Sedimentary ancient DNA reveals the impact of anthropogenic land use disturbance and ecological shifts on fish community structure in small lowland lake.

Freshwater fish biodiversity and abundance are decreasing globally. The drivers of decline are primarily anthropogenic; however, the causative links between disturbances and fish community change are complex and challenging to investigate. We used a suite of sedimentary DNA methods (droplet digital PCR and metabarcoding) and traditional paleolimnological approaches, including pollen and trace metal analysis, ITRAX X-ray fluorescence and hyperspectral core scanning to explore changes in fish abundance and drivers over 1390 years in a small lake. This period captured a disturbance trajectory from pre-human settlement through subsistence living to intensive agriculture. Generalized additive mixed models explored the relationships between catchment inputs, internal drivers, and fish community structure. Fish community composition distinctly shifted around 1350 CE, with the decline of a sensitive Galaxias species concomitant with early land use changes. Total fish abundance significantly declined around 1950 CE related to increases in ruminant bacterial DNA (a proxy for ruminant abundance) and cadmium flux (a proxy for phosphate fertilizers), implicating land use intensification as a key driver. Concurrent shifts in phytoplankton and zooplankton suggested that fish communities were likely impacted by food web dynamics. This study highlights the potential of sedDNA to elucidate the long-term disturbance impacts on biological communities in lakes.

A paleoecological investigation of recent cyanobacterial blooms and their drivers in two contrasting lakes.

Cyanobacterial blooms are one of the most significant threats to global water security and freshwater biodiversity. Interactions among multiple stressors, including habitat degradation, species invasions, increased nutrient runoff, and climate change, are key drivers. However, assessing the role of anthropogenic activity on the onset of cyanobacterial blooms and exploring response variation amongst lakes of varying size and depth is usually limited by lack of historical records. In the present study we applied molecular, paleolimnological (trace metal, Itrax-µ-XRF and hyperspectral scanning, chronology), paleobotanical (pollen) and historical data to reconstruct cyanobacterial abundance and community composition and anthropogenic impacts in two dune lakes over a period of up to 1200 years. Metabarcoding and droplet digital PCR results showed very low levels of picocyanobacteria present in the lakes prior to about CE 1854 (1839-1870 CE) in the smaller shallow Lake Alice and CE 1970 (1963-1875 CE) in the larger deeper Lake Wiritoa. Hereafter bloom-forming cyanobacteria were detected and increased notably in abundance post CE 1984 (1982-1985 CE) in Lake Alice and CE 1997 (1990-2007 CE) in Lake Wiritoa. Currently, the magnitude of blooms is more pronounced in Lake Wiritoa, potentially attributable to hypoxia-induced release of phosphorus from sediment, introducing an additional source of nutrients. Generalized linear modelling was used to investigate the contribution of nutrients (proxy = bacterial functions), temperature, redox conditions (Mn:Fe), and erosion (Ti:Inc) in driving the abundance of cyanobacteria (ddPCR). In Lake Alice nutrients and erosion had a statistically significant effect, while in Lake Wiritoa nutrients and redox conditions were significant.

An integrative approach to assess the impact of disturbance on native fish in lakes.

Freshwater fish are in a perilous state with more than 30% of species considered critically endangered. Yet significant ecological and methodological complexities constrain our ability to determine how disturbances are impacting native fish communities. We review current methods used to assess the responses of fish communities, especially native fish, to disturbances, with a focus on lakes. These methods include contemporary population surveys, manipulative experimental approaches, paleolimnological approaches and Indigenous Knowledge and social histories. We identify knowledge gaps, such as a lack of baseline data for native fish, an inability to assess the impact of historical disturbances, stressor response dynamics in contemporary multi-stressor environments, and natural disturbance regimes. Our assessment of the current methods highlights challenges to filling these knowledge gaps using the reviewed methods. We advocate strongly for the implementation of an integrative approach that combines emerging technologies (i.e. molecular-based techniques in contemporary surveys and paleolimnology) and underutilised knowledge streams (i.e. Indigenous Knowledge and social histories) which should be used in concert with conventional methods. This integrative approach will allow researchers to determine the key drivers of decline and the degree of change, which will enable more informed and successful management actions.

