Establishment of a model for chemoattraction of <Emphasis Type="Italic">Symbiodinium</Emphasis> and characterization of chemotactic compounds in <Emphasis Type="Italic">Acropora tenuis</Emphasis>

Corals harbor symbiotic dinoflagellates, Symbiodinium spp., acquired from surrounding environments. Because Symbiodinium are present at low densities in the water column, corals may attract these symbionts using chemotactic compounds. To examine whether corals contain chemotactic compounds, we established an assay to measure the chemotactic activity for Symbiodinium using an extract of the coral Acropora tenuis, a major reef-building coral in Japan. Our assay revealed that Symbiodinium strain NBRC102920 (clade A), which is taken up by juvenile A. tenuis polyps, is attracted to crude A. tenuis extracts. We found that the chemotactic compounds are water-soluble, heat-labile macromolecules and that the chemotactic activity was inhibited by N-acetyl-d-glucosamine (GlcNAc). We separated the GlcNAc-binding fraction (Fr-ActL) and identified it as the most plausible candidate for the chemoattractant, since the chemotactic activity of the crude A. tenuis extract appeared to be mainly attributable to the activity of Fr-ActL and was also inhibited by the addition of GlcNAc. These results indicate that chemoattraction is mediated via the binding of Symbiodinium to Fr-ActL.     

Effects of the commercial probiotic Lactobacillus casei on the growth,protein content of skin mucus and stress resistance of juveniles of the Porthole livebearer Poecilopsis gracilis (Poecilidae)

A 11‐week feeding trial was carried out to determine the effects of the probiotic bacteria Lactobacillus casei from the commercial product Yakult^® on the growth performance, proximal composition, protein content of skin mucus and stress resistance of juvenile Porthole livebearer Poeciliopsis gracilis. Triplicate groups of 15 juveniles per tank with an initial weight of 47 ± 9 mg (mean ± standard deviation) were fed with Artemia nauplii enriched with the probiotic, by using the bacteria cells plus the fermented milk (group ProN) and the other (group ProC) by using only the bacterial cells, eliminating the fermented milk by centrifugation. A control of fish was set up, by feeding non‐enriched Artemia nauplii. Growth performance and survival rates did not show significantly differences among the treatments and control group, but a slightly tendency of higher values for body weight, weight gain and specific growth rate was observed in the juveniles of ProC treatment. Whole body proximate composition did not show significant differences among the groups, but higher values of protein and lipid contents were observed in the groups fed with the probiotic. Content of protein in the skin mucus were significantly higher in the ProC treatment than control group. Recovery rates after an air‐dive test were significantly higher on the fish fed with the probiotic cells than the control group. These results show that L. casei might be used as a probiotic for fish and would help during the culture of juvenile of the Porthole livebearer P. gracilis.     

Effects of lectin in the scleractinian coral Ctenactis echinata on symbiotic zooxanthellae

We report herein the presence of a lectin in the scleractinian coral Ctenactis (Fungia) echinata. The lectin bound preferentially to lactose, melibiose, and d-galactose. The purified lectin CecL was composed of several isolectins, and it was found to have a molecular mass of 67.4 kDa via gel filtration. Glycopeptidase F-treated CecL showed a single band at 32.5 kDa. The mass/charge ratios of the reduced CecL peaks were equivalent to half those of the native peaks. These results suggest that CecL is composed of two glycosylated polypeptides linked by interchain disulfide bonds. In a biological activity test using a zooxanthellal culture (Dinoflagellate Symbiodinium) clonally isolated from Fungia cf. fungites, CecL transformed the flagellated motile form of Symbiodinium into the nonmotile coccoid form, a form equivalent to the symbiotic stage. The activity of CecL on Symbiodinium cells was concentration dependent, and 100 μg/ml CecL arrested Symbiodinium cells in the coccoid form for 5 days. CecL also suppressed the growth of Symbiodinium cells, unlike the octocoral lectin derived from Sinularia lochmodes, which arrests Symbiodinium cells in the coccoid form but does not affect the growth of the coccoid. This result provides further evidence that coral lectins play a role in symbiont engagement and maintenance in zooxanthellae–coral symbiosis.     

