Sea Squirt Halocynthia Roretzi Research Articles

A Novel Carotenoid with a Unique 2,6-Cyclo-ψ-End Group, Roretziaxanthin, from the Sea Squirt Halocynthia roretzi

A novel carotenoid with a unique 2,6-cyclo-ψ-end group, named roretziaxanthin (1), was isolated from the sea squirt Halocynthia roretzi as a minor carotenoid along with (3S,3'S)-astaxanthin, alloxanthin, halocynthiaxanthin, mytiloxanthin, mytiloxanthinone, etc. This structure was determined to be 3-hydroxy-1',16'-didehydro-1',2'-dihydro-2',6'-cyclo-β,ψ-carotene-4,4'-dione by UV-VIS, MS, and NMR spectral data. The formation mechanism of roretziaxanthin in the sea squirt was discussed.

Growth of the longline-cultured sea squirt Halocynthia roretzi in a temperate bay of Korea: Biochemical composition and physiological energetics

The broad temperature range on the temperate coast of Korea has led fishermen to develop a unique and specialized procedure for the longline culture of ascidians. In Korea, warming of the coastal sea in winter has accelerated over the past few decades. After warmer winters, the rising temperatures of earlier springs have precluded the rearing of the ascidian spat in the culturing area and have imposed spring spat cultivation in colder-water nursery grounds. To examine the seasonal dynamics of energy reserves and the physiological strategies to optimize energy balance for the growth in the cultured sea squirt Halocynthia roretzi, its gross biochemical composition and physiological energetics were investigated monthly over two culturing periods from July 2005 to January 2006 and July 2013 to January 2014 in Geoje-Hansan Bay on the south coast of Korea. No indicators of the growth performance of sea squirts showed differences between the two culture practices (2005–2006 and 2013–2014) carried out using spat reared in different localities, i.e., the grow-out area and the colder nursery grounds. The seasonal patterns in accumulation and utilization of biochemical constituents in the sea squirt tissues were similar between the two periods. During the culturing period in the grow-out area, sea squirts retained physiological functions across the temperature range. Food energy acquisition and metabolic cost were positively related to water temperatures in the field conditions, probably due to the low and narrow range of suspended particulate matter (SPM) concentrations. A less-clear seasonal variability in food consumption rates yielded a seasonal discrepancy in scope for growth (SFG; i.e., negative during summer vs positive during autumn–winter). Consequently, the tissue weight and protein reserves of sea squirts varied concomitantly with the seasonal changes in SFG, supporting a fast growth during autumn–winter. Our results suggest that the spat reared in colder nursery grounds are suitable for cultivating sea squirts in the traditional grow-out area and support their sustainable culturing performance as an adaptation strategy to the winter–spring warming conditions that are unique to coastal seas.

Inhibitory Effects of Vanadium-Binding Proteins Purified from the Sea Squirt Halocynthia roretzi on Adipogenesis in 3T3-L1 Adipocytes.

The inhibitory effects of vanadium-binding proteins (VBPs) from the blood plasma and the intestine of sea squirt on adipogenesis in 3T3-L1 adipocytes were examined. 3T3L-1 cells treated with VBP blood plasma decreased markedly the lipid content in maturing pre-adipocytes in a dose-dependent manner, whereas VBP intestine did not show significant effects on lipid accumulation. Both VBPs did not have significant effect on cell viability. In order to demonstrate the anti-adipogenic effects of VBP blood plasma, the expressions of several adipogenic transcription factors and enzymes were investigated by Reverse Transcriptase-Polymerase Chain Reaction. VBP blood plasma down-regulated the expressions of transcription factors; PPAR-γ, C/EBP-α, SREBP1, and FAS, but did not have significant effects on the expressions of lipolytic enzymes; HSL and LPL. Both the crude and purified VBPs significantly increased the mRNA levels of Wnt10b, FZ1, LRP6, and β-catenin, while decreased the expression of GSK-3β. Hence, VBP blood plasma inhibited adipogenesis by activating WNT/β-catenin pathway via the activation of Wnt10b. Based on the findings, VBP blood plasma decreased lipid accumulation which was mediated by decreasing adipogenesis, not by lipolysis. Therefore, VBP blood plasma could be used to treat obesity.