A taxonomy-free diatom eDNA-based technique for assessing lake trophic level using lake sediments.

Anthropogenic eutrophication is one of the most pressing issues facing lakes globally. Our ability to manage lake eutrophication is hampered by the limited spatial and temporal extents of monitoring records, stemming from the time-consuming and expensive nature of physiochemical and biological monitoring. Diatom-based biomonitoring presents an alternative to traditional eutrophication monitoring, yet it is restricted by the high degree of taxonomic expertise required. Environmental DNA metabarcoding, while providing a promising substitute for diatom community enumeration, is plagued by inadequate taxonomic coverage of reference databases and methodological bias, limiting its use for biomonitoring. Here we show that taxonomy-free diatom-biomonitoring, in which environmental DNA metabarcoding data is utilised but not assigned to specific taxonomic classes, presents an accurate, fast, and relatively automated alternative to taxonomically assigned eutrophication biomonitoring. Our taxonomy-free index accounted for 85% of trophic level variability across 89 lakes and had the lowest average prediction error of the three approaches tested. By not relying on taxonomic identification or metabarcoding reference databases, taxonomy-free biomonitoring maintains diatom diversity that is lost in taxonomic assignment using molecular approaches. Furthermore, by utilising lake sediments, the approach outlined here presents a time-integrated estimation of lake trophic level and thus does not require time-consuming seasonal sampling. Taxonomy-free biomonitoring addresses the limitations of traditional physicochemical eutrophication monitoring and taxonomic biomonitoring alternatives and can be used to extend the spatial and temporal extents of eutrophication monitoring.

Optimal sample type and number vary in small shallow lakes when targeting non-native fish environmental DNA.

Non-native fish have been shown to have deleterious impacts on freshwater ecosystems in New Zealand. Early detection is critical for their effective management. Traditional capture-based techniques may not detect newly introduced fish, especially if they are present in low abundance. Molecular techniques that target environmental DNA (eDNA) have been shown, in many instances, to be more sensitive, cost-effective and require lower sampling effort. However, appropriate sampling strategies are needed to ensure robust and interpretable data are obtained. In this study we used droplet digital PCR assays to investigate the presence of two non-native fish in New Zealand, the European perch (Perca fluviatilis) and rudd (Scardinius erythrophthalmus) in three small lakes. Samples were collected from water and surface sediment at near-shore and mid-lake sites. Probabilistic modelling was used to assess the occupancy of fish eDNA and develop guidance on sampling strategies. Based on the detection probability measures from the present study, at least six sites and five replicates per site are needed to reliably detect fish eDNA in sediment samples, and twelve sites with eight replicates per site for water samples. The results highlight the potential of developing monitoring and surveillance programs adapted to lakes, that include the use of assays targeting eDNA. This study focused on small shallow lakes, and it is likely that these recommendations may vary in larger, deeper, and more geomorphologically complex lakes, and this requires further research.

Human access and deterministic processes play a major role in structuring planktonic and sedimentary bacterial and eukaryotic communities in lakes.

Lakes provide habitat for a diverse array of species and offer a wide range of ecosystem services for humanity. However, they are highly vulnerable as they are not only impacted by adverse actions directly affecting them, but also those on the surrounding environment. Improving knowledge on the processes responsible for community assembly in different biotic components will aid in the protection and restoration of lakes. Studies to date suggested a combination of deterministic (where biotic/abiotic factors act on fitness differences amongst taxa) and stochastic (where dispersal plays a larger factor in community assembly) processes are responsible for structuring biotic communities, but there is no consensus on the relative roles these processes play, and data is lacking for lakes. In the present study, we sampled different biotic components in 34 lakes located on the South Island of New Zealand. To obtain a holistic view of assembly processes in lakes we used metabarcoding to investigate bacteria in the sediment and surface waters, and eukaryotes in the sediment and two different size fractions of the water column. Physicochemical parameters were collected in parallel. Results showed that deterministic processes dominated the assembly of lake communities although the relative importance of variable and homogeneous selection differed among the biotic components. Variable selection was more important in the sediment (SSbact and SSeuks) and for the bacterioplankton (Pbact) while the assembly of the eukaryotic plankton (SPeuks, LPeuks) was driven more by homogeneous selection. The ease of human access to the lakes had a significant effect on lake communities. In particular, clade III of SAR11 and Daphnia pulex were only present in lakes with public access. This study provides insights into the distribution patterns of different biotic components and highlights the value in understanding the drivers of different biological communities within lakes.