Isolation,identification and pathogenicity of Vibrio harveyi,the causal agent of skin ulcer disease in juvenile hybrid groupers Epinephelus fuscoguttatus × Epinephelus lanceolatus

The hybrid grouper, Epinephelus fuscoguttatus (♀) × Epinephelus lanceolatus (♂), is a newly bred cultivated marine fish species of high economic value. However, a skin ulcer disease with high mortality has occurred, and the responsible pathogen remains unknown. In this study, we summarized the epidemic status and external signs of this disease. We screened potential pathogens and finally isolated one bacterial strain ML01 from affected fish. We subjected healthy juvenile hybrid groupers to bacterial challenge tests with the isolate by immersion, immersion after dermal abrasion and intraperitoneal injection, respectively. Within 14 days post‐infection, the isolate ML01 caused mass mortality of juveniles infected via immersion after dermal abrasion or intraperitoneal injection. Diseased juveniles displayed obvious signs of skin ulcers. The median lethal dose of ML01 by intraperitoneal injection was 1.10 × 10⁵ colony‐forming units. ML01 was identified as Vibrio harveyi by bacterial morphology, analytical profile index identification, 16S rDNA sequencing and multilocus sequence analysis. Antibiotic susceptibility tests showed that ML01 was sensitive to ceftriaxone, doxycycline and minocycline. The results of this study suggest that V. harveyi is the causal agent of skin ulcer disease in juvenile hybrid groupers, thus providing a basis for effective control and prevention of this disease.     

Digestive enzymes and in-vitro digestibility of different species of phytoplankton for culture of the freshwater pearl mussel, <Emphasis Type="Italic">Hyriopsis</Emphasis> (<Emphasis Type="Italic">Hyriopsis</Emphasis>) <Emphasis Type="Italic">bialatus</Emphasis>

Hyriopsis (Hyriopsis) bialatus has been cultured during the mussel life cycle from glochidia to the adult stage with a low total survival of 6% up to 130-day-old juveniles. The main digestive enzymes (amylase and proteinases) were not detectable in one-day-old juveniles, and increased during development. The stomach, including digestive glands, was the major digestive organ for both carbohydrate and protein. The optimum conditions for amylase activity were 40°C and pH 7; for acidic proteinases they were 60°C and pH 5. Two main alkaline proteinases were found in the intestine, with optimum conditions of 30°C and pH 8 and 60°C and pH 8. To improve mussel survival by finding suitable phytoplankton species and age as food for juveniles and adults, an in-vitro digestibility test was performed on ten algal species three and seven days old using amylase and proteinases in crude enzyme extracts from different mussel life stages. Among the phytoplankton selected, the three most efficiently digested by juveniles were seven-day-old Chlorella sp.2, seven-day-old Chlorococcum sp. and seven-day-old Kirchneriella incurvata, in the ratio 1:1:3 for 30-day-old juveniles and 3:1:1 for 130-day-old juveniles. For the adult mussel, three-day-old Chlorella sp.2, seven-day-old Coccomyxa sp., and seven-day-old Monoraphidium sp., in the ratio 3:1:1, were the most digestible phytoplankton. Levels of in-vitro digestibility were related to the quality (not the concentrations) of carbohydrate and protein in the phytoplankton mixtures, and protein digestibility seemed to be the key factor determining food quality for the mussel.     

中华鳖dazl基因克隆及在生殖细胞中的表达

为探索龟鳖类生殖细胞的发育分化机制,实验通过特异性引物克隆了中华鳖dazl基因的cDNA片段,长1 007 bp,其中包括5′端非编码区197 bp,3′端非编码区33 bp,开放阅读框777 bp,编码258个氨基酸。氨基酸序列比对显示其与西部锦龟Dazl同源性最高,达96%;与小鼠Dazl同源性达75%。荧光定量PCR分析结果显示,中华鳖dazl mRNA主要在精巢和卵巢中表达,在体细胞组织中仅检测到微量表达。冰冻切片原位杂交结果显示,中华鳖dazl mRNA在生殖细胞中特异表达,且在不同时期的生殖细胞中呈动态表达模式。在精巢中,中华鳖dazl mRNA在初级和次级精母细胞中表达最强,在精原干细胞中表达水平次之,在精子细胞中未检测到信号;在卵巢中,中华鳖dazl mRNA信号在初级卵母细胞胞质中均匀分布且在最早期的初级卵母细胞中信号最强,随着卵母细胞的增大,信号逐渐聚集并逐渐减弱。研究表明,dazl基因可能对中华鳖两性生殖细胞的发生具有重要的调控作用。     

Effect of dietary protein and energy levels on growth,oxygen consumption,haemolymph and digestive gland carbohydrates,nitrogen excretion and osmotic pressure of Litopenaeus vannamei (Boone) and L. setiferus (Linne) juveniles (Crustacea,Decapoda; Penaeidae)

