocellated puffer females (14.6%); the average diameter of ocellated puffer eggs (1.49 ± 0.12 mm) was significantly larger than that of the obscure puffer (1.22 ± 0.08 mm); and obscure puffer females (320.8 oocytes mg−1 somatic wet weight) had significantly higher relative fecundity than ocellated puffer females (125.2 oocytes mg−1 somatic wet weight). These differences in reproductive strategies between two closely related species of the Takifugu genus indicate that both obscure puffer and ocellated puffer fit the r/K dichotomy. Obscure puffer shows K-selected characters with maturity at relatively large size and r-selected characters with relatively many and small offspring, whereas ocellated puffer shows r-selected characters with maturity at a relatively small size and K-selected characters with relatively few and large offspring.

Salinity tolerance of embryos of obscure puffer Takifugu obscurus

Salinity tolerance of embryos of obscure puffer Takifugu obscurus was investigated at salinities of 0, 4, 8, 12, 16, 20, 24, 28, and 32 parts per thousand (ppt), respectively. Survival rates of embryos at 0, 4 and 8 ppt salinities were higher than 90% throughout the experiment, and there were no significant differences between 0 and 8 ppt ( P > 0.05), whereas most embryos died gradually at salinity of 12 ppt or higher after four days. Total hatch rates at 0, 4, and 8 ppt were 95.0%, 95.0%, and 91.0%, respectively. A few embryos hatched at salinities of 12, 16, 20, and 28 ppt; however, all larvae were deformed and died in 24 h post-hatch. Only at salinity of 8.0 ppt or lower, the embryos hatched into viable larvae. Hatching period at 0, 4, and 8 were 171, 182, and 182 h, respectively. Within the tolerable salinity range (0–8 ppt), there were no significant differences in total hatch rates, viability of 24 h post-hatch larvae, and rate of embryonic development ( P > 0.05). We concluded that embryos of obscure puffer could not tolerate salinities of 12 ppt and higher, but could well adapt to salinities below 8 ppt. Thus, it is viable to incubate embryos of obscure puffer under lower salinity conditions.

Effect of temperature on incubation period and hatching success of obscure puffer Takifugu obscurus (Abe) eggs

Artificially fertilized eggs of obscure puffer Takifugu obscurus were obtained by induced spawning of cultured broodstock and incubated at temperatures of 15, 19, 23, and 27 °C. The results showed that the optimal temperature for obscure puffer embryonic development ranged from 19 to 23 °C, based on total hatch rate, viability of newly hatched larvae 24 h post-hatch, and total mortality rate of eggs. At the given temperature range, the times taken for 50% embryos to hatch were 11.3, 6.6, 5.0, and 4.2 days, respectively. There was significant difference in time to 50% hatch among the temperatures used in this experiment. The power law model, quadratic equation, exponential equation, and effective degree-day model all provided good fits for the relationship between incubation temperature and time to 50% hatch, with r 2 values greater than 0.90. The formulae for these were y=1031.7T −1.6885, y=44.721−3.1574 T+0.0615 T 2, y=34.663e −0.0813 T and y=78.905/( T−7.6033), respectively, where y is time to 50% hatch in days, and T is incubation temperature in degrees Celsius. The effective degree-day model was determined to be the best model because of efficient computation, good fit to the experimental data, and most importantly, the derived parameters, k (the sum of effective degree-days) and t 0 (the temperature of biological zero), have important biological meaning. Based on the effective degree-day model, the t 0 and k values were calculated as 7.6033 °C and 78.905 degree-days, respectively.