A bacterial index to estimate lake trophic level: National scale validation.

Lakes and their catchments have been subjected to centuries to millennia of exploitation by humans. Efficient monitoring methods are required to promote proactive protection and management. Traditional monitoring is time consuming and expensive, which limits the number of lakes monitored. Lake surface sediments provide a temporally integrated representation of environmental conditions and contain high microbial biomass. Based on these attributes, we hypothesized that bacteria associated with lake trophic states could be identified and used to develop an index that would not be confounded by non-nutrient stressor gradients. Metabarcoding (16S rRNA gene) was used to assess bacterial communities present in surface sediments from 259 non-saline lakes in New Zealand encompassing a range of trophic states from alpine microtrophic lakes to lowland hypertrophic lakes. A subset of lakes (n = 96) with monitoring data was used to identify indicator amplicon sequence variants (ASVs) associated with different trophic states. A total of 10,888 indicator taxa were identified and used to develop a Sediment Bacterial Trophic Index (SBTI), which signficantly correlated (r2 = 0.842, P < 0.001) with the Trophic Lake Index. The SBTI was then derived for the remaining 163 lakes, providing new knowledge of the trophic state of these unmonitored lakes. This new, robust DNA-based tool provides a rapid and cost-effective method that will allow a greater number of lakes to be monitored and more effectively managed in New Zealand and globally. The SBTI could also be applied in a paleolimnological context to investigate changes in trophic status over centuries to millennia.

Development of droplet digital Polymerase Chain Reaction assays for the detection of long-finned (Anguilla dieffenbachii) and short-finned (Anguilla australis) eels in environmental samples.

Freshwater eels are ecologically, and culturally important worldwide. The New Zealand long-finned eel (Anguilla dieffenbachii) and short-finned eel (Anguilla australis) are apex predators, playing an important role in ecosystem functioning of rivers and lakes. Recently, there has been a national decline in their populations due to habitat destruction and commercial harvest. The emergence of targeted environmental DNA detection methodologies provides an opportunity to enhance information about their past and present distributions. In this study we successfully developed species-specific droplet digital Polymerase Chain Reaction (ddPCR) assays to detect A. dieffenbachii and A. australis DNA in water and sediment samples. Assays utilized primers and probes designed for regions of the mitochondrial cytochrome b and 16S ribosomal RNA genes in A. dieffenbachii and A. australis, respectively. River water samples (n = 27) were analyzed using metabarcoding of fish taxa and were compared with the ddPCR assays. The presence of A. dieffenbachii and A. australis DNA was detected in a greater number of water samples using ddPCR in comparison to metabarcoding. There was a strong and positive correlation between gene copies (ddPCR analyses) and relative eel sequence reads (metabarcoding analyses) when compared to eel biomass. These ddPCR assays provide a new method for assessing spatial distributions of A. dieffenbachii and A. australis in a range of environments and sample types.

Phospholipid fatty acid (PLFA) analysis as a tool to estimate absolute abundances from compositional 16S rRNA bacterial metabarcoding data.

