Thesis (Ph.D.) - James Cook University, 2004. / Typescript (photocopy)

Barramundi (Lates calcarifer) is a centropomid teleost with a wide distribution across the Indo Pacific. In Australia, barramundi are native to the tropical zone from Exmouth Gulf in Western Australia, across the northern part of the continent, to the Mary River in Queensland. Barramundi are protandrous hermaphrodites, and are euryhaline, with a catadromous life history. Barramundi are a valuable Australian resource, with important commercial and recreational fisheries and aquaculture production to the value of $11 million dollars per year. Recent declines in the availability of the fish in some rivers has led to an interest in the possibility of restocking rivers with barramundi from other areas. Determining the genetic structure of barramundi populations in Australia is important for understanding biogeographic history, and appropriate management practices for both aquaculture and recreational and commercial fishing. Previous studies have concentrated on the east coast of Australia, and have largely ignored the western populations. In this study, I obtained DNA data from barramundi populations across the Australian range of the species, as well as populations from Papua New Guinea and Indonesia. The aims of this study were to use the genetic data to determine: 1. if populations in Western Australia show genetic differences between geographic regions 2. if these populations show an ancestral split from populations in the east of Australia and 3. the ancestral origins of Australian barramundi. Previous studies of DNA data from barramundi have discovered an east/west split occurring at the Torres Strait that was assumed to be caused by the closing of the strait during lowered sea levels. However, these studies suffered from a bias in sampling area, concentrating either on the eastern half of the range of barramundi, or on the western tip of the range. Data from these studies were combined and reanalyzed. Two major clades were discovered, with considerable biogeographic structuring, but their geographic locations did not coincide with the reported vicariance event at the Torres Strait. Instead, historical divisions among freshwater drainage systems appeared to have driven the evolutionary history of barramundi in Australia. In order to investigate these historical divisions further, a 290 bp section of the mitochondrial DNA control region was sequenced in 284 barramundi from seven populations across the Australian geographic range of the species and from one population in Papua New Guinea and one population in Indonesia. Analyses of molecular variance within and among populations showed significant geographic structuring, based on biogeographical provinces and drainage divisions. Nested clade analyses indicated that these geographical associations were the result of restricted gene flow, range expansion, and past fragmentation events. I hypothesise that the Ord River area in the west of the continent was the ancestral source population for the rest of the species’ range across Australia, with Indonesia being the most likely origin of this source. Populations of barramundi from the Pilbara region are genetically distinct and geographically isolated, with strong evidence of an ancestral divide along geographical barriers to dispersal. There is a strong association between Papua New Guinea and Australia, although further investigations using the cytochrome b region of mitochondrial DNA indicated a more ancestral divide between the two than is currently evident, which could reflect an ancient geographical divide between the two, or could be evidence of a secondary migration route to Australia. For a more detailed study of evolutionary processes acting on populations of barramundi in Western Australia, allelic diversity was examined at five microsatellite loci. All loci were polymorphic and genotypic frequencies conformed to Hardy-Weinberg expectations, with no significant linkage between loci evident in any population. Measures of within population diversity were significantly related to latitude, suggesting southerly migration from a northern source population. The Ord River was the most genetically diverse population, and the most likely ancestral migration source to the area, with diversity decreasing down the west coast. Although there were significant differences among populations, the nuclear microsatellite data do not indicate the same degree of genetic structuring as is evident in the mitochondrial data. This may be a consequence of rapid evolutionary change at microsatellite loci, with past separations or population differences masked by recombination and back mutation of the microsatellite alleles. However, the nature of nuclear and mitochondrial inheritance may also indicate life history differences between the sexes, where significant genetic contribution to gene flow by males and limited female gene flow may lead to preservation of maternally inherited population substructure. The principal findings from this study are: • There is no genetic evidence for an east/west division of barramundi populations in Australia, as suggested by previous research. • Despite barramundi’s catadromous life history, and ability to disperse through marine waters, the present genetic structure indicates a division principally among river drainages. From a population genetic viewpoint, the species can be regarded as freshwater, rather than marine. • The most likely origin of barramundi in Australia is the Ord River region, with Indonesia as the route of migration. • Differences in the population structure demonstrated by nuclear and mitochondrial data indicate possible life history differences between the sexes. • Barramundi populations in different biogeographical provinces may have been substantially isolated over a long period of time, and may therefore represent independently evolving populations. This has important implications for fishery management and translocation issues for restocking rivers.