Sea Squirt (Halocynthia roretzi) Hydrolysates Induce Apoptosis in Human Colon Cancer HT-29 Cells through Activation of Reactive Oxygen Species

Recent evidence provides that seafood has a lot of health benefits due to its unique bioactive compounds. Sea squirt is widely cultured and consumed as a foodstuff in Korea; however, seldom reports with reference to bioactivities are available until now. In this study, edible part of sea squirt was hydrolyzed by pepsin and its hydrolysates was evaluated for anticancer effect on human colon cancer HT-29 cells. Sea squirt hydrolysates (SSQ) reduced HT-29 cell viability. Treatment with SSQ resulted in the increase in reactive oxygen species (ROS) generation followed by disruption of mitochondrial membrane potential (MMP). Flow cytometry analysis revealed that SSQ induced G2/M phase arrest and apoptosis evidenced by Hoechst 33342 staining. Levels of mRNA expression by real-time polymerase chain reaction (PCR) showed that treatment with SSQ in HT-29 cells upregulated expression of p53, bax, and caspase-3 genes and downregulated expression of bcl-2 gene. Protein level of cytochrome c into cytosol and caspase-3 by Western blotting were also increased by treatment with SSQ in HT-29 cells. These results suggest that SSQ may be useful for functional food ingredients and/or nutraceuticals.

Antioxidant and antidiabetic activities of vanadium binding proteins purified from the sea squirt Halocynthia roretzi.

Sea squirts accumulate vanadium compounds with potent antidiabetic activity, which are involved in immune defense. In this study, vanadium concentrations of fresh blood plasma, intestine, and muscle of the sea squirt Halocynthia roretzi were 6.3, 3.7 and 2.1mg/kg respectively. Two vanadium binding proteins (VBPs) from blood plasma and intestine were purified through (NH4)2SO4 precipitation, and DEAE-Sepharose ion exchange and Sephacryl S-200 HR gel filtration chromatography, in that order. The purity and yield of the intestine and blood plasma vanadium binding proteins, VBPintestine and VBPblood plasma, were 13.4 folds and 7.1%, and 20.9 folds and 6.8%, respectively. There were two protein bands on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with molecular weights of 24.3 and 68.8kDa and one with 96.7kDa on the native-PAGE of VBPblood plasma, whereas only one protein band of VBPintestine on the SDS-PAGE with 26.5kDa. Antioxidant activities of VBPs were lower than that of ascorbic acid. Both VBPs exerted strong inhibitory activity against Saccharomyces cerevisiae and mild against Bacillus stearothermophilus and rat intestinal α-glucosidase. IC50 values of VBPintestine and VBPblood plasma against S. cerevisiae α-glucosidase were 28.34 and 12.60μg/ml, respectively. The Km , Vmax , kcat , and kcat/Km values of VBPintestine and VBPblood plasma were 4.29, 0.036, 6.58 and 1.53 × 103, and 7.63mM, 0.057mM/min, 10.41s-1 and 1.36 × 103 (Msec)-1, respectively. There was a synergistic interaction between VBPblood plasma and VBPintestine on rat intestinal α-glucosidase inhibitory activity.

Shelf life of Bottled Sea Squirt Halocynthia roretzi Meat Packed in Vegetable Oil (BSMO)