Microbial biodiversity monitoring through the analysis of DNA extracted from environmental samples is increasingly popular because it is perceived as being rapid, cost-effective, and flexible concerning the sample types studied. DNA can be extracted from diverse media before high-throughput sequencing of the prokaryotic 16S rRNA gene is used to characterize the taxonomic diversity and composition of the sample (known as metabarcoding). While sources of bias in metabarcoding methodologies are widely acknowledged, previous studies have focused mainly on the effects of these biases within a single substrate type, and relatively little is known of how these vary across substrates. We investigated the effect of substrate type (water, microbial mats, lake sediments, stream sediments, soil and a mock microbial community) on the relative performance of DNA metabarcoding in parallel with phospholipid fatty acid (PLFA) analysis. Quantitative estimates of the biomass of different taxonomic groups in samples were made through the analysis of PLFAs, and these were compared to the relative abundances of microbial taxa estimated from metabarcoding. Furthermore, we used the PLFA-based quantitative estimates of the biomass to adjust relative abundances of microbial groups determined by metabarcoding to provide insight into how the biomass of microbial taxa from PLFA analysis can improve understanding of microbial communities from environmental DNA samples. We used two sets of PLFA biomarkers that differed in their number of PLFAs to evaluate how PLFA biomarker selection influences biomass estimates. Metabarcoding and PLFA analysis provided significantly different views of bacterial composition, and these differences varied among substrates. We observed the most notable differences for the Gram-negative bacteria, which were overrepresented by metabarcoding in comparison to PLFA analysis. In contrast, the relative biomass and relative sequence abundances aligned reasonably well for Cyanobacteria across the tested freshwater substrates. Adjusting relative abundances of microbial taxa estimated by metabarcoding with PLFA-based quantification estimates of the microbial biomass led to significant changes in the microbial community compositions in all substrates. We recommend including independent estimates of the biomass of microbial groups to increase comparability among metabarcoding libraries from environmental samples, especially when comparing communities associated with different substrates.

Investigating variability in microbial community composition in replicate environmental DNA samples down lake sediment cores.

Lake sediments are natural archives that accumulate information on biological communities and their surrounding catchments. Paleolimnology has traditionally focussed on identifying fossilized organisms to reconstruct past environments. In the last decade, the application of molecular methodologies has increased in paleolimnological studies, but further research investigating factors such as sample heterogeneity and DNA degradation are required. In the present study we investigated bacterial community heterogeneity (16S rRNA metabarcoding) within depth slices (1-cm width). Sediment cores were collected from three lakes with differing sediment compositions. Samples were collected from a variety of depths which represent a period of time of approximately 1,200 years. Triplicate samples were collected from each depth slice and bacterial 16S rRNA metabarcoding was undertaken on each sample. Accumulation curves demonstrated that except for the deepest (oldest) slices, the combination of three replicate samples were insufficient to characterise the entire bacterial diversity. However, shared Amplicon Sequence Variants (ASVs) accounted for the majority of the reads in each depth slice (max. shared proportional read abundance 96%, 86%, 65% in the three lakes). Replicates within a depth slice generally clustered together in the Non-metric multidimensional scaling analysis. There was high community dissimilarity in older sediment in one of the cores, which was likely due to the laminae in the sediment core not being horizontal. Given that most paleolimnology studies explore broad scale shifts in community structure rather than seeking to identify rare species, this study demonstrates that a single sample is adequate to characterise shifts in dominant bacterial ASVs.

The Role of Environmental Processes and Geographic Distance in Regulating Local and Regionally Abundant and Rare Bacterioplankton in Lakes.

Bacteria are vital components of lake systems, driving a variety of biogeochemical cycles and ecosystem services. Bacterial communities have been shown to have a skewed distribution with a few abundant species and a large number of rare species. The contribution of environmental processes or geographic distance in structuring these components is uncertain. The discrete nature of lakes provides an ideal test case to investigate microbial biogeographical patterns. In the present study, we used 16S rRNA gene metabarcoding to examine the distribution patterns on local and regional scales of abundant and rare planktonic bacteria across 167 New Zealand lakes covering broad environmental gradients. Only a few amplicon sequence variants (ASVs) were abundant with a higher proportion of rare ASVs. The proportion of locally abundant ASVs was negatively correlated with the percentage of high productivity grassland in the catchment and positively with altitude. Regionally rare ASVs had a restricted distribution and were only found in one or a few lakes. In general, regionally abundant ASVs had higher occupancy rates, although there were some with restricted occupancy. Environmental processes made a higher contribution to structuring the regionally abundant community, while geographic distances were more important for regionally rare ASVs. A better understanding of the processes structuring the abundance and distribution of bacterial communities within lakes will assist in understand microbial biogeography and in predicting how these communities might shift with environmental change.

