Nicholas G. Elliott

Oligonucleotide Primers for PCR Amplification of Coelomate Introns

Abstract Seven novel oligonucleotide primer pairs for polymerase chain reaction amplification of introns from nuclear genes in coelomates were designed and tested. Each pair bound to adjacent exons that are separated by a single intron in most coelomate species. The primer sets amplified introns in species as widely separated by the course of evolution as oysters (Mollusca: Protostoma) and salmon (Chordata: Deuterostoma). Each primer set was tested on a further 6 coelomate species and found to amplify introns in most cases. These primer sets may therefore be useful tools for developing nuclear DNA markers in diverse coelomate species for studies of population genetics, phylogenetics, or genome mapping.

Allozyme and mitochondrial DNA variation in yellowfin tuna (Thunnus albacares) from the Pacific Ocean

Samples of yellowfin tuna (Thunnus albacares) collected in 1991 and 1992 from the western, central and eastern regions of the Pacific Ocean were examined for genetic variability. Four polymorphic allozyme loci (ADA*, FH*, GPI-S* and GPI-F*) were examined in all samples and a fifth polymorphism (GDA*) was examined in western and central samples only. Samples were also screened for mitochondrial DNA variation following restriction analysis by two enzymes (BcII and EcoRI) detecting polymorphic cut sites. Eighteen mtDNA haplotypes were revealed, with an overall nucleon diversity of 0.678. A subset of individuals screened for eight restriction enzymes had an overall nucleon diversity of 0.724 and a mean nucleotide diversity per sample of 0.359%. No significant spatial heterogeneity was detected for alleles at the ADA*, FH*, GPI-S* and GDA* loci nor for the mtDNA haplotypes. Significant heterogeneity was detected for GPI-F*. At this locus, the two eastern samples (southern California and northern Mexico) were not significantly different from each other but were significantly different (P<0.001) from the five western/central samples (Philippines, Coral Sea, Kiribati, Hawaii-91 and Hawaii-92). GPI-F*100 was the most common allele in western and central regions, GPI-F*75 the most common in eastern samples.

Genetic differentiation between Tasmanian cultured Atlantic salmon (Salmo salar L.) and their ancestral Canadian population: comparison of microsatellite DNA and allozyme and mitochondrial DNA variation

Abstract Atlantic salmon ( Salmo salar ) were imported to Australia from the River Philip, Nova Scotia, in the mid-1960s. A population was established in New South Wales, and in the mid-1980s ova from this population were used to found the Tasmanian salmon aquaculture industry. An allozyme and mitochondrial DNA examination of the Tasmanian and parent Canadian populations in 1993 showed some small but significant allele frequency differences between the two samples for one of seven polymorphic allozymes and for mitochondrial DNA haplotypes. However, there was no evidence of reduced genetic variability in the Tasmanian sample. The same individual fish from both populations have now been examined for eight polymorphic microsatellite loci. Small but significant differences in allele frequencies between the two samples were found for four of the eight loci, and there was evidence of a small overall loss of genetic variation (both heterozygosity and alleles) in the Tasmanian sample. Mean heterozygosity per microsatellite locus was more than twice that per allozyme locus: for the Tasmanian fish 0.434 ( n =63) (0.207 for allozymes) and for the Canadian fish 0.509 ( n =63) (0.182 for allozymes). Estimates of per-generation effective population sizes were calculated to be 65.2±19.7 (s.d.) from the microsatellite data, 106.1±74.4 for the allozymes data and 70.1±19.4 for the combined data set, assuming allele frequencies in the River Philip sample represent those in the progenitor population and ten generations of isolation. Microsatellite loci, with higher numbers of alleles and higher heterozygosities, are more sensitive than allozyme loci to changes in effective population size.

Genetic differentiation in the Antarctic coastal krill Euphausia crystallorophias.