No description available.

Secondary salinisation has rendered over 100 million hectares of land throughout the world, and over 5 million hectares in Australia, unsuitable for conventional agriculture. The utilization of salinised land and its associated water resources for mariculture is an adaptive approach to this environmental problem with many potential economic, social and environmental benefits. Despite this, inland mariculture is yet to develop into an industrial-scale, rural enterprise. The main aim of this study was therefore to identify and address some of the technical and biological limitations to the development of an inland finfish mariculture industry. Three technical aspects essential to the development of an Australian inland mariculture industry were reviewed; potential sources of water, the species suitable for culture in these water sources and the production systems available to produce them. Based on factors such as their quantity, quality and proximity to infrastructure, the most appropriate water sources were deemed to be groundwater obtained from interception schemes and waters from operational or disused mines. In terms of species, mulloway (Argyrosomus japonicus) were identified as having many positive attributes for inland mariculture, including being temperate and therefore having the ability to be cultured year-round in the regions where the majority of secondary salinity occurs. Seasonal production of barramundi (Lates calcarifer) in ponds in the temperate climatic zones has potential, but may be more appropriate for those salinised water sources located in the warmer parts of the country. Rainbow trout (Oncorhynchus mykiss) were also identified as having excellent potential provided water temperature can be maintained below the upper lethal limit and also have potential for seasonal production, perhaps in rotation with barramundi. In terms of production systems, pond-based culture methods were found to have many advantages specific to inland mariculture. Static ponds enable culture in areas with low groundwater yield and more cost-effective potassium supplementation compared with flow through ponds. Static ponds also largely overcome the issues associated with the disposal of salt-laden and eutrophied waste water; however yields from static ponds are typically low and limited by the nutrient input into the pond. In response to the yield constraints of static pond culture, a new culture technology known as the Semi-Intensive Floating Tank System (SIFTS) was designed, patented and constructed in collaboration with the aquaculture industry and tested in a static inland saline pond in the wheatbelt of Western Australia. This technology was designed to reduce nutrient input into ponds by the collection of settleable wastes and to provide large volumes of well-oxygenated water to the target species, to ameliorate the loss of fish from low dissolved oxygen during strong microalgal blooms. The three species identified above has having excellent potential for inland mariculture (mulloway, rainbow trout, and barramundi) were grown in SIFTS held within a 0.13 ha static, inland saline water body (salinity 14 ppt) over a period of 292 days, yielding the equivalent of 26 tonnes/ha/year (total for all three species). Rainbow trout were grown with an FCR of 0.97 from 83 to 697 grams over 111 days (SGR, 1.91%/day) between June and September, when average daily water temperatures ranged from 12.3„aC to 18.2„aC. Over the same time period, mulloway grew only from 100 to 116 grams, however, once temperatures increased to approximately 21„aC in October, feed intake increased and mulloway grew to an average size of 384 grams over 174 days with an SGR and FCR of 0.68 %/day and 1.39, respectively. Barramundi stocked in November with an average weight of 40 grams increased to 435 grams in 138 days (SGR 1.73%/day) with an FCR of 0.90. The SIFTS significantly reduced nutrient input into the pond by removing settleable wastes as a thick sludge with a dry matter content of 5 to 10%. The total quantity of dry waste removed over the 292 day culture period was 527 kg (5 tonnes/ha/yr), which was calculated to contain 15 kg of nitrogen (144 kg/ha/yr) and 16 kg of phosphorus (153 kg/ha/yr). The release of soluble nutrients into the pond resulted in blooms of macro- and micro- algae which caused large and potentially lethal diurnal fluctuations in dissolved oxygen within the pond, however, comparatively stable levels of dissolved oxygen were maintained within each SIFT through the use of air lift pumps. It is well documented that saline groundwater is deficient in potassium which, depending on the extent of the deficiency, can negatively impact on the performance of marine species, including fish. The physiological effects of this deficiency on fish, however, have not been previously described. As such, I conducted a bioassay investigating the physiological effects of a hypersaline (45 ppt) groundwater source containing 25% of the potassium found in equivalent salinity seawater (i.e. 25% K-equivalence) on juvenile barramundi. Histopathological examination of moribund fish revealed severe degeneration and necrosis of skeletal muscles, marked hyperplasia of branchial chloride cells and renal tubular necrosis. Clinical chemistry findings included hypernatraemia and hyperchloridaemia of the blood plasma and lowered muscle potassium levels. It was concluded from this study that the principal cause of death of these barramundi was skeletal myopathy induced by unsustainable buffering of blood plasma potassium levels from the muscle. Although such hypokalaemic muscle myopathies have been previously described in mammals and birds, this was the first description of such myopathies in fish. It was hypothesized from the results