Predation on native fish eggs by invasive round goby revealed by species‐specific gut content DNA analyses

1. Conservation of riverine fish often aims to improve access to spawning grounds and restore longitudinal connectivity by removing migration barriers, and involves substantial investments. However, these investments also enable non‐native predators to invade upstream into spawning areas and potentially adversely affect the recruitment of threatened freshwater fish through egg or fry predation.
2. Detecting egg predation is often challenging. Visual inspections of fish gut contents may underestimate predation of soft materials such as eggs and fry, which limits the discovery of predators preying upon these life‐stages. DNA‐based detection assays may offer a more sensitive tool to assess predation of soft materials.
3. A conservation issue was confirmed by developing and applying a species‐specific DNA‐based detection assay: invasive round goby (Neogobius melanostomus) prey on the eggs or fry of the threatened common nase (Chondrostoma nasus) in Switzerland.
4. DNA‐based detection assays were also developed for five other valuable native fish species, including endangered salmonid and cyprinid river spawners. The applicability of the assays was confirmed in a series of laboratory and field feeding experiments involving eggs and fish tissue. In addition, this work provides a guiding framework for conservation managers regarding the use and applicability of different DNA‐based detection approaches for gut content analysis.
5. The results of this study could inform local conservation measures – such as temporary reductions in the density of round goby at spawning sites prior to spawning – and demonstrate how targeted application of species‐specific molecular markers may advance freshwater fish management.

     

Development of a novel PCR assay based on the 16S-23S rRNA internal transcribed spacer region for the detection of Lactococcus garvieae

Lactococcus garvieae is recognized as an emerging pathogen in fish. Several PCR‐based methods have been developed for the detection and identification of L. garvieae; however, the sensitivity of these methods is still in question regarding the discrimination of this organism from other closely related species. Two primers, ITSLg30F and ITSLg319R, were designed from the sequence in the 16S–23S internal transcribed spacer (ITS) region and used for the specific detection of L. garvieae. L. garvieae strains including fish isolates were positive by this method. In contrast, previously developed PCR methods showed false‐positive results with non‐L. garvieae species. Our results indicate that a PCR method using the newly designed ITS primer set provides a sensitive and efficient tool for the detection of L. garvieae in fish and aquaculture environments.     

Multiplex nested-polymerase chain reaction for the simultaneous detection of Aeromonas hydrophila, Edwardsiella tarda, Photobacterium damselae and Streptococcus iniae, four important fish pathogens in subtropical Asia

A multiplex nested-polymerase chain reaction (PCR)-based (m-nested PCR) method was developed for simultaneous detection of four important freshwater/marine fish pathogens in subtropical Asia, including Aeromonas hydrophila, Edwardsiella tarda, Photobacterium damselae and Streptococcus iniae . The specificity of the oligonucleotide primers used for PCR detection was confirmed to generate specific amplicons for the corresponding pathogens. Moreover, non-specific amplicons were observed when the primers were tested using pure DNA extracted from 31 related bacterial strains belonging to 23 species or tissue homogenates of infected tilapia. This m-nested PCR approach could detect 19 colony forming unit (CFU) for A. hydrophila , 62 CFU for E. tarda , 280 CFU for P. damselae subsp. piscicida and 179 CFU for S. iniae in infected tilapia kidney homogenates, consistent with the results derived from bacteriological methods. The assay described in this paper is a sensitive and effective method for simultaneous detection of multiple fish pathogens.     

Sequence and polymerase chain reaction–restriction fragment length polymorphism analysis of the large subunit rRNA gene of bivalve: Simple and widely applicable technique for multiple species identification of bivalve larva

ABSTRACT:   One of the biggest and long-standing difficulties in investigation of larval ecology in the field is species-level identification. In the present study, we developed polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis based on the large subunit (LSU) rRNA gene (rDNA) D1/D2/D3 region for identification of multiple species of bivalve larvae using 14 species of bivalve collected from Maizuru Bay. The LSU rDNA D1/D2/D3 region of all analyzed species could be amplified by PCR using bvD1f/bvD3r primers, and RFLP analysis by Hae III digestion on the PCR products showed species-specific fragment patterns. Furthermore, this analysis applied to single bivalve larvae in Maizuru Bay revealed efficient amplification of the target region and the species-specific pattern from 80% of the larvae, 75% of which showed a pattern that matched a certain pattern of the adult bivalves. In addition, the analysis of inter- and intraspecies variation of the LSU rDNA D1/D2/D3 region using the sequence data of the genus Crassostrea from the DDBJ database showed high applicability of this RFLP analysis on closely related species. Because of the wide applicability and technical simplicity, this method can become the standard for the identification of bivalve larvae species once the sequence data of the LSU rDNA D1/D2/D3 region of many bivalve species have been accumulated.     