The influence of protein and energy levels on growth rate, survival, pre- and post-prandial oxygen consumption, ammonia excretion, haemolymph glucose (HG), glycogen in digestive gland and osmotic pressure (OP) in white shrimp Litopenaeus vannamei (Boone) and L. setiferus (Linne) juveniles was studied. Diets containing a high-quality protein at a protein/energy (P/E) ratio of 16, 26, 31 and 36 mg kJ⁻¹ were fed at 20% of shrimp body weight of two sizes: < 1 g and > 1 g. Both species showed a optimum P/E ratio of 36 mg kJ⁻¹ (33–44% protein and 6–23% carbohydrate) in juveniles < 1 g. For shrimp > 1 g, L. setiferus showed a higher growth rate in the diet with 16 mg kJ⁻¹ (27% protein; 32% carbohydrate) and L. vannamei between 26 and 36 mg kJ⁻¹ (33–44% protein and 6–23% carbohydrate). In both experiments, the growth rate of L. vannamei was 2–3 times that observed in L. setiferus. Routine oxygen consumption and apparent heat increment (AHI) of L. setiferus juveniles was two times higher than that observed in L. vannamei juveniles, which could indicate that L. setiferus has a higher metabolic rate. The O/N ratio varied according to protein level, with higher values (O/N = 180) with a 16-mg kJ⁻¹ diet and lower values (O/N = 73) with a 36-mg kJ⁻¹ diet in L. setiferus juveniles. A similar variation in O/N ratio was obtained in L. vannamei fed with all diets with an interval between 22 and 50. An inverse relation between ammonia excretion and HG, and digestive gland glycogen (DGG) in relation to an increase in the P/E ratio indicate that both shrimp species are well adapted to use carbohydrates and/or proteins from their diet. The higher values of hyper-osmotic capacity (hyper-OC) were observed in L. setiferus < 1 g fed with 36 mg kJ⁻¹ and the lowest in L. vannamei < 1 g fed with 31 mg kJ⁻¹. Intermediate values of hyper-OC were observed in both species fed all diets indicating that osmotic factors of juveniles < 1 g of both species are more affected by the P/E ratio than juveniles > 1 g. All results showed that juveniles > 1 g of both species are less dependent of P/E ratio than juveniles < 1 g. Litopenaeus vannamei is a most tolerant shrimp species with a high capacity to use a wide range of dietary P/E ratios for growth, which may be due to its lower energy requirements. Litopenaeus setiferus showed a lower capacity to accept different P/E ratios but the optimum P/E ratio obtained with this species shows that L. setiferus accept diets with a high carbohydrate level as well. These results demonstrate that there are nutritional and physiological differences that explain the differences that have been observed when both species were cultured in commercial ponds.     

The application of sexual coral recruits for the sustainable management of ex situ populations in public aquariums to promote coral reef conservation — SECORE Project

• 1. SECORE (SExual COral REproduction) Project is an initiative of public aquariums and research institutions to produce and exchange sexual coral recruits for the sustainable management of ex situ populations. Here we present the results of the initial three years (2002–2004).
• 2. Primary polyps (n=501) of corals (Acropora tenuis, Agaricia humilis, Favia fragum) were transported from Rotterdam Zoo to Cologne, Burgers', Hagenbeck and London Zoos, where development of juveniles was monitored for 10 months. All polyps were produced at Rotterdam Zoo from laboratory colonies (A. humilis, F. fragum), and from larvae generated from field collected gametes at Akajima, Okinawa, Japan (A. tenuis). Additionally, planulae of A. tenuis (n=1440) were transported from Rotterdam Zoo to Burgers' Zoo and to London Zoo to obtain primary polyps.
• 3. Larval settlement (A. tenuis) was observed to be 3.00 ± 2.57% (mean ± SD; n=1480) in 2002 and 17.36 ± 6.01% (mean ± SD; n=1480) in 2003, significantly lower compared to settlement at Rotterdam Zoo (57.84 ± 11.01% in 2003; mean ± SD, n=1480). High post‐transport survival rates of 95.18 ± 4.86% (mean ± SD; n=501) were observed in primary polyps of all species.
• 4. Juvenile survival (t=10 months; A. tenuis: 18.4–86.2%; A. humilis: 0–19.7%; F. fragum: 13.3–72.7%) differed significantly between institutions. Mean colony sizes (measured 10 months after transportation) were, in all cases, similar or higher to those reported from literature.
• 5. The results demonstrate the potential of this method to serve as an economical and sustainable alternative to existing mostly exploitative techniques for aquarium stocking. The use of sexual recruits provides an effective and low cost alternative, which is, in principle, applicable to all coral species.
• 6. The project was extended from 9 to 28 institutions across Europe, the USA and Japan in 2004.