described above that the physiological effects of potassium deficiency are dependent on salinity and that they would be ameliorated by potassium supplementation. These predictions were tested in a subsequent study which measured the effects of potassium supplementation between 25% and 100% K-equivalence on the growth, survival and physiological response of juvenile barramundi at hyperosmotic (45 ppt), near-isosmotic (15 ppt) and hyposmotic (5 ppt) salinities. Unlike those juvenile barramundi reared at 45 ppt and 25% K-equivalence in the previous study, those reared in 50% K-equivalence water at 45 ppt in this study survived for four weeks but lost weight; whereas at 75% and 100% K-equivalences fish both survived and gained weight. Homeostasis of blood plasma potassium was maintained by buffering from skeletal muscle. Fish reared in 50% K-equivalence at this salinity exhibited muscle dehydration, increased branchial, renal and intestinal (Na+-K+)ATPase activity and elevated blood sodium and chloride, suggesting they were experiencing osmotic stress. At 15 ppt, equal rates of growth were obtained between all K-equivalence treatments. Buffering of plasma potassium by muscle also occurred but appeared to be in a state of equilibrium. Barramundi at 5 ppt displayed equal growth among treatments. At this salinity, buffering of plasma potassium from muscle did not occur and at 25% K-equivalence blood potassium was significantly lower than at all other K-equivalence treatments but with no apparent effect on growth, survival or (Na+-K+)ATPase activities. These data confirmed the hypothesis that proportionally more potassium is required at hyperosmotic salinities compared to iso- and hypo- osmotic salinities and also demonstrated that barramundi have a lower requirement for potassium than other marine and estuarine species being investigated for culture in inland saline groundwater. In addition to ongrowing fish, saline groundwater has potential for hatchery production. Specific advantages include the vertical integration of inland saline farms and the production of disease-free certified stock through isolation from the pathogens and parasites found naturally in coastal water. To determine the potential of utilizing inland saline groundwater for hatchery production, barramundi larvae were reared from 2 to 25 days post hatch in 14 ppt saline groundwater with either no potassium supplementation (38% K-equivalence) or full potassium supplementation (100% K-equivalence). Growth, survival and swimbladder inflation of these larvae were compared against those grown in control treatments of seawater (32 ppt) and seawater diluted to 14 ppt. Those reared in saline groundwater with 38% K-equivalence exhibited complete mortality within 2 days, whilst those held in groundwater with full supplementation survived at a rate equal to both control treatments (pooled average 51.1 ¡Ó 0.5%). At 25 days post hatch, there was no significant difference in larval length or dry weight between those grown in the 14 ppt control treatment and those in the saline groundwater with full potassium supplementation. There were no significant differences in swim bladder inflation between any of the surviving treatments (average 93.3 ¡Ó 2.5%). This is the first description of rearing barramundi larvae both in low salinity seawater and in saline groundwater, and demonstrates that the requirement for potassium by larval barramundi is higher than for juveniles of the same species. In addition to a deficiency in potassium, saline groundwater in Western Australia often contains an elevated concentration of manganese relative to seawater as a result of anaerobic reduction of manganese oxides or the pedogenic weathering of manganese-bearing rock. The effects of elevated manganese on marine or estuarine fish have not been described and a study was therefore conducted to determine if manganese, at a concentration typical of that found in saline groundwater, has any impact on fish. The effects of 5 mg/L of dissolved manganese on juvenile mulloway at salinities of 5, 15 and 45 ppt were determined by comparing the survival, growth and blood and organ chemistry with those grown at the same salinities without manganese addition. Survival of mulloway at 45 ppt in the presence of 5 mg/L of manganese (73 ¡Ó 13%) was significantly lower than all other treatments, which achieved 100% survival. Those fish grown in seawater without manganese exhibited rapid growth, which was not affected by salinity (SGR = 4.05 ¡Ó 0.29%/day). Those fish grown at 5 ppt and 45 ppt in the presence of manganese lost weight over the two week trial (SGR 0.17 ¡Ó 0.42 and -0.44 ¡Ó 0.83%/day, respectively), whilst those at 15 ppt gained only a small amount of weight (SGR 1.70 ¡Ó 0.20%/day). Growth was therefore affected by manganese and by the interaction of manganese and salinity, but not salinity alone. Manganese was found to accumulate in the gills, liver and muscle of the fish. No gill epithelial damage or other significant histological findings were found, however, significant differences in blood chemistry were observed. Blood sodium and chloride of manganese exposed fish were significantly elevated in hyperosmotic salinity (45 ppt) and depressed at hyposmotic salinity (5 ppt) compared with unexposed fish; consistent with manganese causing apoptosis or necrosis to chloride cells. Blood potassium was significantly elevated and liver potassium significantly reduced at all salinities in the presence of manganese. These findings are consistent with manganese interfering with carbohydrate metabolism. There were no differences in blood sodium, chloride or potassium across salinities in fish not exposed to manganese, demonstrating mulloway are capable of efficient osmoregulation across this salinity range.