Predation on walleye pollock (Theragra chakograrnma) eggs and yolk-sac larvae by pelagic crustacean invertebrates in the western Gulf of Alaska

Immunological detection of yolk protein was used to assess predation by pelagic amphipods (gammarid and hyperiid), mysids, and euphausiids on eggs and yolk-sac larvae of walleye pollock Theragra chalcogramma during 1988 and 1989. Consumption estimates were made on the basis of frequency of positive immunoassays, assay detection times (gut clearance time), predator abundance, and spatial overlap of predators and prey. From our results gammarid amphipods and euphausiids were important predators on eggs and yolk-sac larvae, respectively. Gammarid amphipods alone consumed about 14% of the standing stock of pollock eggs in 1989. These results were compared with those from clearance rate experiments of predators feeding on pollock eggs in 300-1 bags. In general, clearance rate estimates of egg consumption were lower than those determined from gut contents.     

Assessing the potential for fish predation to impact zebra mussels (Dreissena polymorpha): insight from bioenergetics models

Abstract –  Rates of annual food consumption and biomass were modeled for several fish species across representative rivers and lakes in eastern North America. Results were combined to assess the relative potential of fish predation to impact zebra mussels ( Dreissena polymorpha ). Predicted annual food consumption by fishes in southern waters was over 100% greater than that in northern systems because of warmer annual water temperatures and presumed increases in metabolic demand. Although generally increasing with latitude, biomasses of several key zebra mussel fish predators did not change significantly across latitudes. Biomasses of some less abundant fish predators did increase significantly with latitude, but increases were not of the magnitude to offset predicted decreases in food consumption. Our results generally support the premise that fishes in rivers and lakes of the southern United States (U.S.) have inherently greater potential to impact zebra mussels by predation. Our simulations may provide a partial explanation of why zebra mussel invasions have not been as rapid and widespread in southern U.S. waters compared to the Great Lakes region.     

Predation by scavenging amphipods to injured hatchery-raised juvenile Japanese flounder Paralichthys olivaceus under laboratory conditions

ABSTRACT:   The attacking potential of the scavenging amphipod Scopelocheirus onagawae on artificially injured hatchery-raised Japanese flounder Paralichthys olivaceus juveniles was investigated in relation to the degree of injury on the fish. All injured flounder juveniles were attacked by amphipods regardless of the degree of injury, while non-injured juveniles were not attacked. The attack by amphipods on the juveniles generally depended on the amount of glycine, a main feeding stimulant for the amphipod, released from the injury opening. The swimming ability of flounder juveniles was important to avoid the attack of amphipods. Furthermore, an area of injury allowing the amphipods to cling to the fish affects to the vulnerability of juveniles against the predation of amphipods. This study suggests that scavenging amphipods are potentially involved in the rapid reduction of the number of hatchery-raised juveniles.     

Qualitative and quantitative changes of F<Subscript>o</Subscript>F<Subscript>1</Subscript>-ATPase in Japanese flounder and red sea bream associated with rearing temperatures

ABSTRACT:   Fast skeletal muscles of Japanese flounder Paralichthys olivaceus and red sea bream Pagrus major were examined for quantitative and qualitative changes of mitochondrial ATP synthase (F_oF₁-ATPase) in association with rearing temperatures. The specific activities of F_oF₁-ATPase from Japanese flounder reared at 10°C, 15°C and 25°C for 4 weeks were determined to be 81 ± 11, 74 ± 13 and 83 ± 11 nmol/min·mg mitochondrial protein, respectively. The corresponding activity from red sea bream reared at 8°C for 5 weeks was determined to be 65 ± 9 nmol/min·mg mitochondrial protein, which was higher than 33 ± 9 nmol/min·mg mitochondrial protein in fish reared at 23°C. The contents of α- and β-F₁-ATPase in total mitochondrial proteins were not significantly different between fish reared at different temperatures for the two fish species. However, the contents of β-F₁-ATPase in the total fast skeletal muscle extracts, prepared from Japanese flounder reared at 10°C, were 2.1- and 2.9-fold higher than those for fish reared at 15°C and 25°C, respectively. The corresponding content from red seabream reared at 8°C was 2.2-fold higher than that for fish reared at 23°C. Therefore, the changes in F_oF₁-ATPase depending on rearing temperatures were species-specific.     