Copyright © 2006 John Wiley & Sons, Ltd.     

Effects of different dietary vitamin C supplementations on growth performance,mucus immune responses and antioxidant status of loach (Misgurnus anguillicaudatus Cantor) juveniles

A 60‐day feeding trial was conducted to determine the effects of different dietary vitamin C levels on growth performance, immune response and antioxidant capacity of loach juveniles. Six isonitrogenous (58.6% of crude protein), isoenergetic (17.5 kJ g^?1) practical diets supplemented with 0 (VC0), 100 (VC100), 200 (VC200), 500 (VC500), 1000 (VC1000) and 5000 mg kg^?1 (VC5000) of VC (35% ascorbic acid equivalent) were fed to fish (mean initial weight 0.11 ± 0.02 g) in triplicate. Results showed that fish fed VC0 diet had significantly lower body weight gain (BWG) and survival rate (SR). However, BWG and SR improved significantly in fish fed VC100 and VC200 diet respectively. Whole body ascorbic acid concentration increased with incremental dietary VC levels from 0 to 100 mg kg^?1. The activity of mucus alkaline phophatase was significantly increased by the dietary VC level. Incremental levels of VC significantly reduced activities of glutathione peroxidase (GPx) and catalase. Moreover, fish fed diets containing more than 100 mg kg^?1 VC significantly down‐regulated the superoxide dismutase and GPx mRNA expression in liver. Meanwhile, the expressions of liver heat shock protein (HSP70) and nuclear factor‐erythroid 2‐related‐2 (Nrf2) were affected by fish fed diets containing VC from 100 to 5000 mg kg^?1. In conclusion, VC requirement of loach juveniles for optimum growth and functionally preventing lipid peroxidation was more than 200 mg kg^?1 of diet. Moreover, high dose of VC supplementation did not show any detrimental effects on loach growth performance.     

Influence of dietary administration of a probiotic strain Shewanella putrefaciens on senegalese sole (Solea senegalensis,Kaup 1858) growth,body composition and resistance to Photobacterium damselae subsp piscicida

Probiotics are currently being supplemented to cultured fish due to their benefits for fish performance. Herein, we tested the health protection and nutritional effects of probiotic Shewanella putrefaciens (Pdp11 strain), both fresh and lyophilized cells, on Senegalese sole (Solea senegalensis) juveniles. Pdp11 was incorporated into fish feed at concentration of 10⁹ cells g^?1, and then provided to juvenile sole for 2 months. Growth rates were significantly higher in fish fed the fresh probiotic compared with fish fed the control diet. Growth of fish receiving lyophilized bacteria was not improved when compared with controls. Body compositions (protein, total lipids and fatty acids profile) were similar for each dietary treatment. At the end of the study, fish were challenged by intraperitoneal inoculation with a pathogenic strain of Photobacterium damselae subsp. piscicida. Relative percentages of survival (RPS) ranged between 25% and 43.8% for fish receiving both probiotic supplemented diets. Both fresh and lyophilized Pdp11 cells conferred protection against P. damselae subsp. piscicida. However, only fresh Pdp11 enhanced Senegal sole performance. These findings should be taken into account for industry purposes in which lyophilized Pdp11 might be easier to manage.     

Effects of Dietary Vitamin C on Blood Chemistry and Nonspecific Immune Response of Juvenile Red Sea Bream,Pagrus major

A trial was conducted to determine the effect of ascorbyl‐2‐monophosphate Na/Ca (AMP‐Na/Ca) on blood chemistry and nonspecific immune response of red sea bream juveniles. Test diets with three levels of AsA (free, 107, and 325 mg/kg diet) were fed to juvenile red sea bream (36.0 ± 1.3 g) two times a day for 3 wk. There were no significant differences in hematocrit, glucose, and blood urea nitrogen. Total cholesterol and triglyceride in plasma of fish fed AsA‐free diet was significantly (P < 0.05) higher than that of fish fed two other diets. There were no significant differences in serum albumin, total bilirubin, and total serum protein. Glutamyl oxaloacetic transaminase in serum of fish fed diets containing 107 and 325 mg of AsA were significantly (P < 0.05) lower than that of fish fed AsA‐free diet. Serum lysozyme activity (LA) of fish fed diets containing 107 and 325 mg of AsA were significantly (P < 0.05) higher than that of fish fed AsA‐free diet. There was no significant difference in mucus LA. The results mentioned above demonstrated that AMP‐Na/Ca is a bioavailable AsA source for red sea bream juveniles. Supplement of more than 107 mg AsA/kg in diets improved blood chemistry and nonspecific immune function of red sea bream juveniles.     