The population genetics of the Antarctic neritic krill species Euphausia crystallorophias was examined by nucleotide sequence variation in its mitochondrial DNA. A 616 base pair region of the cytochrome c oxidase subunit I (COI) gene was screened for mutations by single-strand conformational polymorphism (SSCP) combined with restriction digestion. E. crystallorophias caught in three different regions of the Antarctic coastline were used – two samples from the Mertz Glacier Polynya and one sample each from the western side of the Antarctic Peninsula and from the Davis Sea. Significant genetic differences between krill samples were identified. However, the extent of these differences did not correlate with the degree of geographic separation between the sampling sites. This suggests that the genetic structuring may be the result of small-scale differentiation rather than differentiation between resident populations in separate parts of the Southern Ocean. The possibility that genetic differences between samples within a region are as important as differences between regions has implications for other studies of krill population genetics.

Unusually high levels of non-saponifiable lipids in the fishes escolar and rudderfish identification by gas and thin-layer chromatography.

Analysis of the non-saponifiable lipids of the fishes Lepidocybium flavobrunneum and Ruvettus pretiosus (escolar), and Centrolophus niger and Tubbia spp. (rudderfish) was performed. The analyses were used to clarify the cause of recent reports of illness (diarrhoea) in Australia from consumption of purported rudderfish. Both escolar and rudderfish contained very high levels of oil (generally between 14 to 25%, as % wet mass) in the fillet and the oil compositions were different to most seafood. Escolar oil contained mainly wax ester (>90% of oil). The oil from five specimens of rudderfish contained mainly diacylglyceryl ether (DAGE, >80% of oil) or hydrocarbon (>80% of oil, predominately squalene). One rudderfish specimen contained mainly polar lipid. Major differences in oil content and composition, including fatty alcohol and glyceryl ether diols (derived from DAGE), were observed between purported individuals of the same species or related species of rudderfish, raising the possibility of geographic or seasonal differences affecting the oil composition. The oil composition of fish fillet samples associated with the health issues were consistent with the profiles for escolar, rather than rudderfish species. These findings, in particular the lipid class and fatty alcohol profiles, were supported by general protein fingerprinting results and were consistent with the samples originating from individuals of the escolar species L. flavobrunneum. The high wax ester content of the escolar group clarifies the reported diarrhoeal effects to consumers. Purgative properties of high wax ester containing fish oils have been reported for escolar and other species. The results highlight the potential for non-saponifiable lipid profiles to be used for identification of fish fillets and oils to at least group level.

Comparison of mitochondrial and nuclear DNA analyses of population structure in the blacklip abalone Haliotis rubra Leach

Genetic variation in five geographically isolated samples of the blacklip abalone, Haliotis rubra, from south-eastern Australia was examined using two molecular techniques. A restriction fragment length polymorphism (RFLP) analysis using six restriction enzymes on the ND3/COIII region of mitochondrial DNA was compared with five independent nuclear DNA microsatellite loci. The results from both techniques suggest restricted gene flow between blacklip abalone separated by Bass Strait, and homogeneity among geographically isolated samples from around the island of Tasmania. Although both techniques showed similar resolving power, microsatellite DNA analysis is the preferred molecular technique for the fine scale investigation of blacklip abalone population structure because it makes possible the examination of numerous independent loci with potentially high levels of polymorphism. Both sample and locus specific homozygote excesses were recorded for the microsatellite loci. The most likely explanation for the locus specific deviations from Hardy-Weinberg expectations is the presence of null alleles.

Allozyme and mitochondrial DNA variation in orange roughy, Hoplostethus atlanticus (Teleostei: Trachichthyidae): little differentiation between Australian and North Atlantic populations

Allozyme and mitochondrial DNA (mtDNA) genetic variation was compared in orange roughy (Hoplostethus atlanticus Collett) collected from waters off southern Australia and from waters about 22 000 km away in the North Atlantic west of Scotland. Samples were screened for 11 polymorphic allozyme loci and with 9 restriction enzymes. Significant heterogeneity between the two areas was detected for three allozyme loci (ADA*, CK* and GPI-1*), and the overall GST (gene-diversity statistic) value of ∼1% was small but significant. Significant mtDNA haplotype heterogeneity was observed after χ2- of haplotype frequencies but not after a GST analysis. Nucleotide sequence-diversity analysis showed very low net divergence (0.0023%) between the two samples. The Australian orange roughy had a lower allozyme heterozygosity and a lower mitochondrial DNA nucleon diversity than the North Atlantic sample. The very limited, although significant, allozyme and mitochondrial DNA heterogeneity between these areas suggests that there is some gene flow between these two populations. The species appears to be widespread, with its presence reported from the southern Pacific, southern Indian, and northern and southern Atlantic Oceans, and it is likely that gene flow between the antipodes is mediated by stepping-stone exchange between adjacent populations rather than by direct migration.