Fresh sea squirt meat requires a modified processing and preservation process because it has a short shelf life due to its high mois ture content and strong proteolytic enzyme activity. In this study, bottled sea squirt meat prepared in vegetable oil (BSMO) to enhance the consumer acceptability was exposed to γ-ray (Co60, 10 kGy/h) irradiation to extend the shelf life without the use of a heating process. Response surface methodology was used to determine the optimal mixing ratio of BSMO containing 5% dehydrated fresh meat. Texture analysis and nutritional evaluation were also performed on a control and BSMO samples. The volatile basic nitrogen (VBN) content and total cell count were measured to determine the shelf life of irradiated BSMO products during chilled storage at 4°C for 60 days. According to a panel of 10 trained tasters (aged 20-29 years), the optimal mixing formulation was 80 g meat in 60 mL of mixed vegetable oil (30 mL of olive oil and 30 mL of sesame oil). The highest rated formulation, according to a panel of nine trained tasters (aged ≥30 years), was 80 g meat in 60 mL of mixed vegetable oil (42 mL of olive oil and 18 mL of sesame oil). Moisture, ash, and protein contents in BSMO did not change significantly ( P < 0.05) compared with the control. A higher lipid content (0.84 ± 0.23 to 2.13 ± 0.61; P < 0.05) was observed due to the presence of vegetable oil on the surface of BSMO. The vegetable oil raised the hardness, springiness, cohesiveness, gumminess, chewiness, and resilience of BSMO. BSMO products remained edible after 50 days of storage at 4°C based on the VBN content (BSMO 1: 27.92 ± 0.96 mg/100 g, BSMO 2: 24.84 ± 1.95 mg/100 g) and total cell count (BSMO 1: 4.60 ± 0.80 log CFU/mL, BSMO 2: 3.65 ± 0.20 log CFU/mL) when compared with standard levels of VBN (25.00 mg/100 g) and total cell count (5 log CFU/mL), respectively. The results showed that irradiated BSMO products could help to expand the processed seafood market and increase the popularity of seafood among the younger generations.

멍게(Halocynthia roretzi) 식해 및 조미 멍게의 영양 및 생리활성 특성

【This study was carried out to investigate nutritional and physiologically active characterizations of sikhae and the seasoned products from the sea squirt Halocynthia roretzi. The total taste values of sikhae fermented for 4 and 5 days were 10.9 and 15.4, respectively, which was lower than for commercially seasoned sea squirts. The sikhaes contained mostly glutamic and aspartic acids. The total amino acid contents of sikhaes fermented for 4 and 5 days were 5.5 and 6.0 g/100 g, respectively, which were lower than those of commercial seasoned-sea squirts or similar. An amount of 100 g of sikhae and its seasoned products contained P, K, Mg and Fe, and these minerals, which are deemed good for our health, were at 10% above the recommended daily requirements. The functional properties of sikhae fermented for 4 and 5 days were as follows: for ACE inhibiting activity, 69 and 69.5%, respectively; for antioxidative activity, 28.9 and 29.3%, respectively; for xanthine oxidase inhibitory activity, 52.8 and 53.1%, respectively; and for ${\alpha}$ -glucosidase inhibitory activity, 2.4 and 1.4%, respectively. Antimicrobial activity of the 5 day fermented sikhae against Vibrio parahaemolyticus and Staphyloccus aureus was detected in 8 mm and in 7 mm against Escherichia coli.】

The kinetoplastid parasite Azumiobodo hoyamushi, the causative agent of soft tunic syndrome of the sea squirt Halocynthia roretzi, resides in the East Sea of Korea

Mass mortality of the edible sea squirt Halocynthia roretzi since the 1990s in the southern and eastern seas of Korea has caused large economic losses. The disease is characterized by symptoms of initially softened and thinned tunics that eventually rupture. Thus, the disease is called soft tunic syndrome (STS); however, the causative agent in these regions is unknown. In the present study, two kinetoplastid organisms were isolated from STS sea squirts collected from culture farms in Tongyeong located in the East Sea of Korea. Phylogenetic analysis of 18S rRNA sequences identified these organisms as Azumiobodo hoyamushi and Procryptobia sorokini. These kinetoplastids were injected into healthy sea squirts and cultured at 15°C for 13days. Sea squirts injected with A. hoyamushi showed 100% STS whereas, P. sorokini did not induce disease, thereby confirming A. hoyamushi as the causative agent of STS. A. hoyamushi flourishes in vitro at 10–15°C, and dies at temperatures below 5°C or above 20°C. The optimum salinity level for growth is 30–35psu, and death occurs below 25psu. These optima coincide with marine temperature and salinity levels between March and June on the southern coasts of Korea, the period when the syndrome occurs at the highest frequency. The identification here of A. hoyamushi as the causative agent of STS and our findings regarding its optimum growth conditions should lead to methods for reducing the incidence of STS.