Genetic variations within Symbiodinium clade C among zooxanthellate corals (Scleractinia) in the temperate zone of Japan

In a recent study most of the temperate-living corals were found to harbor stress-sensitive Symbiodinium clade C, which is common in the tropical zone. In this study, we investigated the genetic variations within Symbiodinium clade C among corals living in temperate waters of Japan (17 genera; 26 species) using rDNA internal transcribed spacers (ITSs) to clarify the genetic differences between climatic zones. We also focused on two widespread coral species, Acropora hyacinthus and Pocillopora damicornis, to compare the genetic differences of Symbiodinium clade C between subtropical and temperate regions. We found multiple haplotypes of Symbiodinium ITSs in clade C, which formed nine subclades. Our data indicate that five genera (Acropora, Cyphastrea, Stylocoeniella, Porites, Pocillopora) contained host-specific Symbiodinium, while members of the family Faviidae and others contained several Symbiodinium ITS haplotypes from two or three subclades. A comparative analysis using Acropora and Pocillopora also revealed that the temperate zone coral population formed a single subclade of Symbiodinium clade C that was genetically isolated from those of subtropical populations. In summary, the majority of corals living in temperate waters of Japan, such as faviid corals, have several ITS haplotypes which possibly are utilized as an opportunistic survival strategy under severe environmental conditions, whereas Acropora and Pocillopora at the very least harbor temperate zone-specific Symbiodinium.     

Kinetics of adhesion of Listonella anguillarum to the mucus of gilt-head seabream, and the implication of surface components

The kinetics of adhesion of Listonella anguillarum cells to skin, intestinal and gill mucus suspensions of gilt‐head seabream was studied. A Langmuir adsorption isotherm was calculated, and the results obtained indicated that this microorganism showed a saturation kinetics. The pretreatment of the mucus suspensions with a surface extract obtained from L. anguillarum cells promoted a very important inhibition of the number of adhered bacteria to the different mucus suspensions. The potential role of surface structures on the adhesion of L. anguillarum to mucus of gilt‐head seabream is discussed.     

Dietary supplementation of host‐associated lactic acid bacteria modulates growth,metabolic activities,and immune‐related gene expression in giant freshwater prawn,Macrobrachium rosenbergii

The present study was carried out to evaluate the dietary effects of host‐associated lactic acid bacteria on growth performance, metabolic enzyme activities, and immune response of Macrobrachium rosenbergii juveniles. To formulate the test diets, a control (Con) diet was supplemented with a commercial probiotic and three host‐derived bacteria Enterococcus faecalis (EC), Lactococcus lactis I (LC‐I), and L. lactis II (LC‐II), which were previously isolated from the gastrointestinal tract of adult individuals of M. rosenbergii. Juvenile M. rosenbergii (0.65 ± 0.008 g) were randomly stocked at 20 individuals/100 L of fiberglass tanks with three replications for each test diet. After 50 days, juveniles fed the diets LC‐I and LC‐II showed significantly higher (p < .05) weight gain, specific growth rate, and the lowest feed conversion ratio. The analyses of glutamic oxaloacetate transaminase and glutamic pyruvate transaminase in muscle and hepatopancreas revealed significantly (p < .05) reduced values in LC‐I fed juveniles. The total hemocyte count and phenoloxidase activity were significantly increased (p < .05) in LC‐I and LC‐II fed juveniles. The relative mRNA expression patterns of immune‐related α2‐M, LGBP, ProPO, Cu, Zn‐SOD, TG, PE, AKP, and ACP genes were significantly (p < .05) upregulated in juveniles fed with LC‐I followed by the diet LC‐II. Finally, the study suggests that the growth performance and immune response of M. rosenbergii can be improved through supplementation of host‐associated L. lactis bacteria for its higher production.     

