Development of a chub mackerel with less-aggressive fry stage by genome editing of arginine vasotocin receptor V1a2.

Genome editing is a technology that can remarkably accelerate crop and animal breeding via artificial induction of desired traits with high accuracy. This study aimed to develop a chub mackerel variety with reduced aggression using an experimental system that enables efficient egg collection and genome editing. Sexual maturation and control of spawning season and time were technologically facilitated by controlling the photoperiod and water temperature of the rearing tank. In addition, appropriate low-temperature treatment conditions for delaying cleavage, shape of the glass capillary, and injection site were examined in detail in order to develop an efficient and robust microinjection system for the study. An arginine vasotocin receptor V1a2 (V1a2) knockout (KO) strain of chub mackerel was developed in order to reduce the frequency of cannibalistic behavior at the fry stage. Video data analysis using bioimage informatics quantified the frequency of aggressive behavior, indicating a significant 46% reduction (P = 0.0229) in the frequency of cannibalistic behavior than in wild type. Furthermore, in the V1a2 KO strain, the frequency of collisions with the wall and oxygen consumption also decreased. Overall, the manageable and calm phenotype reported here can potentially contribute to the development of a stable and sustainable marine product.

Two vitellogenins in the loliginid squid Uroteuthis edulis: Identification and specific expression in ovarian follicles.

Vitellogenenesis is a physiological process common in oviparous animals. The molecular profile, modifications, and utilization of vitellogenin (VTG), a precursor of yolk protein, have been characterized in various taxa to understand oogenesis within different modes of reproduction. Hormonal regulation of VTGs has been investigated in invertebrates, such as insects and crustaceans; conversely, little is known for cephalopods. In this study, we isolated two VTG genes (ue-VTG1 and ue-VTG2) from the loliginid swordtip squid, Uroteuthis edulis, via a comprehensive survey of a transcriptome database and subsequent cDNA cloning. Structural analysis of the two ue-VTGs revealed their unique features, namely the absence of two domains usually found in VTGs from other organisms: the von Willebrand factor D domain (vWD) and the domain of unknown function 1943 (DUF1943). Levels of ue-VTG1 and ue-VTG2 transcripts in the ovary, specifically in follicular cells, increased during the late-vitellogenic phase, suggesting that yolk accumulation progresses via paracrine interactions involving follicular cells and oocytes. N-terminal amino acid sequencing of biochemically purified yolk protein revealed its origins from these two VTGs, indicating that both are functional precursors of yolk protein. These results provide information that is essential to understanding the physiological pathway of yolk synthesis, accumulation, and storage in loliginid squids.

Degradation of Distillery Lees (Shochu kasu) by Cellulase-Producing Thraustochytrids.

Single cell oils produced by oleaginous microorganisms have attracted increasing interests as a petroleum alternative energy. Marine eukaryotes, thraustochytrids were heterotrophic, and can grow rapidly and accumulate large amount of lipids containing functional fatty acids, such as docosahexaenoic acid (DHA) in their cells body. In this investigation, thraustochytrids isolated from marine environment were cultured in the medium containing an industrial waste and an unused resource, distillery lees (Shochu kasu) to produce biofuel or functional fatty acids by microorganisms. Sixty-nine thraustochytrids and Schizochytrium aggregatum ATCC 28209 were screened for cellulase production, and the activities were detected using sodium carboxymethyl cellulose (CMC) as a substrate. Based on the screening test, strain TM02Bc identified to Schizochytrium sp. was selected for the Shochu kasu degradation test and compared with S. aggregatum ATCC 28209 previously known as a cellulase-producing thraustochytrid. Strains TM02Bc and ATCC 28209 were cultured in artificial seawater containing Shochu kasu for 15 days. The two strains could degrade Schochu kasu, especially that from sweet potato Shochu (Imo Shochu). Cellulase (CMCase) and protease activities were detected in culture supernatant of both strains, and the ratio of polyunsaturated fatty acids (PUFAs) significantly increased as a result of incubation of Shochu kasu with two strains. This preliminary study indicated that strain TM02Bc was a potent candidate for Shochu kasu treatment and fatty acid production.

Possible role of the leptin system in controlling puberty in the male chub mackerel, Scomber japonicus.

