Comparative characterization of a temperature responsive gene (lactate dehydrogenase-B,ldh-b) in two congeneric tropical fish,Lates calcarifer and Lates niloticus

The characterization of candidate loci is a critical step in obtaining insight into adaptation and acclimation of organisms. In this study of two non-model tropical (to sub-tropical) congeneric perciformes (Lates calcarifer and Lates niloticus) we characterized both coding and non-coding regions of lactate dehydrogenase-B (ldh-b), a locus which exhibits temperature-adaptive differences among temperate and sub-tropical populations of the North American killifish Fundulus heteroclitus. Ldh-b was 5,004 and 3,527 bp in length in L. calcarifer and L. niloticus, respectively, with coding regions comprising 1,005 bp in both species. A high level of sequence homology existed between species for both coding and non-coding regions of ldh-b (> 97% homology), corresponding to a 98.5% amino acid sequence homology. All six known functional sites within the encoded protein sequence (LDH-B) were conserved between the two Lates species. Ten simple sequence repeat (SSR) motifs (mono-, di-, tri- and tetranucleotide) and thirty putative microRNA elements (miRNAs) were identified within introns 1, 2, 5 and 6 of both Lates species. Five single nucleotide polymorphisms (SNPs) were also identified within miRNA containing intron regions. Such SNPs are implicated in several complex human conditions and/or diseases (as demonstrated by extensive genome-wide association studies). This novel characterization serves as a platform to further examine how non-model species may respond to changes in their native temperatures, which are expected to increase by up to 6°C over the next century.     

Variable growth rates of the tropical estuarine fish barramundi Lates calcarifer (Bloch) under different freshwater flow conditions

Relationships between freshwater flows and growth rates of the opportunistic predatory finfish barramundi Lates calcarifer in a dry tropical estuary were examined using data from a long-term tag-recapture programme. Lagged effects were not investigated. After accounting for length at release, time at liberty and seasonal variation ( e.g. winter, spring, summer and autumn), growth rates were significantly and positively related to fresh water flowing to the estuary. Effects were present at relatively low levels of freshwater flow ( i.e. 2·15 m³ s⁻¹, the 5th percentile of the mean flow rate experienced by fish in the study during time at liberty). The analysis, although correlative, provides quantitative evidence to support the hypothesis that freshwater flows are important in driving the productivity of estuaries and can improve growth of species high in the trophic chain.     

Streptococcus iniae, a bacterial infection in barramundi Lates calcarifer.

The cause of ongoing mortality in barramundi Lates calcarifer (Bloch) in seawater culture was identified as Streptococcus iniae by biochemical and physiological tests. This is the first published record of this bacterial species in Australia and the first confirmed report of S. iniae causing mortality in barramundi. The bacterium was highly pathogenic for barramundi when challenged by bath exposure. The pathogen was found to have a LD50 of 2.5 x 10(5) and 3.2 x 10(4) colony-forming units at 48 h and 10 d respectively. Experimental challenge of barramundi resulted in high levels of mortality (> 40%) within a 48 h period. Ten days after the challenge, S. iniae could not be isolated from kidney, spleen, liver or eye of surviving fish. However, the organism was easily isolated from the brain of both moribund and healthy fish, indicating that barramundi can carry the bacterium asymptomatically.     

The gene expression response of the catadromous perciform barramundi Lates calcarifer to an acute heat stress

The acute heat-shock response of the tropical estuarine fish species barramundi Lates calcarifer as indicated by the expression of genes within stress (hsp 90AA, hsp 90AB, hsp 70 and hsc 70), metabolic (cisy, cco II and ldh) and growth (igf1 and mstn 1) related pathways was examined following an increase in water temperature from 28 to 36° C over 30 min. Lates calcarifer were maintained at the acute stress temperature of 36° C for 1 h before being returned to 28° C and allowed to recover at this temperature for a further 2 weeks. Muscle tissue sampling over the experimental period allowed for the expression quantification of stress, metabolic and growth-related genes via quantitative real-time polymerase chain reaction (qrt-PCR) where a robust and reliable normalization approach identified both α-tub and Rpl8 as appropriate genes for the analysis of gene expression in response to an acute heat stress. hsp90AA and hsp70 of the inducible heat-shock response pathway showed a massive up-regulation of gene expression in response to heat stress, whilst the constitutive heat-shock genes hsp90AB and hsp70 showed no change over the course of the experiment and a small increase after 2 weeks of recovery, respectively. Of the three genes representing the metabolic pathway (cisy, cco II and ldh) only cco II changed significantly showing a decrease in gene expression, which may suggest a small suppression of aerobic metabolism. igf1 of the growth pathway showed no significant differences in response to an acute heat stress, whilst mstn1 increased at the beginning of the heat stress but returned to basal levels soon after. Overall, the results demonstrate that an acute heat stress in L. calcarifer caused a significant increase in the expression of genes from the stress response pathway and a possible decrease in aerobic metabolism with only relatively minor changes to the growth pathway highlighting the hardy nature of L. calcarifer and its resilience in coping with sudden temperature changes routinely encountered within its natural environment.     