Likelihood of bottleneck event in the history of the Australian population of Atlantic salmon (Salmo salar L.)

Abstract Previous microsatellite DNA analyses suggested a small overall loss of genetic variation in Tasmanian cultured Atlantic salmon when compared to a sample from the progenitor Canadian population. Here, 15 loci (eight microsatellite and seven allozyme) were examined in an additional sample from Tasmania and a sample from New South Wales (site of original importation to Australia in the mid-1960s from which the Tasmanian population was founded). Significant allele frequency differences were observed between samples for both microsatellite and allozyme loci. The greater allelic variation at microsatellite loci provided more information than allozyme loci on differences between samples from the progenitor and derived populations. A significant reduction in microsatellite heterozygosity, but non-significant loss of microsatellite alleles was observed between the Canadian and Australian samples. Comparison of observed heterozygosities with that expected under a two-phased model of mutation did not support the hypothesis of a severe bottleneck in the Australian population. However, low estimates of per-generation effective population sizes (80–90 over 11 generations) are consistent with a short-term moderate bottleneck in the Australian population of Atlantic salmon early in its introduction. Despite this the breeding population has been sufficiently large to maintain most pre-existing genetic variation in the Australian population.

The base composition of the krill genome and its potential susceptibility to damage by UV-B

We have determined the base composition (percentage of guanine-cytosine base pairs, GC%) of total DNA from Euphausia superba to be 32% ± 0.5%. This is the lowest GC% recorded for a metazoan. Low GC% DNA has high concentrations of thymine (T) residues and consequently a greater abundance of adjacent T residues [T(n) arrays]. Ultraviolet B (280–320 nm, UV-B) radiation damages DNA primarily at (T)n arrays, so we suggest that krill DNA may be more susceptible to damage from increased levels of UV-B radiation over the Southern Ocean than the DNA of other Antarctic organisms.

Variation in lipid composition of some deep-sea fish (Teleostei: Oreosomatidae and Trachichthyidae)

The lipid, fatty acid and fatty alcohol compositions were determined for muscle samples from six species of deep-sea oreo collected from Australian waters; namely Neocyttus rhomboidalis, Neocyttus sp., Allocyttus verrucosus, Allocyttus niger, Pseudocyttus maculatus, and Oreosoma atlanticum. Neocyttus helgae, landed in North Atlantic waters, was also analysed. Similar analyses were also carried out on the muscle and swim bladder of the orange roughy Hoplostethus atlanticus from both Australian and North Atlantic waters. Orange roughy is currently a major commercial species in southern Australia and is a new-fishery in the North Atlantic; there are four species of oreo of increasing commercial significance in Australia due to orange roughy quota reductions. It is therefore necessary to determine if the oreo fishing industry is capable of supplementing the current orange roughy requirements with respect to muscle and oil demand. In the oreos, the mean lipid content ranged from 0.5 to 3% of wet weight, with a mixed lipid composition including wax ester, triacylglycerol, sterol and polar lipid. The ratio of the monounsaturated fatty alcohols 22:1 to 20:1 allowed samples from the two geographical regions to be distinguished. Total wax ester in muscle from North Atlantic male orange roughy was much higher than in Australian fish (27 vs. 8.5% wet weight, respectively); females from both locations contained similar amounts of wax ester (4.5 vs. 3.3%, respectively). Selected swim bladders from North Atlantic and Australian orange roughy show similar wax ester content (90 vs. 82%, respectively). The ratio of 22:1 to 20:1 fatty alcohols in orange roughy from the two regions was 0.5 (Australian) and 1.4 (North Atlantic). Indeed differences exist between oreos from the two locations, but not between orange roughy and this requires further investigation. With respect to the nutritional value, the oreos are more attractive than the orange roughy however lipid levels remain much lower compared with other popular species.