Comparison of visual acuity and visual axis of three flatfish species with different ecotypes

ABSTRACT:   The visual acuity, visual axis and visual accommodation of pointhead flounder, slime flounder, and red halibut were determined to obtain basic knowledge for developing appropriate fishing gear and fishing methods for sustainable fisheries. Each of these species has a different ecotype in terms of habitat, depth and prey species. Thus, it was hypothesized that they may differ in terms of visual acuity, visual axis and visual accommodation. Few studies have compared these characters in flatfishes from different ecotypes. We used histological methods to determine visual acuity (i.e. cone cell density) and visual axis (i.e. cone cell distribution) in each of these species. The maximum visual acuity was 0.127 in pointhead flounder (total length, TL 344 mm), 0.092 in slime flounder (TL 372 mm) and 0.109 in red halibut (TL 336 mm). Based on the cone cell distribution in the retina, the visual axis was upward and forward in pointhead flounder, forward and downward in slime flounder, and downward in red halibut. Finally, the mean angle of lens movement was −2° in pointhead flounder, −13° in slime flounder and −32° in red halibut. This measurement of lens movement indicated that the average near-point distance was 0.87 × TL in pointhead flounder, 0.65 × TL in slime flounder and 1.02 × TL in red halibut. At similar TL (336–355 mm), the visual acuity of these species differs depending on the direction in which they are looking.     

Identification and detection of Pseudomonas plecoglossicida isolates with PCR primers targeting the gyrB region

Pseudomonas plecoglossicida is the agent of bacterial haemorrhagic ascites (BHA) in freshwater fish farming in Japan. To develop a rapid identification and detection method for P. plecoglossicida, a PCR amplification technique targeting the chromosomal DNA region coding the B subunit of the DNA gyrase (gyrB) was used. The nucleotide sequences of gyrB were determined in nine isolates of P. plecoglossicida and two other Pseudomonas species. On the basis of these determined sequences and the gyrB sequences of other Pseudomonas species or fish pathogenic bacteria deposited in international nucleotide sequence databases (GenBank/EMBL/DDBJ), PCR primers PL-G1F, PL-G1R, PL-G2F and PL-G2R were designed for specific amplification of the partial gyrB of P. plecoglossicida. The specificity of these primers in amplifying the gyrB of P. plecoglossicida was verified using selected strains of related bacterial species. The nested PCR technique was used to detect P. plecoglossicida from kidney and intestine of ayu. Primer pair PL-G1F and PL-G1R was used for the external PCR, and primer pair PL-G2F and PL-G2R for the internal PCR. Of 10 ayu juveniles, expected size PCR products were observed from intestine and kidney samples in one and two specimens, respectively. The PCR technique with primers based on the gyrB sequence is thus useful for the diagnosis of BHA.     

Jack mackerel <Emphasis Type="Italic">Trachurus japonicus</Emphasis> juveniles use jellyfish for predator avoidance and as a prey collector

ABSTRACT:   Juveniles of carangid fishes including jack mackerel Trachurus japonicus are known to associate with jellyfishes. The function of this association behavior was studied through rearing experiments and underwater visual observations. Association behavior of jack mackerel with moon jellyfish in experimental tanks was more frequent in the presence compared to the absence of predators (chub mackerel Scomber japonicus ). In the experimental tanks, the presence of jellyfish, however, did not mitigate predation by these predators. Although jack mackerel did not feed on the jellyfish itself, they frequently fed on the captured prey ( Artemia nauplii) whilst in the gut cavity of the jellyfish. Underwater observations of giant jellyfish Nemopilema nomurai off Kyoto and Fukui prefectures revealed that approximately 30% of these jellyfish were accompanied by jack mackerel juveniles with body sizes ranging 10–45 mm standard length (SL). Considering that jack mackerel juveniles found in subtidal rocky reefs ranged 40–120 mm SL, we considered that jack mackerel from 10 to 45 mm SL associate with jellyfish as a hiding place as well as a food collector, until they find a suitable reef habitat when they attain approximately 40 mm SL.     