Structure and development of free neuromasts in barramundi, Lates calcarifer (Block)

This study was conducted to clarify the development of free neuromasts with growth of the barramundi, Lates calcarifer. A pair of free neuromasts was observed behind the unpigmented eyes in newly hatched eleutheroembryos with a mean total length of 1.93 mm, and two-hour-old eleuthero-embryos could respond to an approaching pipette. At 2 days after hatching, the egg yolk sac was mostly consumed, the eyes were pigmented, and the larvae commenced feeding on rotifers. Free neuromasts increased in number with growth and commenced developing into canal neuromasts in barramundi 15 days old with a mean total length of 8.07 mm. The average length of the major axis of the trunk free neuromasts attained approximately 12.9-15.5 microm, and the number of sensory cells was 15.4-17.5 at 15-20 days old. Developed cupulae of free neuromasts were observed in 1-day-old eleutheroembryos. The direction of maximum sensitivity of free neuromasts, determined from the polarity of the sensory cells, coincided with the minor axis of the lozenge-shaped outline of the apical surface of the free neuromasts. The polarity of trunk neuromasts was usually oriented along the antero-posterior axis of the fish body, but a few had a dorso-ventral direction. On the head, free neuromasts were oriented on lines tangential to concentric circles around the eye.     

Whole‐genome profiling and shotgun sequencing delivers an anchored,gene‐decorated,physical map assembly of bread wheat chromosome 6A

Bread wheat (Triticum aestivum L.) is the most important staple food crop for 35% of the world's population. International efforts are underway to facilitate an increase in wheat production, of which the International Wheat Genome Sequencing Consortium (IWGSC) plays an important role. As part of this effort, we have developed a sequence‐based physical map of wheat chromosome 6A using whole‐genome profiling (WGP^?). The bacterial artificial chromosome (BAC) contig assembly tools fingerprinted contig (fpc ) and linear topological contig (ltc ) were used and their contig assemblies were compared. A detailed investigation of the contigs structure revealed that ltc created a highly robust assembly compared with those formed by fpc . The ltc assemblies contained 1217 contigs for the short arm and 1113 contigs for the long arm, with an L₅₀ of 1 Mb. To facilitate in silico anchoring, WGP^? tags underlying BAC contigs were extended by wheat and wheat progenitor genome sequence information. Sequence data were used for in silico anchoring against genetic markers with known sequences, of which almost 79% of the physical map could be anchored. Moreover, the assigned sequence information led to the ‘decoration’ of the respective physical map with 3359 anchored genes. Thus, this robust and genetically anchored physical map will serve as a framework for the sequencing of wheat chromosome 6A, and is of immediate use for map‐based isolation of agronomically important genes/quantitative trait loci located on this chromosome.     

Cloning and characterization of the calreticulin gene in Asian seabass (Lates calcarifer)

Calreticulin (CRT) is a Ca2+-binding molecular chaperone in the endoplasmic reticulum. We cloned and characterized the CRT gene in an important marine food fish species Asian seabass (Lates calcarifer). The full-length DNA of the CRT gene was 2194 bp, including a complete open reading frame encoding 420 amino acid residues, a 113 bp 5'-untranslated region and an 818 bp 3'-untranslated region. The CRT gene contained nine exons and eight introns covering a total of 2772 bp genomic DNA from the start to stop codon. Ten single nucleotide polymorphisms (SNPs) were detected in introns and an exon in six individuals collected from five different locations. The CRT gene was assigned to linkage group 4 of the linkage map of Asian seabass. Quantitative real-time PCR revealed that the CRT gene was highly expressed in liver at the age of 1, 3 and 7 months under normal conditions, whereas its expression in liver reduced sharply after 0.5 to 2 h cold challenge at 16°C, and then increased slowly. A preliminary association analysis showed a significant (P < 0.001) association between the SNP6 in the CRT gene and the mortality after cold challenge at 16°C. Our results suggest that the CRT gene is associated with cold tolerance of Asian seabass and further investigation will be necessary to illustrate the underlying mechanisms.     

