RETRACTION: Nonlethal Age Estimation of Three Threatened Fish Species Using DNA Methylation: Australian Lungfish, Murray Cod and Mary River Cod.

Age‐based demography is fundamental to management of wild fish populations. Age estimates for individuals can determine rates of change in key life‐history parameters such as length, maturity, mortality and fecundity. These age‐based characteristics are critical for population viability analysis in endangered species and for developing sustainable harvest strategies. For teleost fish, age has traditionally been determined by counting increments formed in calcified structures such as otoliths. However, the collection of otoliths is lethal and therefore undesirable for threatened species. At a molecular level, age can be predicted by measuring DNA methylation. Here, we use previously identified age‐associated sites of DNA methylation in zebrafish (Danio rerio) to develop two epigenetic clocks for three threatened freshwater fish species. One epigenetic clock was developed for the Australian lungfish (Neoceratodus forsteri) and the second for the Murray cod (Maccullochellapeelii) and Mary River cod (Maccullochellamariensis). Age estimation models were calibrated using either known‐age individuals, ages derived from otoliths or bomb radiocarbon dating of scales. We demonstrate a high Pearson's correlation between the chronological and predicted age in both the Lungfish clock (cor = .98) and Maccullochella clock (cor = .92). The median absolute error rate for both epigenetic clocks was also low (Lungfish = 0.86 years; Maccullochella = 0.34 years). This study demonstrates the transferability of DNA methylation sites for age prediction between highly phylogenetically divergent fish species. Given the method is nonlethal and suited to automation, age prediction by DNA methylation has the potential to improve fisheries and other wildlife management settings.

Because of the high costs of collecting field data, many species recovery and management plans do not include a monitoring feedback component to measure the success of interventions and refine management strategies. Here, we demonstrate how leveraging existing monitoring data can provide broad-scale, cost-effective information about a threatened fish species, the Murray Cod Maccullochella peelii, which is of cultural and recreational importance in Australia. We applied a Bayesian hierarchical model of abundance to Murray Cod catch data collected as part of broad-scale, general condition monitoring in the Murray–Darling Basin. The model uses replicated sampling at spatially independent sites to disentangle the confounding effects of detection probability and abundance on catch data. We demonstrate the reliability of the analysis for determining trends in abundance with a simulation study, and we show that basinwide abundance of Murray Cod declined by over 50% between 2010 and 2013. We found that detection probability of Murray Cod can vary substantially across space and through time, suggesting that accounting for variable detection will be important in any future evaluation of Murray Cod populations. This study highlights variable detection as an issue in monitoring regimes and demonstrates a method for the cost-effective use of existing monitoring data to evaluate species abundance trends.

Worldwide, riverine fish are the target of environmental water because populations have declined in lotic river habitats following river regulation. Murray cod is an endangered Australian riverine fish with remaining populations associated with lotic river reaches with instream habitat, including some creeks operated as part of irrigation systems. Our objectives were to develop a life history model, apply the building block method of environmental flows to enhance the abundance of juvenile Murray cod and promote population recovery. From 2008 to 2018 we evaluated changes to Murray cod juvenile abundance before and after implementation of a perennial environmental flow regime that began in 2013. During the first year of the environmental flow, larvae were collected as evidence of spawning. Murray cod abundance can be enhanced with environmental flows that target: (1) an annual spring spawning or recruitment flow with no rapid water level drops; (2) maximising hydrodynamic complexity (i.e. flowing habitats that are longitudinally continuous and hydrodynamically complex); and (3) an annual base winter connection flow. Recognition that incorporating hydraulics (water level and velocity) at fine and coarse time scales, over spatial scales that reflect life histories, provides broader opportunities to expand the scope of environmental flows to help restore imperilled fish species in regulated ecosystems.

Early ontogeny-related changes of the fatty acid composition in the Percichthyid fishes trout cod, Maccullochella macquariensis and Murray cod, M. peelii peelii

One of the most iconic Australian fish is the Murray cod, Maccullochella peelii (Mitchell 1838), a freshwater species that can grow to ∼1.8 metres in length and live to age ≥48 years. The Murray cod is of a conservation concern as a result of strong population contractions, but it is also popular for recreational fishing and is of growing aquaculture interest. In this study, we report the whole genome sequence of the Murray cod to support ongoing population genetics, conservation, and management research, as well as to better understand the evolutionary ecology and history of the species. A draft Murray cod genome of 633 Mbp (N50 = 109 974bp; BUSCO and CEGMA completeness of 94.2% and 91.9%, respectively) with an estimated 148 Mbp of putative repetitive sequences was assembled from the combined sequencing data of 2 fish individuals with an identical maternal lineage; 47.2 Gb of Illumina HiSeq data and 804 Mb of Nanopore data were generated from the first individual while 23.2 Gb of Illumina MiSeq data were generated from the second individual. The inclusion of Nanopore reads for scaffolding followed by subsequent gap-closing using Illumina data led to a 29% reduction in the number of scaffolds and a 55% and 54% increase in the scaffold and contig N50, respectively. We also report the first transcriptome of Murray cod that was subsequently used to annotate the Murray cod genome, leading to the identification of 26 539 protein-coding genes. We present the whole genome of the Murray cod and anticipate this will be a catalyst for a range of genetic, genomic, and phylogenetic studies of the Murray cod and more generally other fish species of the Percichthydae family.