Predation on glass eels of Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis> in the Tone River Estuary,Japan

Aquaculture of Japanese eel Anguilla japonica relies upon the natural recruitment of their glass eels (juveniles); however, predation that could influence glass eel recruitment remains unknown. In the present study, we aimed to elucidate the proportion of predation on A. japonica glass eels through stomach content analysis of predatory fishes collected in the estuary region of the Tone River system and its vicinity in Japan. Species of the predated glass eels were identified by DNA barcoding. A total of 270 predatory fishes of 15 taxa was collected over 2 years. The overall proportion of predation on glass eels, genetically identified as Japanese eel, was 0.7%, but this rose to 2.0% when data were limited to fishes caught during the peak months of glass eel recruitment. A glass eel was found in the stomach contents of a channel catfish Ictalurus punctatus, an invasive species in this river system, and a blackfin sea bass Lateolabrax latus. These fishes are therefore considered potential predators of A. japonica glass eels. However, as the proportion of predation was low, and the glass eels represented only small proportions of predator stomach contents, further investigation is needed for a better understanding of predation on A. japonica glass eels, and its effects on the early life stages of this endangered species.     

牙鲆和半滑舌鳎5S rDNA基因的染色体定位及鲽形目5种鱼类的分子系统学分析

利用荧光原位杂交(FISH)技术将5S rDNA基因定位在牙鲆和半滑舌鳎染色体上,结果表明,5S rDNA基因在雌、雄性半滑舌鳎的一对同源染色体上分别存在2个杂交信号位点;在二、三倍体牙鲆的同源染色体上分别存在2个和3个杂交信号位点,杂交信号明显且特异。本研究首次将5S rDNA基因定位在半滑舌鳎和三倍体牙鲆的染色体上,为牙鲆及半滑舌鳎倍性鉴定、染色体鉴别提供了有效方法。此外,根据牙鲆5S rDNA基因编码区序列设计引物,扩增出大菱鲆和半滑舌鳎5S rDNA基因编码区序列,与鲽、塞内加尔鳎、大口黑鲈、七鳃鳗的5SrDNA基因序列进行同源性比较后发现,5种鲽形目鱼类5S rDNA基因序列同源性高达98.3%,系统发生分析结果显示5种鲽形目鱼类聚为一支;各序列中GC含量均显著高于AT含量。     

Effect of taurine supplemented practical diet on growth performance and taurine contents in whole body and tissues of juvenile Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

ABSTRACT:   The effect of dietary taurine on juvenile Japanese flounder was determined by feeding three taurine-supplemented experimental diets (TAU) and a commercial diet (CD) to evaluate a practical diet for juvenile Japanese flounder. Juvenile Japanese flounder were reared on the three experimental diets supplemented with taurine at 0, 0.5, 1.0% and CD. These diets were fed to juvenile Japanese flounder of an initial mean body weight of 0.2 g for 6 weeks at 20°C and the taurine contents of the whole body and tissues were analyzed. The final average body weight of juvenile Japanese flounder fed the 1.0% TAU was significantly higher than that of the other groups. Taurine contents in the whole body and tissues increased with the increase in dietary taurine level. These results indicate that juvenile Japanese flounder require at least 15 mg/g taurine in the diet, even though a combined mix of fish, krill and squid meal was the main protein source in the experimental diets.     

Genetic population evaluation of two closely related flatfish species, the rare barfin flounder and spotted halibut, along the Japanese coast

ABSTRACT:   Barfin flounder and spotted halibut have been selected as target species for stock enhancement in Japan. Understanding the genetic condition of the wild stock is a principal requirement in any stock enhancement program. The genetic variability of barfin flounder and spotted halibut, and the population structure of spotted halibut were evaluated using microsatellite DNA markers (msDNA) and the control region of the mitocondrial DNA (mtDNA). Barfin flounder and spotted halibut showed high genetic variability at the msDNA level. Barfin flounder A was 16.7 and H _e was 0.860; spotted halibut A _n ranged from 7.7 to 10.2 and H _e ranged from 0.710 to 0.774. At the mtDNA level, high haplotype ( h  = 0.922) and low nucleotide (π = 0.002) diversities were observed for barfin flounder; however, low haplotype and nucleotide diversities ( h  = 0.603–0.620 and π = 0.001–0.002), and very low haplotype and nucleotide diversities ( h  = 0.193 and π = 0.0003) were observed for spotted halibut in the north and south locations, respectively. Slight genetic differentiation among spotted halibut sampling locations was observed from the msDNA. MtDNA analyses showed genetic differentiation between north and south locations, but not within them. The designation of north-specific and south-specific management units in the future stock enhancement activities of spotted halibut is recommended.     