Barramundi (Lates calcarifer) aquaculture is expanding throughout Australia and the Asia-Pacific region. The Department of Primary Industries, Queensland have estimated that the production from this industry could reach $30 million per annum in Australia by 2010. However, current production is severely impeded by outbreaks of Streptococcus iniae, which causes a fatal septicaemia in barramundi. S. iniae is a Gram positive bacterium which infects both humans and fish and was first reported in Australia in the 1980s in Queensland, but has rapidly disseminated to other states in Australia (Western Australia, Northern Territory, South Australia). Globally, there appears to be little geographical restriction to the distribution of S. iniae and infection occurs in temperate, sub-tropical and tropical, marine and fresh water fish with no evidence of species specificity. Outbreaks have been reported in North America, Middle East, Europe and Asia-Pacific, including Australia, Indonesia, Malaysia, Korea, China, Taiwan and Japan. Understanding distribution and spread of S. iniae is confounded by a number of factors. Firstly, identification of S. iniae is not straightforward, thus isolates often remain ‘unidentified’, as this bacterium is not included in commercial databases. In other cases it is misidentified as other bacteria such as S.uberis, S. dysgalactiae subsp. equisimilis and S. anginosus. Furthermore, variability in phenotypic traits has led to difficulty in identifying isolates using standard commercial diagnostic kits. Additionally, there is tangible evidence of geographic diversity and endemism with strain variability having been reported from fish isolates in Japan, USA and Israel, and in human isolates from Canada, USA and SE Asia. Understanding strain diversity amongst S. iniae is critically important in terms of managing the disease. Ability to track routes of distribution of the pathogen in imported fish, including ornamentals and food fish has implications for better biosecurity. Perhaps most importantly, strain diversity has been reported as a cause of vaccine failure in trout in Israel and in barramundi in Northern Territory, Australia. To date, very little information exists on strain diversity in S. iniae and no research has been conducted on the diversity amongst Australian isolates within the barramundi industry. The aim of this thesis is to develop reliable methods for identification of differing strains of S. iniae and to investigate antigenic diversity in order to better inform both vaccine design and biosecurity procedures with which to manage this important disease in Australia and globally. To achieve this, a collection of more than 100 isolates from Australia and throughout the world has been created and stored at the University of Queensland. In the first chapter of my thesis, routine confirmatory diagnosis using amplification of the lactate oxidase gene was performed to support biochemical and physiological identification provided by the supplying laboratories and veterinarians. During this initial screen, two important discoveries were made. Firstly, S. iniae isolates can be divided into two groups based on the different sizes of PCR product obtained, 869 bp (now named Type 1) and 921 bp (now named Type 2). This difference was only found in isolates from Northern Territory, Australia. In light of this, identity was further confirmed by the results of partial sequencing of the 16S rRNA gene with the 530F primer and submission to the BLAST server (http://www.ncbi.nlm.nih.gov/BLAST), which returned identities of 100% to S. iniae ATCC 29178. Sequence analysis of the lctO gene in isolates representing both the normal (lctO type 1) and higher molecular weight (lctO type 2) revealed that there is an insertion of 51 bp of repeat sequence in lctO type 2. Apart from the insertion sequence found in the 3' end of the gene in some isolates, three nucleotides in positions 211-213, not previously detected when the gene was described previously, resulted in an inserted valine residue in the translated product from all isolates. I also note an apparent error in the primary sequence and translation of the GenBank sequence (Y07622). This is likely to be due to an inserted C nucleotide at position 1148 at the far 3’ end of the gene sequence (inside the LOX-1, LOX-2 priming region) that has altered the reading frame. This means that the expected PCR product size of 870 bp is incorrect and is actually 869 bp. To determine the phenotypic relevance of the variation in lox gene product size, the lactate oxidase enzyme was extracted from cell lysates and assayed for activity. The two variant genes were each cloned and expressed in E. coli. Lactate oxidase enzyme activity also showed that there were differences in enzyme activity between the two gene products with strains expressing the