Comparing sediment DNA extraction methods for assessing organic enrichment associated with marine aquaculture.

Marine sediments contain a high diversity of micro- and macro-organisms which are important in the functioning of biogeochemical cycles. Traditionally, anthropogenic perturbation has been investigated by identifying macro-organism responses along gradients. Environmental DNA (eDNA) analyses have recently been advocated as a rapid and cost-effective approach to measuring ecological impacts and efforts are underway to incorporate eDNA tools into monitoring. Before these methods can replace or complement existing methods, robustness and repeatability of each analytical step has to be demonstrated. One area that requires further investigation is the selection of sediment DNA extraction method. Environmental DNA sediment samples were obtained along a disturbance gradient adjacent to a Chinook (Oncorhynchus tshawytscha) salmon farm in Otanerau Bay, New Zealand. DNA was extracted using four extraction kits (Qiagen DNeasy PowerSoil, Qiagen DNeasy PowerSoil Pro, Qiagen RNeasy PowerSoil Total RNA/DNA extraction/elution and Favorgen FavorPrep Soil DNA Isolation Midi Kit) and three sediment volumes (0.25, 2, and 5 g). Prokaryotic and eukaryotic communities were amplified using primers targeting the 16S and 18S ribosomal RNA genes, respectively, and were sequenced on an Illumina MiSeq. Diversity and community composition estimates were obtained from each extraction kit, as well as their relative performance in established metabarcoding biotic indices. Differences were observed in the quality and quantity of the extracted DNA amongst kits with the two Qiagen DNeasy PowerSoil kits performing best. Significant differences were observed in both prokaryotes and eukaryotes (p < 0.001) richness among kits. A small proportion of amplicon sequence variants (ASVs) were shared amongst the kits (~3%) although these shared ASVs accounted for the majority of sequence reads (prokaryotes: 59.9%, eukaryotes: 67.2%). Differences were observed in the richness and relative abundance of taxonomic classes revealed with each kit. Multivariate analysis showed that there was a significant interaction between "distance" from the farm and "kit" in explaining the composition of the communities, with the distance from the farm being a stronger determinant of community composition. Comparison of the kits against the bacterial and eukaryotic metabarcoding biotic index suggested that all kits showed similar patterns along the environmental gradient. Overall, we advocate for the use of Qiagen DNeasy PowerSoil kits for use when characterizing prokaryotic and eukaryotic eDNA from marine farm sediments. We base this conclusion on the higher DNA quality values and richness achieved with these kits compared to the other kits/amounts investigated in this study. The additional advantage of the PowerSoil Kits is that DNA extractions can be performed using an extractor robot, offering additional standardization and reproducibility of results.

Predicting cyanobacterial biovolumes from phycocyanin fluorescence using a handheld fluorometer in the field.

Toxic cyanobacterial blooms are becoming more prevalent in freshwater systems, increasing the need for monitoring to protect human health. Phycocyanin fluorescence sensors have been developed as tools for providing fast and cost-effective proxy measurements for cyanobacterial biomass. However, poor precision and low sensitivity in many of the probe sensors assessed to-date has restricted their potential for practical application in cyanobacterial monitoring programmes. In the present study, the sensitivity and accuracy of a handheld fluorometer, the CyanoFluor, was assessed using 12 cyanobacterial strains and samples from four different lakes collected weekly for 12 weeks. After the initial measurements, the samples were lysed by sonication, which we hypothesised would reduce inter and intra-specific differences. The CyanoFluor displayed high sensitivity (limit of quantification = 3.5 µg L-1 of phycocyanin) and was able to detect cyanobacterial biovolumes to levels much lower than the threshold levels in current recreational guidelines worldwide. There were strong and significant phycocyanin to biovolume relationships (r2 ≥ 0.88, P < 0.05) for all 12 cyanobacterial cultures. Collectively, strong relationships between phycocyanin fluorescence and cyanobacterial biovolumes were also identified in environmental samples (r2 ≥ 0.78, P < 0.001), although weaker relationships were identified when lakes were analysed separately (r2 = 0.06 - 0.90). There were differences in phycocyanin per biovolume between both cultured strains and lakes, highlighting innate interspecific differences that exist between cyanobacterial species. Lysis of samples consistently reduced variability between technical replicates, in cyanobacteria cultures (up to 87% reduction in sample variability) and environmental samples (71 - 93% reduction), indicating that it would be a useful methodological step to improve the repeatability of results. When guideline thresholds (aligned with currently enforced risk assessment categories) were modelled based on the most successful linear regression model, 74% of samples were assigned to the correct risk category. The sensitivity of the CyanoFluor and accuracy of the phycocyanin threshold models, indicates high potential for this method to be integrated into cyanobacterial monitoring programmes.