Leptin directly regulates kisspeptin neurons in the hypothalamus and gonadotropin secretion from the pituitary, making it a central player in the onset of mammalian puberty. Recently, we identified two leptin genes (lepa and lepb) and a single leptin receptor (lepr) in the marine perciform fish chub mackerel; however, the expression of these genes did not correlate with the expression of important reproductive genes or ovarian stage during female puberty. Here, we expand upon these initial observations by evaluating the expression of lepa, lepb, and lepr during pubertal transition and under differential feeding conditions in the male chub mackerel. Reverse transcription-polymerase chain reaction (RT-PCR) showed that lepa was primarily expressed in the liver of pubertal and gonadal recrudescence adults, as well as in the brain of adult fish; lepb was primarily expressed in the brain of all fish tested; and lepr was widely expressed in a variety of tissues. qRT-PCR analyses revealed significant increases in the hepatic expression of lepa in accordance with testicular stage, whereas pituitary follicle-stimulating hormone (fshß) expression increased in unison with hepatic lepa. In contrast, expression of both brain lepa and lepb dramatically decreased during pubertal transition, with brain kisspeptin 1 (kiss1) expression strongly correlating with leptin expression patterns. In pre-pubertal males, lepa, lepb, and lper gene expression in the brain, pituitary gland, and liver decreased in fish given a high feed diet, relative to the controlled feeding group. Taken together, these results indicate high sexual specificity of leptin expression, suggesting a possible role for leptin signaling in endocrine and neuroendocrine functions during spermatogenesis in the pubertal male chub mackerel.

Two leptin genes and a leptin receptor gene of female chub mackerel (Scomber japonicus): Molecular cloning, tissue distribution and expression in different obesity indices and pubertal stages.

Leptin is a hormone produced by fat cells that regulates the amount of fat stored in the body and conveys nutritional status to the reproductive axis in mammals. In the present study we identified two subtypes of leptin genes (lepa and lepb) and a leptin receptor gene (lepr) from chub mackerel (Scomber japonicus) and there gene expression under different feeding conditions (control and high-feed) and pubertal development stages was analyzed using quantitative real-time PCR. The protein lengths of LepA, LepB and LepR were 161 amino acids (aa), 163 aa and 1149 aa, respectively and both leptin subtypes shared only 15% similarity in aa sequences. In pubertal females, lepa was expressed in the brain, pituitary gland, liver, adipose tissue and ovary; however, in adult (gonadal maturation after the second in the life) females, lepa was expressed only in the liver. lepb was expressed primarily in the brain of all fish tested and was expressed strongly in the adipose tissue of adults. lepr was characterized by expression in the pituitary. The high-feed group showed a high conditioning factor level; unexpectedly, hepatic lepa and brain lepr were significantly more weakly expressed compared with the control-feed group. Furthermore, the expression levels of lepa, lepb and lepr genes showed no significant differences between pre-pubertal and post-pubertal fish. On the other hand, pituitary fshß and lhß showed no significant differences between different feeding groups of pre-pubertal fish. In contrast, fshß and lhß expressed abundantly in the post-pubertal fish of control feed group. Based on these results, whether leptin plays an important role in the nutritional status and pubertal onset of chub mackerel remains unknown.

Subcutaneous administration of Kiss1 pentadecapeptide accelerates spermatogenesis in prepubertal male chub mackerel (Scomber japonicus).

Kisspeptins, encoded by kiss genes, have emerged as critical regulator of reproductive function in vertebrates. Our previous studies demonstrated that the chub mackerel (Scomber japonicus) brain expresses kiss1 and kiss2 and peripheral administration of synthetic Kiss1 pentadecapeptide (Kiss1-15) but not Kiss2 dodecapeptide (Kiss2-12) induces spermiation in sexually immature adult chub mackerel. In the present study, we evaluated the potency of Kiss1-15, Kiss2-12, and GnRH analogue (GnRHa) to induce pubertal onset in prepubertal chub mackerel. Peptides were administered through subcutaneous injection for three times (bi-weekly) over 6weeks. Interestingly, gonadosomatic index (GSI) of Kiss1-15 treated fish increased significantly in comparison to other treatments. Histologically, 66.7% of Kiss1-15 treated fish exhibited presence of spermatozoa (SPZ) in the testes with only 28.6% of GnRHa treated fish. However, Kiss2-12 treated fish showed only spermatocytes (SC) as the advanced germ cells in the testes. In contrast, only spermatogonia (SPG) were observed in the testes of control fish. Changes in the number of testicular germ cells among treatments revealed a significantly higher number of SC, spermatids and SPZ in the Kiss1-15 treated fish. Gene expression analyses revealed no significant changes in gnrh1 in the telencephalon-preoptic region of the brain, including fshß and lhß in the pituitary of experimental fish. However, GnRHa treated fish showed significantly higher lhß expression. Levels of sex steroids, 11-ketotestosterone and estradiol-17ß were significantly higher in Kiss1-15 treated fish. These results indicate application of Kiss1-15 peptides for accelerating pubertal onset in chub mackerel.

Effect of trace elements on growth of marine eukaryotes, tharaustochytrids.