The Murray cod (Maccullochella peelii peelii) is a large fish species keenly sought by anglers. However, this species has declined in distribution and abundance and is now listed nationally as vulnerable. This study was undertaken in the Ovens and Murray rivers, to collect larvae and age-0 Murray cod and determine the distribution of larval Murray cod around the mid-Murray River irrigation storage of Lake Mulwala. Murray cod larvae were collected from 17 of 18 sites: main channels and flowing anabranch channels of regulated and unregulated rivers, sites upstream and downstream of the lake, in the upper and lower reaches of the lake, and in the outflowing Yarrawonga irrigation channel. Larval Murray cod were collected only by methods that sampled drift in flowing waters. Age-0 Murray cod were collected by electrofishing in the main river, but not in off-channel waters, suggesting that cod are likely to settle into habitats in the main channel at a post-larval stage. The widespread occurrence of drifting larvae suggests that this species may be subject to previously unrecognised threats as they pass through hydro-electric power stations or become stranded in anabranch and irrigation channels. Results of this study are likely to be applicable to other species with drifting larval stages, and are relevant to other locations in the Murray–Darling Basin.

Wild fish as reservoirs of parasites on Australian Murray cod farms

Variation in river flow is a strong behavioural determinant for the movement of many freshwater fish species and often is linked to key aspects of their life cycle. The alteration of natural flow regimes to meet human water demands can result in changes to this variability, and cause declines in water‐dependent biota. Environmental flows are used as a remediation tool in some regulated rivers with the intention of restoring aspects of the natural flow regime to benefit riverine species, although empirical data are required to inform the efficacy of these interventions. Using acoustic telemetry, we quantified the movement responses of two large‐bodied native fish species (freshwater catfish, Tandanus tandanus and Murray cod, Maccullochella peelii) to variations in river flow over 4 years in two intermittent regulated rivers in the northern Murray–Darling Basin, Australia. Both rivers received periodic environmental flow releases and also there were several large natural flow events during the study period. Both species displayed a range of intra‐specific movement behaviours. Analysis of individuals' movements revealed five distinct functional groups, which were represented in both species. We found that periods of environmental flow delivery played an important role in the movement behaviour of both species. Murray cod were more likely to move during periods of environmental flow releases, whereas freshwater catfish were less likely to move on an environmental release following higher antecedent flows. No large‐scale philopatric movements were observed, yet Murray cod were more likely to move during the breeding period, indicative of nest site selection. We also found that the likelihood of movement in both species was higher in the smaller of the two rivers for a given magnitude of flow. Our results suggest that environmental flows may benefit certain fish species by facilitating, rather than cueing breeding behaviours, allowing individuals improved access to, and provision of, higher‐quality nesting habitats. These findings will aid water managers in creating economical and targeted environmental flow releases, timing larger flow pulses for the species with flow‐cued reproductive strategies, and providing a continuity of smaller baseflows for species whose reproduction is not dependent on flow cues but may be enhanced by greater connectivity.

Diet composition and preference of juvenile Murray cod, trout cod and Macquarie perch (Percichthyidae) reared in fertilised earthen ponds

Cytochemical characterisation of the leucocytes and thrombocytes from Murray cod ( Maccullochella peelii peelii, Mitchell)

Murray cod (Maccullochella peelii peelii) is an iconic native Australian freshwater fish and an ideal species for ecotoxicological testing of environmental pollutants. The species is indigenous to the Murray-Darling basin, which is the largest river system in Australia but also the ultimate sink for many environmental pollutants. The organotins tributyltin (TBT) and dibutyltin (DBT) are common pollutants of both freshwater and marine environments and are also known for their immunotoxicity in both mammals and aquatic organisms. In this study, TBT and DBT were used as exemplar immunotoxins to assess the efficiency of immune function assays (i.e., mitogen-stimulated lymphoproliferation, phagocytosis in head kidney tissue, and serum lysozyme activity) and to compare the sensitivity of Murray cod to other fish species. The organotins were lethal to Murray cod at concentrations previously reported as sublethal in rainbow trout (i.e., intraperitoneal [i.p.] lethal dose to 75% of the Murray cod [LD75] = 2.5 mg/kg DBT and i.p. lethal dose to 100% of the Murray cod [LD100] = 12.5 mg/kg TBT and DBT). In vivo TBT exposure at 0.1 and 0.5 mg/kg stimulated the phagocytic function of Murray cod (F = 6.89, df = 18, p = 0.004), while the highest concentration of 2.5 mg/kg TBT decreased lymphocyte numbers (F = 7.92, df = 18, p = 0.02) and mitogenesis (F = 3.66, df = 18, p = 0.035). Dibutyltin was the more potent immunosuppressant in Murray cod, causing significant reductions in phagocytic activity (F = 5.34, df = 16, p = 0.013) and lymphocyte numbers (F = 10.63, df = 16, p = 0.001).