수온과 체중을 고려한 멍게(Halocynthia roretzi)의 여수율

In order to understand the co-culture impact of the sea squirt Halocynthia roretzi on the Pacific oyster Crassostrea gigas, we examined the filtration rate of the sea squirt in relation of water temperature () and body weight. The filtration rate increased in relation to body weight and water temperature, indicating a clear positive correlation with the two variables. Due to the clear positive correlation between filtration rates at each temperature groups for acceptable collinearity, each constants at regression equation was further analyzed for a unified model of filtration rate. Therefore the filtration rate of H. roretzi was estimated as: Fr (L/h/animal)

멍게의 물렁증 발생에 미치는 용존산소의 영향

멍게의 물렁증 발생에 미치는 용존산소의 영향

Colwellia meonggei sp. nov., a novel gammaproteobacterium isolated from sea squirt Halocynthia roretzi

A Gram-negative, non-spore-forming, aerobic, motile and rod-shaped or ovoid bacterial strain, designated MA1-3(T), was isolated from a sea squirt (Halocynthia roretzi) collected from the South sea in South Korea. Strain MA1-3(T) was found to grow optimally at 25 °C, at pH 7.0-8.0 and in the presence of 2.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences revealed that strain MA1-3(T) fell within the clade comprising Colwellia species, clustering coherently with the type strains of Colwellia aestuarii, Colwellia polaris and Colwellia chukchiensis, showing sequence similarity values of 97.2, 96.4 and 95.6 %, respectively. It exhibited 16S rRNA gene sequence similarity values of 93.9-96.1 % to the type strains of the other Colwellia species. Strain MA1-3(T) was found to contain Q-8 as the predominant ubiquinone and C16:1 ω7c and/or C16:1 ω6c, C16:0 and C16:1 ω9c as the major fatty acids. The DNA G+C content of strain MA1-3(T) was determined to be 39.1 mol% and its mean DNA-DNA relatedness value with the type strain of C. aestuarii was 13 ± 5.4 %. The differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that the novel strain is separated from other Colwellia species. On the basis of the data presented, strain MA1-3(T) is considered to represent a novel species of the genus Colwellia, for which the name Colwellia meonggei sp. nov. is proposed. The type strain is MA1-3(T) (=KCTC 32380(T) = CECT 8302(T)).

Tenacibaculum halocynthiae sp. nov., a member of the family Flavobacteriaceae isolated from sea squirt Halocynthia roretzi

A Gram-negative, non-spore-forming, aerobic, non-flagellated, non-gliding and rod-shaped bacterial strain, designated P-R2A1-2(T), was isolated from sea squirt (Halocynthia roretzi) collected from the South Sea, Korea. It grew optimally at 25-28 °C, at pH 7.0-8.0 and in the presence of 2 % (w/v) NaCl. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences revealed that the strain fell within the clade comprising Tenacibaculum species. Strain P-R2A1-2(T) exhibited the highest 16S rRNA gene sequence similarity values of 97.6, 97.2 and 97.0 % to Tenacibaculum aestuarii SMK-4(T), T. lutimaris TF-26(T) and T. aiptasiae a4(T), respectively, and of 94.5-96.8 % to the type strains of the other Tenacibaculum species. Strain P-R2A1-2(T) contained MK-6 as the predominant menaquinone and C16:1 ω7c and/or iso-C15:0 2-OH, iso-C15:0 3-OH and iso-C15:0 as the major fatty acids. The DNA G + C content of strain P-R2A1-2(T) was 30.7 mol % and its DNA-DNA relatedness values with the type strains of T. aestuarii, T. lutimaris and T. aiptasiae were 17 ± 4.2, 21 ± 6.1 and 16 ± 5.2 %, respectively. Differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that the novel strain is separate from other Tenacibaculum species. On the basis of the data presented, strain P-R2A1-2(T) is considered to represent a novel species of the genus Tenacibaculum, for which the name Tenacibaculum halocynthiae sp. nov. is proposed. The type strain is P-R2A1-2(T) (=KCTC 32262(T )= CCUG 63681(T)).