We determined the effect of trace elements on the growth of thraustochytrids. The growth of the strains cultured with the trace elements was much higher than that of the strains cultured without any trace element. Iron and zinc were particularly important to obtaining the optimum growth of thraustochytrids.

Versatile transformation system that is applicable to both multiple transgene expression and gene targeting for Thraustochytrids.

A versatile transformation system for thraustochytrids, a promising producer for polyunsaturated fatty acids and fatty acid-derived fuels, was established. G418, hygromycin B, blasticidin, and zeocin inhibited the growth of thraustochytrids, indicating that multiple selectable marker genes could be used in the transformation system. A neomycin resistance gene (neo(r)), driven with an ubiquitin or an EF-1α promoter-terminator from Thraustochytrium aureum ATCC 34304, was introduced into representatives of two thraustochytrid genera, Aurantiochytrium and Thraustochytrium. The neo(r) marker was integrated into the chromosomal DNA by random recombination and then functionally translated into neo(r) mRNA. Additionally, we confirmed that another two genera, Parietichytrium and Schizochytrium, could be transformed by the same method. By this method, the enhanced green fluorescent protein was functionally expressed in thraustochytrids. Meanwhile, T. aureum ATCC 34304 could be transformed by two 18S ribosomal DNA-targeting vectors, designed to cause single- or double-crossover homologous recombination. Finally, the fatty acid Δ5 desaturase gene was disrupted by double-crossover homologous recombination in T. aureum ATCC 34304, resulting in an increase of dihomo-γ-linolenic acid (C(20:3n-6)) and eicosatetraenoic acid (C(20:4n-3)), substrates for Δ5 desaturase, and a decrease of arachidonic acid (C(20:4n-6)) and eicosapentaenoic acid (C(20:5n-3)), products for the enzyme. These results clearly indicate that a versatile transformation system which could be applicable to both multiple transgene expression and gene targeting was established for thraustochytrids.

Detection of genes involved in fatty acid elongation and desaturation in thraustochytrid marine eukaryotes.

Heterotrophic marine protists known as thraustochytrids can synthesize polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA). The biosynthetic pathways of PUFAs in thraustochytrids are poorly understood, however. In this study, we attempted to reveal the enzymes involved in DHA synthesis in thraustochytrids. Nine thraustochytrid strains representing 3 genera (Aurantiochytrium, Schizochytrium, and Thraustochytrium) were used for PCR-based detection of the genes encoding Δ5-elongase and Δ4-desaturase and for fatty acid analysis. The degenerate primers were designed to amplify the Δ5-elongase and Δ4-desaturase genes, and the partial sequences of the enzymes were obtained from the genera Thraustochytrium and Schizochytrium. These fragments were identical to those of known Δ5-elongase and Δ4-desaturase. Neither Δ5-elongase nor Δ4-desaturase was detected in the strains belonging to the genus Aurantiochytrium, however, suggesting that this group likely synthesizes DHA not via the elongation/desaturation pathway but via an alternate pathway such as the polyketide synthase pathway. The fatty acid profiles of thraustochytrids were consistent with the presence of genes involved in PUFA biosynthesis in thraustochytrid genera. Thus, our findings suggest that two biosynthetic pathways for PUFAs exist in these organisms.

Effects of cold shock treatment on total lipid content and fatty acid composition of Aurantiochytrium limacinum strain mh0186.

To examine the effect of cold shock treatment on the fatty acid composition of Aurantiochytrium limacinum strain mh0186, a marine thraustochytrid, we cultivated this strain at 28°C for 72 h with shaking and stored the obtained biomass at 10°C for 72 h. A growth experiment was carried out for comparison, wherein strain mh0186 was grown at 10 and 15°C for 72 h with shaking, and it was found that the unsaturation of fatty acids was accelerated relative to that at 28°C. In the cold shock experiment, the total lipid content significantly increased during storage at 10°C for 72 h. Overall, the percentage of unsaturated fatty acids such as docosahexaenoic acid was almost stable while that of n-6 docosapentaenoic acid decreased slightly, but significantly, relative to that in the growth experiment.

Effect of Tween 80 on the growth, lipid accumulation and fatty acid composition of Thraustochytrium aureum ATCC 34304.