Local factors drive bacterial and microeukaryotic community composition in lake surface sediment collected across an altitudinal gradient.

Lake surface sediments are dominated by microorganisms that play significant roles in biogeochemical cycling within lakes. There is limited knowledge on the relative importance of local environmental factors and altitude on bacterial and microeukaryotic community richness and composition in lake sediments. In the present study, surface sediment samples were collected from 40 lakes along an altitude gradient (2-1215 m). Microbial communities were characterized using 16S (bacteria) and 18S (microeukaryotes) rRNA gene metabarcoding. Bacterial and microeukaryotic richness were not correlated with altitude but instead to environmental variables (e.g. area of water in the catchment (bacteria: R = -0.43). For both bacteria and microeukaryotes, dissimilarity in the community structure had a higher correlation to combined environmental variables (without altitude) (bacteria: R = 0.53; microeukaryotes: R = 0.55) than altitude alone (bacteria: R = 0.34; microeukaryotes: R = 0.47). Sediment sulfur and productive grassland were important variables in determining the relative abundance of sulfate reducing bacteria. Nitrospira, was positively related to altitude but negatively to water column total organic carbon and the proportion of productive grassland in the catchment. Little overlap in amplicon sequence variants was shown amongst lakes. This has important considerations for management decisions, suggesting that to protect biodiversity, conservation of numerous lakes and lake types is required.

Broad and Fine Scale Variability in Bacterial Diversity and Cyanotoxin Quotas in Benthic Cyanobacterial Mats.

Benthic proliferations of Microcoleus autumnalis (basionym Phormidium autumnale) and closely related taxa are being reported with increasing frequency in streams and rivers worldwide. This species commonly produces the potent neurotoxin anatoxin, and exposure to this has resulted in animal fatalities and human health concerns. Bacterial communities within cyanobacterial assemblages can facilitate processes such as nutrient cycling and are posited to influence cyanobacterial growth and function. However, there is limited knowledge on spatial variability of bacterial communities associated with benthic cyanobacteria and anatoxin content and quotas. In this study, M. autumnalis-dominated mat samples were collected from six sites in two New Zealand streams. Associated bacterial communities were characterized using 16S rRNA metabarcoding, anatoxin content by liquid chromatography-mass spectrometry and anaC copies using droplet digital PCR. Bacterial assemblages differed significantly when amplicon sequence variants were compared between streams and most sites within streams. These differences were associated with conductivity, DRP, DIN, temperature, anatoxin concentration, and quota. Despite the differences in bacterial community composition; at phyla, class and order levels there was high similarity across spatial scales, with Bacteroidetes (ca. 67%) and Proteobacteria (ca. 25%) dominant. There was significant variability in total anatoxin concentrations between sites in both streams (p < 0.001). When the data were converted to anatoxin quotas variability was reduced, suggesting that the relative abundance of toxic genotypes is a key driver of total anatoxin concentrations in mats. This study demonstrates the complexity of microbial communities within M. autumnalis-dominated mats and highlights their likely important role in within-mat nutrient cycling processes.

Effects of estradiol and 11-ketotestosterone pre-treatment on artificial induction of maturation in silver female shortfinned eels (Anguilla australis).