멍게(Halocynthia roretzi) 식해의 개발 및 특성

This study was conducted to develop and characterize sea squirt Halocynthia roretzi sikhae. According to the results for pH, total acidity, lactic acid bacteria, amino nitrogen and sensory evaluation of sea squirt sikhae during fermentation for 6 days at <TEX>$15^{\circ}C$</TEX>, the optimum fermentation periods were 4 days for sourness-disliking customers and 5 days for sour-disliking customers. No differences in the proximate compositions of sea squirt sikhaes fermented for 4 days (4D) and for 5 days (5D) were found. There was a difference in the eproximate compositions of commercial seasoned sea squirts, 4D and 5D, sea squirt sikhaes. The results of salinity, total acidity, amino nitrogen and sensory evaluation of two kinds of sikhae suggest that the taste was stronger for 5D than for 4D, both of which were superior to commercial seasoned sea squirts. There was, however, no difference in color of 4D, 5D and commercial seasoned sea squirts. The results of E. coli analyses suggest that sea squirt sikhae is a safe food in terms of sanitation.

시판 조미 멍게(Halocynthia roretzi)의 맛, 영양 및 건강기능 특성

This study examined the taste, nutritional and functional characterizations of commercial seasoned sea squirt Halocynthia roretzi (CSS). Total taste values of CSS ranged from 7.6 to 69.5 and the major free amino acids were glutamic acid and aspartic acid. Total contents of amino acids in CSS ranged from 5.91 to 7.59 g/100 g and the major amino acids were also glutamic acid and aspartic acid. When taking 100 g of CSS, the minerals that could be expected to have functional health effects (minerals whose levels were above 10% of the recommended daily requirements) were P, Mg and Fe. Other minerals were also present in non-negligible quantities. In terms of the functional properties of CSS, ACE inhibitory activity was 21.2-37.1%, antioxidative activity was 55.4-90.4%, xanthine oxidase inhibitory activity was 52.9-76.6% and -glucosidase inhibitory activity was 0-32%. Antimicrobial activity against Vibrio parahaemolyticus was not detected, but activity against Staphylococcus aureus, groups such as KB, GG, CY, DN, HC and KH, and against Escherichia coli groups such as SF, WD, KB and GG, was detected.

시판 조미 멍게(Halocynthia roretzi)의 식품학적 품질 특성

This study investigated the food biochemical characterization of commercial seasoned sea squirt Halocynthia roretzi (CSS). The proximate composition of CSS was 77.2-82.7% moisture, 7.1-9.1% crude protein, 0.3-2.6% crude lipid and 3.5-6.3% ash. Taste compound contents of CSS were 2.3-5.4% salinity (saltiness), 0.42-1.12 g/100 g total acidity (sourness) and 114.9-330.2 mg/100 g amino nitrogen (taste intensity). The Hunter color values of CSS were 23.79-32.50 for lightness, 9.97-20.45 for redness, 14.01-20.96 for yellowness and 64.50-76.63 for color difference. The odor intensity of CSS was 35.0-62.0. According to these results, there were large differences in proximate composition, taste compounds, Hunter color values and odor intensity of CSS. Viable cell counts ranged from 6.20 to 7.69 log (CFU/g), and most of the viable cells comprised of lactic acid-forming bacteria. CSS was not detected in the coliform group.

Effects of Heavy Metals on Clearance and Oxygen Consumption Rates of the Sea Squirt Halocynthia roretzi According to Various Body Sizes

Effects of Heavy Metals on Clearance and Oxygen Consumption Rates of the Sea Squirt Halocynthia roretzi According to Various Body Sizes

Identification of cellulolytic bacteria associated with tunic softness syndrome in the sea squirt, Halocynthia roretzi

The edible ascidian, sea squirt Halocynthia roretzi (Drasche) is marine invertebrate that is a valuable source of foods and bioactive compounds. A severe disease of the sea squirt characterized by degeneration of tunic fibers formed of bundled cellulose microfibrils has occurred. We hypothesized that bacteria lyse the cellulose fibril structure, cellulase activity may be a causative agent of the disease. Among the bacteria isolated from diseased sea squirt, Pseudoalteromonas sp. NO3 had cellulase activity based on a Congo red overlay assay and starch-reducing activity. Sea squirts exhibited 40–100% cumulative mortality after injection with Pseudoalteromonas sp. NO3 using doses of 2×106−2×108 colony forming unit (CFU)/individual. Dead sea squirts possess thinner and ruptured tunics, which were similar to natural outbreaks. These results suggest that Pseudoalteromonas sp. NO3 possessing cellulase activity is one of the causes of tunic softness syndrome in sea squirt.

