Validation of eDNA as a viable method of detection for dangerous cubozoan jellyfish

Stings from certain species of cubozoan jellyfish are dangerous to humans and their seasonal presence in tropical marine waters poses a significant risk to coastal communities. The detection of cubozoans is difficult due to high spatial and temporal variation in their occurrence and abundance. Environmental DNA (eDNA) has the potential to detect rare species and therefore offers potential to detect cubozoans, not only pelagic medusae, but presence of cryptic polyp life stages. The objective of this study was to validate the use of eDNA as a viable detection method for four cubozoan species (Chironex fleckeri, Copula sivickisi, Carybdea xaymacana, and Carukia barnesi). Species‐specific primers were developed for each of these four cubozoans and an eDNA approach validated utilizing both laboratory and field trials. Laboratory DNA degradation experiments demonstrated that C. sivickisi DNA degraded quickly but could still be detected in sea water for up to 9 days post‐jellyfish removal. Positive detection was found for C. fleckeri, C. xaymacana, and C. sivickisi medusae in the waters surrounding Magnetic Island, Queensland, in the Austral spring/summer (September‐January). Based on visual surveys, there was a poor relationship between concentration of eDNA and abundance of jellyfish. Positive eDNA amplification was also found from water sampled near the substratum when C. sivickisi medusae were out of season and absent. This suggests the eDNA analysis was likely detecting C. sivickisi polyps located within the substratum. Consequently, eDNA is an effective tool to detect both the medusae and likely polyps of cubozoans. This approach provides the means to reduce the risk of envenomation to swimmers and enhance our knowledge of cubozoan ecology

Sex-specific dmrt1 and cyp19a1 methylation and alternative splicing in gonads of the protandrous hermaphrodite barramundi.

Epigenetics is involved in sex differentiation of gonochoristic and hermaphroditic fish species, whereby two genes dmrt1 (pro-male) and cyp19a1 (pro-female) are known to play major roles. Barramundi, Lates calcarifer, is an important tropical aquaculture species that undergo natural and permanent male to female sex change, a process for which the exact underlying molecular mechanisms are still unknown. To elucidate whether DNA methylation is involved in sex control of barramundi, a next-generation bisulfite amplicon sequencing approach was used to target 146 CpG sites within proximal promoters and first exons of seven sex-related genes (dmrt1, cyp19a1, amh, foxl2, nr5a2, sox8 and sox9) of 24 testis and 18 ovaries of captive and wild adult barramundi. Moreover, comparative expression profiles of the key dmrt1 and cyp19a1 genes were further investigated using RT-qPCR and Sanger sequencing approaches, whereas expression levels of remaining targeted genes were based on available literature for the species. Results showed that cyp19a1 and amh were more methylated in males, whereas dmrt1 and nr5a2 were more methylated in females (P 0.05). Sex-biased promoter DNA methylation was inversely related to gene expression only for dmrt1 and nr5a2, and directly related to amh expression, whereas no differences in cyp19a1 expression were found between testes and ovaries. Notably, unique sex-specific alternative splicing of dmrt1 and cyp19a1 were discovered, whereby males lacked the full-length aromatase coding cyp19a1 mRNA due to partial or total exon splicing, and females lacked the dmrt1 exon containing the DM-domain sequence. This study advances the current knowledge aiming to elucidate the genetic mechanisms within male and female gonads of this large protandrous hermaphrodite by providing the first evidence of epigenetics and alternative splicing simultaneously affecting key genes (cyp19a1 and dmrt1) central to sex differentiation pathways

Genotype by environment interactions of harvest growth traits for barramundi (Lates calcarifer) commercially farmed in marine vs. freshwater conditions