Our previous work documented significant advancements in steroid-induced progression of oogenesis, demonstrating that co-treatment of female eels with 11-ketotestosterone (11KT) and estradiol-17ß (E2) successfully induced uptake of vitellogenin by oocytes. Here we evaluate the effects of this steroid co-treatment on subsequent time to ovulation and egg quality in shortfinned eels artificially matured by hypophysation. Co-treatment with 11KT (1 mg) and E2 (0.2 or 2 mg) significantly reduced time to ovulation and therefore, the amount of pituitary homogenate required, without any detrimental effects on gonadosomatic index, oocyte diameter or the total weight of stripped eggs. E2 treatment resulted in promising increases in fertilization rates. These indicators suggest that co-treatment with 11KT and E2 holds promise for future artificial maturation practices in terms of minimising fish handling and stress, and of reducing the need for expensive pituitary preparations.

Synergistic effects of estradiol and 11-ketotestosterone on vitellogenin physiology in the shortfinned eel (Anguilla australis).

Estradiol-17ß (E2) and 11-ketotestosterone (11KT) have been implicated in vitellogenesis and in regulating expression of the follicle-stimulating hormone receptor (fshr), respectively. To override the captivity-induced reproductive block in shortfinned eel, Anguilla australis, we hypothesized that in combination, 11KT and E2 would stimulate ovarian uptake of vitellogenin (Vtg). Early pubertal eels received hormone implants containing varying concentrations of E2 (0, 0.2, 2, 5 mg) with or without 11KT (1 mg). Vtg levels were determined in plasma, liver, and ovarian tissues by histological examination, qPCR, immunoblotting, or single radial immunodiffusion. The expression of gonadotropin-beta subunits and gonadotropin receptors in the pituitary and ovary, respectively, were analyzed to determine mechanisms by which steroid effects may be exerted. When administered alone, E2 increased hepatic production and plasma levels of Vtg. In contrast, 11KT decreased plasma levels of Vtg, seemingly reducing its production. Neither 11KT nor E2 could induce uptake of Vtg into oocytes, although E2 treatment appeared necessary for uptake to occur. This was the case despite 11KT dramatically increasing both oocyte size and fshr mRNA levels. Astonishingly, the uptake of Vtg was successfully induced by co-treatment with 11KT and E2, suggesting that 11KT might facilitate the incorporation of Vtg into the developing oocyte. These results highlight the potential of sex steroid co-treatment, an approach aimed at mimicking oogenesis in wild eels, to induce vitellogenesis, specifically ovarian yolk deposition, even in the absence of exogenous gonadotropin treatment.

The effects of migratory stage and 11-ketotestosterone on the expression of rod opsin genes in the shortfinned eel (Anguilla australis).

The androgen 11-ketotestosterone (11KT) can induce many of the changes associated with silvering, i.e., the transformation of a non-migrating 'yellow' eel into a migrating 'silver' eel. We posited that plasticity in spectral sensitivity of the eye, accompanied by expression of different opsins in the retina during silvering, is controlled by 11KT. To test this hypothesis, mRNA levels of freshwater (fwo) and seawater (swo) opsins and of the two androgen receptors (ara and arb) in retinas of wild-caught female shortfinned eels, Anguilla australis were compared. Swo expression was much higher (3-4 orders of magnitude) and fwo expression substantially lower in silver than in yellow eels, whereas mRNA levels of both ars did not differ between stages. Yellow eel retinas exposed to 11KT in vitro exhibited a robust dose-dependent increase in swo, but weak decreasing effects on fwo transcript abundance were inconsistent. Similarly, increased retinal swo expression was seen after in vivo treatment of yellow eels with 11KT implants, whereas expression of fwo remained unaffected. Lastly, co-treatment with 11KT and the androgen receptor blocker flutamide was undertaken to determine whether 11KT exerts its effects through nuclear androgen receptors. Flutamide did not block 11KT-affected expression of any target gene, neither in vivo nor in vitro. We conclude that 11KT greatly increases the abundance of swo, identifying the androgen as an important regulator of the opsin switch during silvering in freshwater eels.