Genetic Variation in Wild and Cultured Populations of the Sea Squirt Halocynthia roretzi Inferred from Microsatellite DNA Analysis

We used nine microsatellite DNA markers to estimate genetic variation among wild and cultured populations of the sea squirt Halocynthia roretzi. The loci were polymorphic, with 6-32 alleles, and allelic richness ranged from 6.0 to 26.1 in each population. The wild and the cultured populations had similar mean heterozygosities (<TEX>$H_O$</TEX> and <TEX>$H_E$</TEX>), allele numbers, and allelic richness. One cultured population with softness syndrome had a lower mean in the observed heterozygosity (<TEX>$H_O$</TEX> = 0.57) and higher mean inbreeding coefficient (<TEX>$F_{IS}$</TEX> = 0.261) than any other populations. This suggests that the loss of genetic variation in the diseased population might be due to increased inbreeding. A neighbor-joining tree and pairwise population estimates of <TEX>$F_{ST}$</TEX> showed moderate genetic differentiation between the wild and the cultured populations. Additionally, the softness syndrome population was genetically divergent from wild populations, but it was genetically close to the cultured populations.

Ruegeria halocynthiae sp. nov., isolated from the sea squirt Halocynthia roretzi

A Gram-negative, non-motile, rod-shaped bacterial strain, designated MA1-6T, was isolated from a sea squirt (Halocynthia roretzi) collected from the South Sea, Korea, and was characterized to determine its taxonomic position. Strain MA1-6T grew optimally at pH 7.0-8.0, at 30 °C and in the presence of 2-3 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain MA1-6T fell within the clade comprising Ruegeria species and exhibited 95.3-96.5 % similarity to the type strains of recognized Ruegeria species. Strain MA1-6T contained Q-10 as the predominant ubiquinone and C18:1ω7c as the major fatty acid, which is consistent with data for Ruegeria species. The major polar lipids detected in strain MA1-6T and Ruegeria atlantica KCTC 12424T were phosphatidylcholine, phosphatidylglycerol, an unidentified aminolipid and an unidentified lipid. The DNA G+C content of strain MA1-6T was 58.6 mol%. Differential phenotypic properties, together with phylogenetic distinctiveness, demonstrated that strain MA1-6T can be distinguished from recognized Ruegeria species. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain MA1-6T is considered to represent a novel species of the genus Ruegeria, for which the name Ruegeria halocynthiae sp. nov. is proposed; the type strain is MA1-6T (=KCTC 23463T=CCUG 60744T).

난질과 유생발생을 이용한 4개 멍게(Halocynthia roretzi) 어미계군 특성 비교

To compare four populations of sea squirt Halocynthia roretzi adults, their egg quality and larval development were investigated in the hatchery. The populations were: south sea wild (SSW), south sea cultured (SSC), east sea wild (ESW) and Iwate Japan cultured (IJC). Egg quality and larval development were compared using 13 factors (fertilization rate and diameter, proximate composition, amino acid and fatty acid contents of eggs, hatching rate and various sizes of tadpole larvae) which were obtained from each population. Fertilized egg diameter, hatching rate and size of tadpole larvae were significantly different among the four populations (P<0.05). Eggs were produced with higher crude protein (<TEX>$5.20{\pm}0.00%$</TEX> SSW, <TEX>$4.71{\pm}0.01%$</TEX> ESW, <TEX>$4.66{\pm}0.01%$</TEX> SSC and <TEX>$3.96{\pm}0.01%$</TEX> IJC) and lipid (<TEX>$1.22{\pm}0.01%$</TEX> ESW, <TEX>$1.01{\pm}0.00%$</TEX> SSW, <TEX>$0.77{\pm}0.01%$</TEX> SSC and <TEX>$0.69{\pm}0.00%$</TEX> IJC,) contents from domestic wild populations than from Japanese or cultured populations. Also amino acid and fatty acid contents were different. The extent of similarity between domestic and Japanese populations (30.5% IJC:SSW, 34.3% IJC:SSC and 40.7% IJC:ESW) was relatively low but was very high between SSW and SSC (73.9%). These results may have been due to differences in the abundance of food types and environmental conditions in the four localities and consequent differences in the diets of the sea squirts.