Open-top static respirometry is a reliable method to determine the routine metabolic rate of barramundi,Lates calcarifer

Closed-system respirometry is a standard technique used to determine aerobic metabolism of aquatic organisms. Open-top systems are rarely used due to concerns of gas exchange across the air–water interface. Here, we evaluated an open-top respirometry system by comparing the mass-specific routine metabolic rate (RMR) of the tropical diadromous finfish barramundi, Lates calcarifer, in both closed-top and open-top respirometers. The RMR of 190?g barramundi was determined across broad temperatures ranging from 18 to 38?°C. There was no significant difference in RMR between barramundi in either closed- or open-top respirometers at any temperature (p?>?0.05). To ensure RMR measurements were not an artifact of the respirometry system, barramundi were reciprocally transplanted into either respective closed-top or open-top respirometer and oxygen consumption re-measured at each temperature treatment. The RMR of transplanted barramundi was found to be virtually identical in either respirometer. RMR increased linearly with increasing temperature; the relationship between RMR and temperature (T; 18–38?°C) can be described as 3.658T?36.294?mg?O₂?kg^?0.8?h^?1. The daily energetic cost of RMR was 1.193T?11.838?kJ?kg^?0.8?day^?1. Q₁₀ for barramundi increased significantly with increasing temperature (p?Q_10(18–28) was the lowest at 1.7 and Q_10(28–38) the highest at 1.9, over the whole experiment temp range Q_10(18–28) was 1.8. The current study demonstrates that open-top respirometry is a reliable and practical alternative to closed-top respirometry for accurate determination of the aerobic metabolism of barramundi and has potential application for a number of different aquatic organisms.     

Tyrosine hydroxylase and insulin-like growth factor II but not insulin are adjacent in the teleost species barramundi, Lates calcarifer

In humans, the genes encoding tyrosine hydroxylase (TH), insulin and insulin-like growth factor II (IGF-II) form an extremely tight linkage group on chromosome 11p15. Characterisation of the homologous genomic region of a teleost, the barramundi Lates calcarifer , revealed tight linkage of the TH and IGF-II genes, and the absence of the gene encoding insulin.     

Seaweed extracts as a natural control against the monogenean ectoparasite,Neobenedenia sp., infecting farmed barramundi (Lates calcarifer)

Aqueous extracts from common tropical seaweeds were evaluated for their effect on the life cycle of the commercially important ectoparasite, Neobenedenia sp. (Platyhelminthes: Monogenea), through the survival of attached adult parasites, period of embryonic development, hatching success and oncomiracidia (larvae) infection success. There was no significant effect of any extract on the survival of adult parasites attached to fish hosts or infection success by oncomiracidia. However, the extracts of two seaweeds, Ulva sp. and Asparagopsis taxiformis, delayed embryonic development and inhibited egg hatching. The extract of A. taxiformis was most effective, inhibiting embryonic development of Neobenedenia sp. and reducing hatching success to 3% compared with 99% for the seawater control. Furthermore, of the 3% of eggs that hatched, time to first and last hatch was delayed (days 14 and 18) compared with the seawater control (days 5 and 7). Asparagopsis taxiformis shows the most potential for development as a natural treatment to manage monogenean infections in intensive aquaculture with the greatest impact at the embryo stage.     

Isolation and characterization of microsatellite DNA loci in sea bass,Lates calcarifer Bloch

Polymerase chain reaction (PCR)‐based isolation of microsatellite arrays (PIMA) technique was used to isolate seven polymorphic microsatellite loci in sea bass, Lates calcarifer Bloch. A total of 62 samples of wild and cultivated sea bass collected from a few populations within Peninsular Malaysia were used in the study. For seven polymorphic loci, the number of alleles ranged from four to nine and locus heterozygosities ranged from 0.710 to 1.000. The loci will be useful for studying population structure, genetic variability of wild and hatchery stocks of L. calcarifer and broodstock management purposes.     