Gene transfer for Japanese flounder fertilized eggs by particle gun bombardment

ABSTRACT:   Fish transgenesis has progressed considerably. However, the technique of gene transfer for most marine aquaculture species has not been established. A method to introduce foreign genes into fertilized eggs of Japanese flounder Paralichthys olivaceus by particle gun bombardment was developed by the authors. A recombinant plasmid which contains the Japanese flounder keratin gene promoter linked to the green fluorescence protein (GFP) gene was introduced into fertilized eggs by particle gun bombardments twice at 250 psi. In each experimental group, 25 000–35 000 eggs were treated. However, the survival rate was 12.8% which is lower than that of the control groups (56.8%). Of 2606–5205 the embryos survived for 24 h, 43–61 GFP positive embryos were obtained in one experiment, giving a final gene transfer efficiency of 1.4%. All GFP-positive embryos developed and hatched normally. GFP expression was observed in epithelial tissue throughout the developmental stages. At 3 months after gene transfer, foreign DNA was detected by genome polymerase chain reaction analysis in 37 of 69 fry (53.6%). These results suggest that the particle gun method is an effective method to use with fertilized Japanese flounder eggs.     

Analysis of the diet of the long‐snouted seahorse Hippocampus guttulatus by 18SrDNA amplification of prey in faeces

Breeding in captivity for research or exhibition (e.g. in aquaria) can replace the capture of wild specimens of endangered species and allow controlled reinforcement of wild populations. With this aim, diet analysis and establishing the adequate prey are determinant factors for breeding success. However, non‐invasive approaches such as faecal DNA analysis are advisable for analysing the diet of these species. Therefore, the aim of the present study was to demonstrate the usefulness of faecal DNA analysis by specific PCR amplification of prey DNA for assessing the diet of the seahorse Hippocampus guttulatus. In a comparison of the suitability of different genes (COI, 18SrRNA and 28SrRNA), 18SrRNA was found to be the most suitable for designing specific primers for the prey types fed to seahorses (Artemia, Palaemonetes and Mysidae). The technique was assessed in feeding experiments in which prey ingestion was recorded daily, and faeces were collected for DNA extraction and presence/absence PCR analysis. Amplification of the prey DNA in faeces was consistent with the sequence of prey supplied (prey eaten the day before was always detected). Differences in the time between feeding and detection in faeces suggested prey‐specific gut passage times, which were shorter for Palaemonetes than for Mysidae. This fact highlights the importance of feeding trials to avoid overestimating the consumption of prey with long gut retention when PCR‐based methods are used. This molecular technique is thus applicable for studying the feeding behaviour of captive seahorses and could be adapted for use in other marine species.     

Application of the rpoS gene for the detection of Vibrio anguillarum in flounder and prawn by polymerase chain reaction

Vibrio anguillarum , an opportunistic fish pathogen, is the main species responsible for vibriosis, a disease that affects feral and farmed fish and shellfish, and causes considerable economic losses in marine aquaculture. In this study, we used polymerase chain reaction (PCR) to detect V. anguillarum . PCR specificity was evaluated by amplifying the rpoS gene, a general stress regulator, in six strains of V. anguillarum and 36 other bacterial species. PCR amplified a species-specific fragment (689 bp) from V. anguillarum . Furthermore, the PCR assay was sensitive enough to detect rpoS expression from 3 pg of genomic DNA , or from six colony-forming units (CFU) mL⁻¹ of cultured V. anguillarum . However, the assay was less sensitive when genomic DNA from the infected flounder and prawn was used (limit of detection, 50 ng and 10 ng g⁻¹ tissue, respectively). These data demonstrate that PCR amplification of the rpoS gene is a sensitive and species-specific method to detect V. anguillarum in practical situations.     

Habitat use by the African anabantid fish Ctenopoma muriei: implications for costs of air breathing

Abstract –  This study examined whether habitat use by the African anabantid fish Ctenopoma muriei is consistent with predictions relating to two costs of air breathing (surface travel and aerial predation). We predicted that C. muriei would be most abundant in habitats that minimize potential costs of air breathing. We quantified environmental correlates of C. muriei abundance within a series of wetland lagoons in Uganda and found that C. muriei was more abundant in shallow waters of the deepest lagoon, in vegetated habitats, and at a depth 15–30 cm below the surface. These locations may minimize cost of travel to the surface for aerial respiration, while maintaining a reasonable distance from the surface to avoid detection by aerial predators. In addition, we experimentally tested effectiveness of vegetative cover in reducing mortality of C. muriei by aerial predators (pied kingfishers) using enclosures with and without vegetation cover in a swamp pool. We found that the presence of vegetative cover significantly reduced susceptibility of C. muriei to predation by pied kingfishers when dissolved oxygen in the water was low and air-breathing frequency was potentially high.