higher molecular weight enzyme variant exhibiting higher enzyme activity. This suggests that positive selection may apply in favour of the larger gene in situations where lactate is the most readily available carbon source. However, no variation was detected in the lactate permease gene lctP, for any of the strains analysed. Whilst there was no difference in the Minimum Inhibitory Concentration Test (MIC) using different concentration of lactate there were differences detected in the growth rate of QMA0165 and QMA0177. Significant inhibition on growth rate of QMA0165 was detected with a 0.3% and 0.5% of lactate while there was no significant inhibition in QMA0177 with the same concentration. Prediction on three dimensional protein structure using PyMol based on Aerococcus viridans 3-D protein structure showed that there was an additional loop in lctO type 2 which suggested that it might play a role in enhancing enzyme activity of the binding site. The environment in barramundi farms in Northern Territory where the lctO type 2 isolates were isolated has 9 metre tides resulting in water flows in excess of 3km/h. It is likely that the resulting enforced swimming of the fish host has led to selection and maintenance of a gene encoding the higher efficiency enzyme in S. iniae. As diversity has led to reported vaccine failure in Israel, and antigenic diversity has been recorded in Japan and in isolates from the USA, the second data chapter of this thesis explores surface antigenic diversity of Australian S. iniae isolates from barramundi using a whole cell ELISA using a suite of antibodies raised in barramundi against four S. inaie isolates from differing habitats (freshwater and marine) and states (Western Australia and Queensland) in Australia. Forty-one isolates predominantly from farmed fish throughout Australia between 1995 and 2006 were serotyped and compared with reference isolates from the USA and Canada. Multiple serotypes were identified using polyclonal sera raised in barramundi against four different Australian S. iniae isolates i.e. anti QMA0072 (Queensland), anti QMA0074 (Queensland), anti QMA0083 (Western Australia) and anti QMA0087 (Western Australia). Different serotypes were often isolated from the same sites either simultaneously or within short time periods, indicating potential coexistence of multiple isolates in a particular geographic location or habitat. Electrophoretic profiles of whole cell proteins and integral membrane proteins were similar amongst isolates when analysed by SDS- PAGE, regardless of serotype. The results presented here suggest that surface serotypic variability of S. iniae is complex and multifactorial involving capsular carbohydrate and some surface proteins. As raising consistent antiserum in barramundi is almost impossible, and rabbit antisera invariably recognise more epitopes than teleost fish, a more consistent molecular method of typing was investigated in the third data chapter. In this chapter, the whole genome of S. iniae was digested using SmaI and separated using Pulsed Field Gel Electrophoresis (PFGE). Twenty four isolates representing different geographical origin and host were analysed using this method. Reference isolates from dolphins, fish and humans were obtained from the Centers for Disease Control for comparison. PFGE profiles indicated at least 4 distinct groups amongst the Australian isolates, but these did not correlate with surface serotype. Interestingly, whilst there have been no reports of human cases of S. iniae infection in Australia, many of the isolates examined had closely related PFGE profile with reference human isolates from USA and Canada, but were markedly different from the type isolates isolated from dolphins. One of the major difficulties associated with PFGE is between lab variability, hence the requirement for inclusion of large numbers of reference isolates on each gel. Recently, multilocus sequence typing (MLST) has been developed for epidemiological studies in a variety of human pathogens. MLST is based on sequencing of 8 ubiquitous housekeeping genes, genes which evolve slowly, allowing clustering of isolates. As sequencing is consistent between laboratories, results can be posted on a website and compared internationally without requiring transfer of strains overseas. The fourth data chapter in my thesis develops for the first time an MLST scheme for S. iniae. Primers for eight housekeeping genes were designed and annealing temperature for amplification were optimized. The selected housekeeping genes were: adhP (Alcohol dehydrogenase), pheS (Phenylalanyl tRNA syhthetase), atr (Amino acid transporter), glnA (Glutamine synthetase), sdhA (Serine dehydrogenase), glcK (Glucose kinase) and tkt (Transketolase). However, glnA was dropped from the analysis because of the inconsistent PCR product. Thirty seven