The use of freshwater for human consumption and agriculture has resulted in many wetland ecosystems being some of the most seriously impacted ecosystems in the world. In attempts to rehabilitate wetland ecosystems environmental flows are used to restore parts of the hydrological regime altered by human water use. The use of environmental water intends to improve ecosystem health, but frequently aims to have specific outcomes for populations of higher-order consumers such as iconic fish and bird species. To date, research and monitoring has mainly focused on understanding ‘flow-ecology’ relationships, without investigating the mechanisms underlying them. We sought to understand the importance of different basal food sources to the growth of the endangered Murray cod in temporary wetland systems using fatty acid biomarkers. We flooded replicate mesocosms with two different wetland soils to produce sufficient zooplankton prey to sustain and grow Murray cod larvae for approximately 2 weeks. The fatty acid profiles of Murray cod and percentages of different biomarkers were compared at the start and finish of the experiment and our results suggest that the most important basal food source is green algae. However, the biomarkers of diatoms, cyanobacteria and bacteria also increased and differed between wetlands with different hydrological regimes. It is unclear if our results can be extended to other wetland systems and we encourage further research both into the relationship between length of wetland flooding and invertebrate densities in other systems. We also encourage research into the mechanistic pathways in which green algae carbon is transferred through food webs to higher order consumers in wetland systems to help generalise our results to other wetlands and support the management of wetlands through the timing and duration of flooding from environmental water.

The Australian lungfish, Neoceratodus forsteri, exists as remnant natural populations in two rivers of south-east Queensland, Australia, and several translocated populations. Lungfish habitats have been impacted by agriculture and forestry, alien plants and fish and by river impoundment and regulation of flows. The species has been listed as vulnerable under Australian Commonwealth legislation. A proposal to construct Traveston Crossing Dam on the free-flowing main channel of the upper Mary River could seriously threaten the lungfish. The dam can be stopped by Commonwealth legislation if important populations of lungfish in the Mary River are likely to be significantly impacted by the new dam. This paper assembles evidence that impoundment of the Mary River and regulation of river flows are likely to decrease and fragment important lungfish populations, disrupt the breeding cycle, reduce juvenile recruitment, and isolate and decrease habitat availability/quality to such an extent that the species is likely to decline. Proposed mitigation strategies include fish transfer facilities, provision of flow releases from the dam (environmental flows) to sustain lungfish habitat and breeding downstream, and translocation of hatchery-reared juvenile lungfish into suitable natural habitats. These mitigation efforts may not be sufficient to secure the genetic diversity and long-term viability of lungfish populations in the Mary River.

Conservation of Murray cod (Maccullochella peelii), a large endangered fish species of Australia’s Murray–Darling Basin, relies on a detailed understanding of life history, including movement patterns and habitat use. We used radio-tracking to investigate the movement of 36 Murray cod in main channel and anabranch habitats of the lower River Murray during a flood and associated hypoxic blackwater event. During a flood peak of ~93 000 ML day–1, dissolved oxygen decreased to 1.2 mg L–1. Four movement types were observed: (1) localised small-scale movement, (2) broad-scale movement within anabranch habitats, (3) movement between anabranch and main channel habitats, and (4) large-scale riverine movement. Murray cod exhibited high fidelity to anabranch habitats but also moved extensively between anabranches and the main channel. Fish were consistently located in the main channel or permanent anabranches, suggesting that use of ephemeral floodplain habitats is limited, and highlighting the importance of connectivity between off-channel and main channel habitats. Mortality of radio-tagged fish was considerable (25%) in association with low dissolved oxygen concentrations, indicating that hypoxic blackwater may have had a substantial impact on Murray cod populations in the lower River Murray.

Karyotypic data from Australian native freshwater fishes are scarce, having been described from relatively few species. Golden perch (Macquaria ambigua) and Murray cod (Maccullochella peelii) are two large-bodied freshwater fish species native to Australia with significant indigenous, cultural, recreational and commercial value. The arid landscape over much of these fishes’ range, coupled with the boom and bust hydrology of their habitat, means that these species have potential to provide useful evolutionary insights, such as karyotypes and sex chromosome evolution in vertebrates. Here we applied standard and molecular cytogenetic techniques to characterise karyotypes for golden perch and Murray cod. Both species have a diploid chromosome number 2n = 48 and a male heterogametic sex chromosome system (XX/XY). While the karyotype of golden perch is composed exclusively of acrocentric chromosomes, the karyotype of Murray cod consists of two submetacentric and 46 subtelocentric/acrocentric chromosomes. We have identified variable accumulation of repetitive sequences (AAT)10 and (CGG)10 along with diverse methylation patterns, especially on the sex chromosomes in both species. Our study provides a baseline for future cytogenetic analyses of other Australian freshwater fishes, especially species from the family Percichthyidae, to better understand their genome and sex chromosome evolution.