Barramundi (Lates calcarifer), also known as Asian seabass, is a commercially important tropical aquaculture species farmed in diverse culture production systems and salinities (marine to freshwater). Despite adaptability to different culture conditions, selective breeding programs to improve growth rates in barramundi should consider the impact of genotype by environment (GxE) interactions on genetic gains. Barramundi juveniles from 144 families, originating from 24 dams and 54 sires were farmed in a seawater (SW) raceway in Bowen (QLD, Australia) and a freshwater (FW) pond environment in Townsville (QLD, Australia) - both operated under commercial culture conditions. Fish were sampled at 15 months post-hatch (mph) in the SW raceway (mean 1718 ± 309 g weight (W), 454 ± 28 mm total length (Lₜ) and 141 ± 11 mm body depth (BD) (n = 752)) and at 21 mph in the FW pond (mean 1905 ± 426 g W and 451 ± 39 mm Lt and 144 ± 15 mm BD (n = 752)). DNA parentage analyses were used to assign progeny to their respective parents, and the final dataset comprised of 1116 offspring. Moderate-low heritability estimates were found for body traits (W h² = 0.46 ± 0.10; Lt h² = 0.41 ± 0.12; BD h² = 0.49 ± 0.13; body shape H h² = 0.41 ± 0.12; and Fulton's K condition factor h² = 0.15 ± 0.07). Deformities (Def) were observed in 1.8% of fish in SW and 25.1% of fish in FW, although negligible additive genetic effects were evident (Def h² = 0.05 ± 0.04). GxE interactions were found to be moderate for harvest growth traits (W GxE rg = 0.81 ± 0.11; Lt GxE rg = 0.64 ± 0.18; BD GxE rg = 0.78 ± 0.13; H GxE rg = 0.71 ± 0.17), and high for Fulton's K condition factor (K GxE rg = 0.36 ± 0.31; P > 0.05). This study reveals the presence of weak to moderate re-ranking of genotypes for harvest growth traits in L. calcarifer farmed in marine and freshwater conditions, suggesting that GxE interactions should be taken into account in a breeding program servicing multiple environments. Incorporation of sib-information from extreme salinity environments into the selection criteria of a breeding program may therefore optimize the realization of genetic gains across distinct commercial conditions

Environmental DNA detects critically endangered largetooth sawfish in the wild

Environmental DNA (eDNA) is a relatively new tool for the detection of rare, threatened and invasive species in water bodies. In this study we investigated the utility of an eDNA approach in detecting the Critically Endangered largetooth sawfish Pristis pristis in freshwater habitats in northern Australia. Water samples were collected from large aquaria mesocosms containing largetooth sawfish and other aquatic species, and floodplain waterholes and the main river channel of the Daly River, Northern Territory. Water samples were filtered using a 20 mu m nylon filter. DNA was extracted from filters and analysed with PCR using species-specific mitochondrial cytochrome c oxidase subunit I (COI) primers designed to amplify only largetooth sawfish DNA. PCR products were cleaned and the COI gene sequenced to confirm the species identity. Using 3 aquaria, with one containing a largetooth sawfish, this method positively identified sawfish only in the correct aquarium. In the field water samples, 7 of 8 floodplain waterholes produced a sawfish eDNA PCR product, while eDNA was not detected in the main river channel. Based on gillnet sampling and traditional ecological knowledge, largetooth sawfish were known to occur at half of the waterhole and floodplain sites that tested positive for sawfish eDNA. These results demonstrated that an eDNA approach to detecting largetooth sawfish can produce reliable outcomes and can be used as a survey tool to help with conservation efforts for this and other threatened elasmobranchs

Multimethod approach to advance provenance determination of fish in stocked systems

Fish stocking occurs in aquatic systems for conservation purposes, to create or enhance recreational fisheries and to enhance wild-catch commercial fisheries. Identifying and quantifying the contribution of stocking efforts to wild populations is crucial to informing these management objectives. Provenance determination methods trade off accuracy, replicability, and cost-effectiveness at fishery-relevant scales. We present and assess multiple methods for provenance determination using a case study of barramundi (Lates calcarifer) in the Dry Tropics region of northern Australia. A novel application of near-infrared spectroscopy (NIRS) is compared to two established methods for fish provenance, otolith microchemistry and genetic parentage analysis using microsatellites. The otolith microchemistry method was able to provide extremely high provenance resolution (>99% accuracy). The microsatellite parentage analysis method had a slightly lower overall accuracy (95%), likely as a result of genetic introgression in this region. Provenance determination using otolith NIRS had the lowest overall accuracy (76%). Once limitations regarding spectral noise, image resolution, and sample size are addressed, NIRS may have potential for cost-effectively determining provenance in fish