isolates were selected representing the Australian isolates and other international isolates from United States, Canada, Israel, Thailand, Reunion Island with different hosts i.e. Amazon freshwater dolphin, human, flying fox and different species of fish (Channa striata, Oncorhynchus mykiss and Lates calcarifer). As there is no database available for S. iniae in the MLST database yet, only limited isolates from the global collection that can be analysed with the Australian isolates. The present study found that MLST results are less discriminative when compared to PFGE, but were very useful in pinpointing origin to a particular country, perhaps indicating little transfer of isolates between nations. MLST grouped together Australian, Thailand (QMA0187 and QMA0190), human isolates from Canada and USA regardless the geographic origin (QMA0130, QMA0133 and QMA0137) and also fish isolate from Canada (QMA0139). The similarity of the human isolates with the Australian isolates supporting PFGE results, which had a similar SmaI PFGE profiles to many of the fish isolates from Australia. However, MLST managed to distinguish isolates QMA0140 (dolphin/ USA), QMA0141 (dolphin/ USA), QMA0136 (human/ USA) and other international isolates from Israel (QMA0186 and QMA0188), Reunion Island (QMA0189). Despite the degree of heterogeneity in other methods used (serotyping, PFGE), MLST method showed a high homogeneity amongst S. iniae from Australia, perhaps reflecting the slow evolution of these genes and no accidental import of isolates. During the 12 years of isolates covered by our strain collection, there would appear to have been no evolution of these highly conserved genes within Australia. The variation in serotype within a single Sequence Type showed that there may be frequent horizontal gene transfer, or more rapid evolution of genes involved in synthesis and transport of capsular polysaccharide or other surface features. Moreover, the PFGE results indicate that there is more genetic plasticity in amongst the genome of S. iniae than indicated by then MLST. In order to gain a more discriminative epidemiological perspective of S. iniae, the results presented in this thesis suggest combination of different typing methods such as PFGE and MLST, with the latter providing an accurate means of determining nation of origin of strains (and therefore of great potential for biosecurity purposes) whilst PFGE may provide better discrimination of movement of local isolates within Australia.

The global aquaculture industry is expanding to support increased demand in global markets and supplement traditional fisheries. The rapid increase of aquaculture production relies on introducing and using species outside their native range, posing significant regional environmental and socio-economic risks. Non-native species are selected based on transferable large-scale production protocols, fast growth, and existing market demand. Aquaculture is an important sector within Saudi Arabia’s Vision 2030 (anticipated >400% production increase by 2030). Barramundi (Lates calcarifer) was introduced into the Red Sea for open sea-cage farming in 2008, with large-scale production beginning in 2014. Regionally, there are numerous anecdotal reports of escape events of this species, including an incident in 2015 involving the release of ~300,000 fish. Since then, local fishers have caught up to 15 wild individuals per month in Al Lith, central Saudi Arabia. This study presents the first biological information on L. calcarifer aquaculture escapees in the Saudi Arabian Red Sea. Wild-caught L. calcarifer (n=5) were collected from a local fish landing and measured up to 10 kg, eight years of age, were sexually mature, and consumed benthic and coral reef fishes. In contrast, individuals in aquaculture facilities are harvested at < 1 kg and < 400 mm. In their native range, L. calcarifer can reach up to 200 cm and 60 kg. To assist in detecting and monitoring escapees within the Red Sea, I designed and validated a species-specific SYBR-based environmental DNA quantitative PCR assay targeting a 16S mitochondrial region of L. calcarifer rRNA (Barramundi_16S assay). Preliminary results, using DNA metabarcoding and environmental (seawater) samples, detected L. calcarifer near active/historical aquaculture farms and north/south of areas where this species has not been reported previously in the Red Sea (10–250 km from aquaculture facilities). In the future, the Barramundi_16S assay can screen additional eDNA samples (n=250) collected for this study to delineate the geographic range of barramundi in the Red Sea. Cumulatively, this study highlights the need to consider the ecological impacts of aquaculture escapees and provides managers and industry with valuable information and a novel molecular monitoring tool for detecting aquaculture escapees.