The effect of high water temperatures on the allometric scaling effects of energy and protein starvation losses in juvenile barramundi, Lates calcarifer

This study was undertaken to examine the effects of high water temperatures on the allometric scaling effects of energy and protein starvation losses by juvenile barramundi, Lates calcarifer. The somatic energy and protein loss was examined in fish of varying sizes when starved for 24 or 25 days at temperatures ranging from 23 °C to 38 °C. The amount of energy and protein lost varied according to both size and temperature and was consistent with the function of a*W(b), where a is a temperature dependent coefficient, W the animal's weight and b an exponent relating energy loss to live-weight. The coefficient for energy or protein loss varied and was described by a polynomial function, with a general increase from 23 °C to 32 °C, before a dramatic decline after 35°C. In contrast, the exponent for energy loss was relatively constant between 23 °C and 35 °C, but showed a rapid increase at 38 °C. Both the coefficients and exponents of protein loss mirrored that of energy loss. Analysis of the protein and lipid contributions to energy loss shows that typically lipid loss accounts for the greater part of energy loss (~67%), but also shows that above 35 °C protein energy loss increases (from ~33% to ~39%) while the losses seen from lipid catabolism remain the same. These results show that one of the main nutritional issues associated with heat stress in fish is a dramatic increase in endogenous loss of protein.     

Whole‐exome targeted sequencing of the uncharacterized pine genome

The large genome size of many species hinders the development and application of genomic tools to study them. For instance, loblolly pine (Pinus taeda L.), an ecologically and economically important conifer, has a large and yet uncharacterized genome of 21.7 Gbp. To characterize the pine genome, we performed exome capture and sequencing of 14 729 genes derived from an assembly of expressed sequence tags. Efficiency of sequence capture was evaluated and shown to be similar across samples with increasing levels of complexity, including haploid cDNA, haploid genomic DNA and diploid genomic DNA. However, this efficiency was severely reduced for probes that overlapped multiple exons, presumably because intron sequences hindered probe:exon hybridizations. Such regions could not be entirely avoided during probe design, because of the lack of a reference sequence. To improve the throughput and reduce the cost of sequence capture, a method to multiplex the analysis of up to eight samples was developed. Sequence data showed that multiplexed capture was reproducible among 24 haploid samples, and can be applied for high‐throughput analysis of targeted genes in large populations. Captured sequences were de novo assembled, resulting in 11 396 expanded and annotated gene models, significantly improving the knowledge about the pine gene space. Interspecific capture was also evaluated with over 98% of all probes designed from P. taeda that were efficient in sequence capture, were also suitable for analysis of the related species Pinus elliottii Engelm.     

Isolation, characterization, and linkage analyses of 74 novel microsatellites in Barramundi (Lates calcarifer).

Barramundi (Lates calcarifer) is an important marine food fish species in Southeast Asia and Australia. Seventy-four novel microsatellites were isolated from a genomic DNA library enriched for CA repeats and were characterized in 24 unrelated individuals. Among the 74 microsatellites, 71 were polymorphic, with an average allele number of 7.0 +/- 3.6/locus. The average expected heterozygosity of these polymorphic markers was 0.66. Sixty-three of the 71 polymorphic microsatellites conformed to Hardy-Weinberg equilibrium. Linkage analyses were conducted in a reference family, leading to the assignment of 34 novel microsatellites and 16 published markers in 16 linkage groups. The novel microsatellites developed in this study will contribute significantly to the construction of a first-generation linkage map for mapping of quantitative trait loci in Barramundi, and supply a large choice of markers for studies on population genetics, stock management, and pedigree reconstruction.     

The food of barramundi, Lates calcarifer (Bloch), in coastal and inland waters of Van Diemen Gulf and the Gulf of Carpentaria, Australia

Examination of the diet of Lates calcarifer , based on 3684 specimens of 4–1200 mm total length (T.L.), from two localities in north Australia showed it is an opportunistic predator with an ontogenetic progression in its diet from microcrustacea to macrocrustacea to fish. The increasing importance of fish in the diet of L. calcarifer in salt water (up to 87% of food consumed) involved a dietary shift from feeding on bottom-dwelling forms to pelagic forms. There were few differences in the diet of L. calcarifer between the two localities. Cannibalism was rarely encountered in the Gulf of Carpentaria although it was significant (11–4% of the diet of L. calcarifer of 1001–1200 mm T.L.) in Van Diemen Gulf. A significant correlation between predator and prey length was found for Ariidae, Dorosomidae, Engraulidae, L. calcarifer , Mugilidae and Polynemidae. The maximum prey/predator length ratio observed was 0–61 for the dorosomid, Nematolosa erebi.