No significant effect of additive ratios of docosahexaenoic acid to eicosapentaenoic acid on the survival and growth of cobia (Rachycentron canadum) juvenile

An experiment was conducted in the laboratory to investigate the effects of additive ratios of docosahexaenoic acid (DHA) to eicosapentaenoic acid (EPA) on the growth and survival of cobia (Rachycentron canadum) juveniles from August to October 2005. Three hundred and eighty cobia juveniles (56 days of age, body weight 6.9 ± 0.1 g, body length 9.2 ± 0.1 cm) were selected and 20 of them were freely taken for initial sample analysis in the week 0. Additional 360 juveniles were randomly assigned into eight groups with triplicate, total 24 tanks with 15 fish each. Cobia juveniles were reared in glass‐steel tanks (200‐L volume per tank) using filtered seawater with temperature 26–30.5 °C, salinity 25.4–33.0 g L^?1 and pH 7.8–8.0. Cobia juveniles were fed for 8 weeks using seven treatment diets (D‐1 to D‐7) with the same amount of DHA and EPA (15.0 ± 1.2 g kg^?1 of dried diet), but varying ratios of DHA to EPA (0.9, 1.1, 1.3, 1.5, 1.7, 1.9, 2.1, respectively) and a control diet (D‐0, DHA + EPA = 8.0 g kg^?1 of dried diet, DHA/EPA = 1.3). Five juveniles per tank were randomly taken for sample analysis at the end of weeks 4 and 8, respectively. The highest protein efficiency rate (PER; 1.5 in mean), average body weight (BW; 73.3 g per fish in mean) and the lowest feed conversion ratio (FCR; 1.6 in mean) were obtained in cobia juveniles fed the control diet at the end of week 8. These parameters were significantly different (P < 0.05) among juveniles fed the control and treatment diets; however, no significant difference (P > 0.05) was found among juveniles fed the treatment diets evaluated in this study. It was concluded that the survival and growth of cobia juveniles were not greatly influenced by additive ratios of DHA to EPA in our experimental conditions.     

Is the turbot,Scophthalmus maximus (L.), intestine a portal of entry for the fish pathogen Vibrio anguillarum?

The role of the intestinal tract in Vibrio anguillarum infection of turbot, Scophthalmus maximus (L.), fingerlings was investigated in two in vivo models and the possible mechanisms involved were studied in vitro. Viable V. anguillarum cells were detected in spleens from more than 50% of the fish administered the pathogen orally or rectally, suggesting that the intestinal tract is a portal of entry for V anguillarum. In transmission electron micrographs, V. anguillarum- like cells were seen close to the rectal epithelium, suggesting penetration of the mucus layer, but no epithelial cell penetration or endocytosis was evident. Attachment to intact turbot intestines was investigated, and 80% or more of the bacterial cells still remained attached after serial washings. A significantly higher number of cells attached to rectal segments than to the other intestinal segments. In vitro, V. anguillarum cells did not adhere specifically to intestinal mucus, but rather accumulated close to intestinal mucus interfaces and subsequently penetrated them. It is proposed that the intestinal ttact of tutbot is a portal of entry for V. anguillarum and that the cells penetrate the intestinal mucus overlaying the epithelial cells.     

Genome-wide SNP genotyping reveals hidden population structure of an acroporid species at a subtropical coral island: Implications for coral restoration

1. It is essential to consider genetic composition for both conventional coral restoration management and for initiating new interventions to counter the significant global decline in living corals. Population genetic structure at a fine spatial scale should be carefully evaluated before implementing strategies to achieve self-sustaining ecosystems via coral restoration.
2. This study investigated the population genetic structure of two acroporid species at Kume Island, Okinawa, Japan. There were 140 colonies of Acropora digitifera collected from seven study sites, and 81 colonies of Acropora tenuis from six sites. In total, 384 single nucleotide polymorphism (SNP) loci for A. digitifera and 470 SNPs for A. tenuis were obtained using a comparatively economical technique, Multiplexed ISSR Genotyping by sequencing.
3. Observed heterozygosity was significantly lower than expected heterozygosity at all SNP sites in both acroporid species, suggesting deficient genetic diversity possibly caused by past massive coral bleaching. Even though both species are broadcast spawners, the population structure was different in the two species. No detectable structure was evident in A. digitifera, but two distinct clades were found in A. tenuis. The genetic homogeneity of A. digitifera at Kume Island suggests that this species could be used as a focal species for active restoration in terms of genetic differentiation at this island. By contrast, A. tenuis unexpectedly included two distinct clades with little or no admixture within a small study area, possibly representing two reproductively isolated cryptic species. Thus, when using A. tenuis, it would be prudent to avoid disturbing the genetic composition of wild populations until this question is answered.