Thraustochytrium aureum ATCC 34304 was grown in the presence and absence of polyoxyethylene sorbitan monooleate (Tween 80). The aim of this work was to obtain basic knowledge about the effect of Tween 80 on growth, lipid accumulation and fatty acid composition in T. aureum. The addition of Tween 80 to a culture medium significantly enhanced the growth of T. aureum, and the biomass increased with an increase of Tween 80 content. Total lipid content and total fatty acid content were significantly higher in 1.0% Tween 80 in comparison with the control (absence of Tween 80). The fatty acid profile showed that the content of C18:1n-9 (oleic acid) significantly increased as a result of the addition of Tween 80. These results indicated that part of the Tween 80 added to the medium was utilized as a carbon source or that the oleate included in Tween 80 was directly incorporated into T. aureum cells as a fatty acid. Neither the DHA content nor the percentage of DHA did not change in spite of the addition of Tween 80. However, the DHA yield significantly increased because the biomass increased due to the addition of Tween 80.

The distribution of extracellular cellulase activity in marine Eukaryotes, thraustochytrids.

Cellulolytic ability was evaluated in 19 strains of thraustochytrids, representing nine genera, using carboxymethylcellulose (CMC) as a substrate. Extracellular cellulolytic enzyme activity was determined in the culture supernatants during cell growth. CMC hydrolysis was observed in 14 out of the 19 strains examined. These belonged to the genera Aplanochytrium, Botryochytrium, Oblongichytrium, Parietichytrium, Schizochytrium, Sicyoidochytrium, Thraustochytrium and Ulkenia. On the other hand, cellulolytic enzyme activity was not detected in any strains belonging to the genus Aurantiochytrium.

Use of an antifungal drug, amphotericin B for isolation of thraustochytrids.

The inhibitory effect of amphotericin B (AMPH) on the growth of fungi during the isolation of thraustochytrids was examined. The growth of fungi was significantly inhibited by addition of AMPH, and therefore colonies of thraustochytrids were not overlaid with fungal mycelia, which resulted in increased efficiency of thraustochytrids isolation.

Optimization of culture conditions for growth and docosahexaenoic acid production by a marine thraustochytrid, Aurantiochytrium limacinum mh0186.

The effects of carbon sources, seawater concentration and seawater component in a culture medium were investigated to optimize culture conditions for growth by a marine thraustochytrid Aurantiochytrium limacinum strain mh0186. Strain mh0186 could utilize D-glucose, D-fructose and D-mannose as carbon sources. Seawater concentrations between 12.5 - 200% were required for good growth, and a single omission of magnesium sulfate from the seawater reduced the growth of the cells. Jar fermentor trials were carried out for the purpose of docosahexaenoic acid (DHA) production by strain mh0186. The total fatty acid content of the cell was 466.5 mg/g dry cells, and biomass and DHA yield were estimated as 23.1 g/L and 4.3 g/L, respectively, at 26 h. The daily production of DHA by the strain was 4.0 g/L/d, suggesting that the higher DHA production rate of our strain mh0186 should be appropriate for industrial production of DHA.

Extracellular enzymes produced by marine eukaryotes, thraustochytrids.

Extracellular enzymes produced by six strains of thraustochytrids, Thraustochytrium, Schizochytrium, and Aurantiochytrium, were investigated. These strains produced 5 to 8 kinds of the extracellular enzymes, depending on the species. Only the genus Thraustochytrium produced amylase. When insoluble cellulose was used as substrate, cellulase was not detected in the six strains of thraustochytrids. This study indicates that marine eukaryotes, thraustochytrids, produced a wide variety of extracellular enzymes.

Influences of culture temperature on the growth, lipid content and fatty acid composition of Aurantiochytrium sp. Strain mh0186.

The growth, lipid content, and fatty acid composition of Aurantiochytrium sp. strain mh0186 at different temperatures were investigated. Strain mh0186 grew well at 15-30 degrees C, but weakly at 10 degrees C. The biomass at 15-30 degrees C was significantly higher than at 10 and 35 degrees C, and the total lipid at 15-35 degrees C was significantly higher than that at 10 degrees C. The amount of DHA in the total fatty acid was highest at 10 degrees C and decreased in response to temperature increase. The content of DHA (mg/g-dry cell weight) at 15-30 degrees C were significantly higher than those at 35 degrees C and those at 15-25 degrees C were significantly higher than those at 10 and 35 degrees C. The DHA yield at 15-35 degrees C was significantly higher than those at 10 and 35 degrees C. Unsaturation of fatty acid was regulated by temperature and was enhanced in response to temperature decrease. The ratio of DHA to DPA varied at different temperatures.

Effect of addition of Tween 80 and potassium dihydrogenphosphate to basal medium on the isolation of marine eukaryotes, thraustochytrids.

Tween 80, KH(2)PO(4) and tomato juice were added to basal medium for the isolation of thraustochytrids. By the addition of Tween 80 and KH(2)PO(4), the number of thraustochytrids isolated from seawater increased. KH(2)PO(4) and Tween 80 were considered to be useful for isolating thraustochytrids.