N-Amino-l-Proline Methyl Ester from an Australian Fish Gut-Derived Fungus: Challenging the Distinction between Natural Product and Artifact

Further investigation into a fish gut-derived fungus Evlachovaea sp. CMB-F563, previously reported to produce the unprecedented Schiff base prolinimines A–B (1–2), revealed a new cryptic natural product, N-amino-l-proline methyl ester (5)—only the second reported natural occurrence of an N-amino-proline, and the first from a microbial source. To enable these investigations, we developed a highly sensitive analytical derivitization methodology, using 2,4-dinitrobenzaldehyde (2,4-DNB) to cause a rapid in situ transformation of 5 to the Schiff base 9, with the latter more readily detectable by UHPLC-DAD (400 nm) and HPLC-MS analyses. Moreover, we demonstrate that during cultivation 5 is retained in fungal mycelia, and it is only when solvent extraction disrupts mycelia that 5 is released to come in contact with the furans 7–8 (which are themselves produced by thermal transformation of carbohydrates during media autoclaving prior to fungal inoculation). Significantly, on contact, 5 undergoes a spontaneous condensation with 7–8 to yield the Schiff base prolinimines 1–2, respectively. Observations made during this study prompted us to reflect on what it is to be a natural product (i.e., 5), versus an artifact (i.e., 1–2), versus a media component (i.e., 7–8).

Behavioural plasticity in a native species may be related to foraging resilience in the presence of an aggressive invader

Competition between invasive and native species can result in the exploitation of resources by the invader, reducing foraging rates of natives. However, it is increasingly recognized that multiple factors can enhance the resilience of native species competing for limiting resources with invaders. Although extensively studied in terrestrial species, little research has focused on behavioural plasticity in aquatic ecosystems and how this influences native species resilience. Here, we examined the role of behavioural plasticity in interactions between a native Australian fish, Pseudomugil signifer, and a widespread invasive fish, Gambusia holbrooki . To determine whether P. signifer displays behavioural plasticity that may mitigate competition with G. holbrooki , we first quantified social behaviours (aggression, submission and affiliation) and shoal cohesion for each species in single- and mixed-species groups. Second, we compared the feeding rates of both species in these groups to ascertain if any modulation of social behaviours and cohesion related to foraging success. We found that aggressive and submissive behaviours of G. holbrooki and P. signifer showed plasticity in the presence of heterospecifics, but social affiliation, shoaling and, most importantly, foraging, remained inflexible. This variation in the degree of plasticity highlights the complexity of the behavioural response of a native species and suggests that both behavioural modulation and consistency may be related to sustaining foraging efficiency in the presence of an invader.

Cryopreservation of testicular tissue from Murray River Rainbowfish, Melanotaenia fluviatilis

Globally, fish populations are in decline from overfishing, habitat destruction and poor water quality. Recent mass fish deaths in Australia’s Murray–Darling Basin highlight the need for improved conservation methods for endangered fish species. Cryopreservation of testicular tissue allows storage of early sperm precursor cells for use in generating new individuals via surrogacy. We describe successful isolation and cryopreservation of spermatogonia in an Australian rainbowfish. Testis histology showed rainbowfish spermatogonia are large (> 10 μm) and stain positive for Vasa, an early germ line-specific protein. Using size-based flow cytometry, testis cell suspensions were sorted through “A” (> 9 μm) and “B” gates (2–5 μm); the A gate produced significantly more Vasa-positive cells (45.0% ± 15.2%) than the “B” gate (0.0% ± 0.0%) and an unsorted control (22.9% ± 9.5%, p < 0.0001). The most successful cryoprotectant for “large cell” (> 9 μm) viability (72.6% ± 10.5%) comprised 1.3 M DMSO, 0.1 M trehalose and 1.5% BSA; cell viability was similar to fresh controls (78.8% ± 10.5%) and significantly better than other cryoprotectants (p < 0.0006). We have developed a protocol to cryopreserve rainbowfish testicular tissue and recover an enriched population of viable spermatogonia. This is the first step in developing a biobank of reproductive tissues for this family, and other Australian fish species, in the Australian Frozen Zoo.

Low light levels increase avoidance behaviour of diurnal fish species: Implications for road culverts

Inadequately designed culverts are known to pose hydraulic barriers to fish passage, but they may also be behavioural barriers if they adversely affect light levels within them. To test this, we performed a choice experiment and quantified the amount of time individuals of four Australian fish species spent in darkened and illuminated areas of an experimental swimming fume. Behavioural responses were reflective of the species’ diel activity patterns; diurnal species preferred illuminated regions, while nocturnal species preferred the darkened region. We then determined a threshold light level of only ~100-200 lux (c.f. midday sunlight ~100,000 lux) was required to overcome the behavioural barrier in ~ 70% of the diurnal fish tested. Placing these threshold values into field context, 100% of culverts sampled recorded inadequate light levels. Attention is required to better understand the impacts of low light levels in culverts on fish passage and to prioritise restoration.

Swimming performance traits of twenty-one Australian fish species: a fish passage management tool for use in modified freshwater systems

Freshwater ecosystems have been severely fragmented by artificial in-stream structures designed to manage water for human use. Significant efforts have been made to reconnect freshwater systems for fish movement, through the design and installation of dedicated fish passage structures (fishways) and by incorporating fish-sensitive design features into conventional infrastructure (e.g. culverts). Key to the success of these structures is making sure that the water velocities within them do not exceed the swimming capacities of the local fish species. Swimming performance data is scarce for Australian fish, which have a reduced swimming capacity when compared to many North American and European species. To help close this knowledge gap and assist fisheries management and civil engineering, we report the swimming performance capacities of twenty-one small-bodied fish and juveniles (< 10 cm) of large bodied species native to Australia as measured by critical swimming speed (Ucrit) and burst swimming speed (Usprint) in a recirculating flume. This data is complemented by endurance swim trials in a 12-meter hydraulic flume channel, and by measures of flume traverse success. Building on the utility of this dataset, we used a panel of morphological, behavioural and ecological traits to first assess their relative contributions to the observed swimming performance data, and second, to determine if they could be used to predict swimming performance capacity – a useful tool to assist in the management of species of conservation concern where access to swimming performance data may be limited. We found that body length combined with depth station (benthic, pelagic or surface) explained most of the interspecific variation in observed swimming performance data, followed by body shape and tail shape. These three traits were the most effective at predicting swimming performance in a model/unknown fish. This data will assist civil engineers and fisheries managers in Australia to mitigate the impact of in-stream structures on local fish populations.

Complete Genome Sequences of Betanodavirus from Australian Barramundi (Lates calcarifer)

The complete RNA-1 and RNA-2 genome sequences of Betanodavirus were obtained from Australian barramundi (Lates calcarifer). Phylogenetic analyses revealed that the sequences have closest homology to the red spotted grouper nervous necrosis virus (RGNNV) species and share between 91 and 98% homology with the other two published complete/near-complete sequences of isolates from Australian fish.