Limnology and Oceanography: Methods最新文献

We present a newly developed design for a self-contained benthic chamber for conducting in situ ecosystem experiments in streams, with a focus on biogeochemical processes such as ecosystem metabolism and nutrient cycling. Our design expands upon smaller, portable chamber designs and is meant to answer questions at larger scales. These new chambers allow for a high level of experimental control in the field and can be used to generate spatially explicit data regarding ecosystem processes and to test mechanistic hypotheses. They are built to be deployed within the stream over periods of weeks to months and to withstand natural hydraulic forces of the benthic zone. First, we describe the materials and steps that are needed to construct these chambers in detail. Then, we report the methods and results of a multi-part, diagnostic field study meant to demonstrate the performance and utility of the design. We quantified solute dynamics using a conservative tracer injection, then we estimated ecosystem metabolism across the study site and performed nutrient additions. We detected asymptotic declines in tracer concentrations, calculated nutrient removal rates, and mapped hotspots of ecosystem metabolism. Flow velocity and water depth imposed limitations, but with appropriate methodological forethought these limitations can be minimized. The capacity of our design to accommodate complex, three-dimensional habitats and macrofauna, along with the capability to generate spatially explicit data, are the main advances we present. These advances provide a novel method whereby motivated users can connect mechanistic hypothesis testing with natural ecological processes through ecosystem-level field experiments.

Volatile fatty acids (VFAs) are key intermediates in carbon transformation in marine environments and feature widely in models for a hydrothermal origin of life. Quantifying VFAs in hydrothermal fluids is challenging due to their trace concentrations and the high inorganic ion loads of these matrices. Previous methods often rely on manual sample pre-treatment or complex instrumentation (e.g., mass spectrometry), increasing contamination risks, consumable use, and costs. To circumvent such challenges, we developed a simplified quantification method for trace VFAs in seawater-like matrices using a modern high-pressure ion chromatography (HPIC) system. This approach utilizes single-dimension ion exchange chromatography with conductivity detection alone, and a choice of two analytical column options to separate formate, acetate, propionate, butyrate, valerate, pyruvate, and lactate (measured as ∑anion) from inorganic anions. Modern HPIC systems, in addition to being versatile for other analytes (e.g., cations, nutrients), enable higher peak resolution and increased ion exchange capacity, and our tests show this allows for significantly greater trace VFA sensitivity than previous techniques. With careful sample handling and contamination control, our method achieves better absolute limits of detection for smaller sample requirements (≤ 0.3 mL), statistically determined to be below 10 ppb (~ 0.05 μmol/kg for ∑formate, ~ 0.03 μmol/kg for ∑acetate). Additionally, our study provides detailed insights into limiting VFA contamination sources, as well as their stability in storage. Initial analysis of hydrothermal fluids from the Arctic Mid-Ocean Ridges reveals formate (0.6–7.3 μmol/kg) controlled by metastable CO₂–H₂–HCOOH equilibrium, and unexpectedly low acetate (2.6–5.8 μmol/kg), likely reflecting competition between thermogenic formation and stability.

Ciliates are widespread and play a major role in ecosystems as they form an important link between primary producers and higher trophic levels. They have been used as a classic model to study predator–prey interactions of co-evolutionary processes. In our experimental system, predators and prey interact dynamically, with prey exhibiting predator-induced defenses and predators potentially adapting through offensive strategies, both of which influence population dynamics. When analyzing population dynamics in similar ciliate species, individuals must be accurately identified and counted. Morphologically, the genus Euplotes is generally identified by the arrangement of cilia and cirri and the position of the macronucleus and micronucleus. This requires expensive and laborious cell counters and time-consuming staining methods. Furthermore, staining methods are not ideal for determining cell numbers, as individual cells may be lost during staining processes. As ciliates are unicellular organisms, we used DNA quantity to determine the number of individuals. We identified unique sequences of three Euplotes species: Euplotes octocarinatus, Euplotes daidaleos, and Euplotes aediculatus using random amplified polymorphic DNA (RAPD) fingerprinting. Using these sequences, we designed species-specific primers for quantitative polymerase chain reaction and generated corresponding standard curves based on microscopic cell counts. Using this method, we are now able to determine cell counts in unknown samples of different Euplotes species within a single experimental system and monitor population growth rates of one or even several species simultaneously. Additionally, using RAPD fingerprinting enables the identification of unique genetic sequences, allowing differentiation between clones of the same species and facilitating measurement of their population growth rates in mixed experiments.

Imaging methods have developed rapidly in recent decades, opening new opportunities for taxonomy and biodiversity studies of marine organisms. In particular, the microscopic size range, which used to be challenging to study due to time-consuming preparation and observation steps, now benefits from high-throughput quantitative imaging methods and the development of fast high-resolution microscopy approaches. Meiofauna, interstitial sediment animals ranging from 20 μm to 1 mm, are important components of ecosystems. These organisms can serve as bioindicators, and the group as a whole holds immense potential for the discovery of new species. However, protocols for studying meiobenthos are highly time-consuming, which helps explain why this group is understudied. We tested five imaging techniques, ranging from low to high resolution, that could accelerate hard-bodied meiofauna studies, both for ecology and species description, and address the gap in our understanding of this part of marine life. Thus, two flow imaging modalities (in line holographic microscopy and classic optic microscopy), a semi-automated microscopy acquisition procedure, and two three-dimensional (3D) fluorescence microscopy protocols were used. We examined the classic compromises of imaging, including resolution, throughput, and data volume, to evaluate the potential benefits of using such techniques for meiofaunal studies. For ecological surveys, flow imaging could benefit meiobenthos studies, but resolution remains a limiting factor. For taxonomic description, 3D fluorescent imaging added relevant information, considering the time required for preparation and acquisition. The semi-automated motorized microscopy procedure could be used for both purposes according to the versatility of the system.

Retrospective isotope analysis using metabolically inert tissues is a powerful tool for reconstructing historical environmental conditions experienced by animals. Although this technique has been successfully applied to fish and squids using eye lenses, its applicability to metamorphosing organisms, such as amphibians, across multiple life-history stages has not been thoroughly assessed. In this study, we examined whether stable isotope ratios in frog eye lenses reflect dietary data from both the larval and post-metamorphic stages through feeding trials of two species, Rana ornativentris and Babina subaspera, using diets with distinct δ¹³C values. Additionally, we analyzed samples collected from the wild and assessed the potential future use of this method. Our feeding trials demonstrated that δ¹³C values in the central and outer lens sections were closely aligned with the larval and adult food, respectively, confirming the applicability of the method. The transition point of stable isotope ratios was closely aligned with the actual metamorphosis point, indicating that this method can effectively identify metamorphosis. However, the two wild specimens exhibited no discernible patterns in isotope ratios across their life stages. This indicates that applying this method in the field requires careful selection of the environmental conditions and a comprehensive understanding of the stable isotope ratios of potential food sources.

Aquatic biodiversity assessments are often labor-intensive due to the large size of the equipment and the complex logistics of sea vessel operations. Traditional drift and drop cameras are typically tethered to the surface, causing cable and line clutter on sea vessels. At the same time, landers rely on auto-release mechanisms that use costly acoustic signals or inaccurate galvanic reactions. We introduce a reusable, novel, and low-cost Multipurpose Auto-Release System, a versatile and programmable solution for diverse payloads and applications in shallow and mesophotic waters. Building on existing drop-cam and Baited Remote Underwater Video System techniques, we enhance them with natural ballasts and an electronically controlled timed-release mechanism, which is programmed via a smartphone app using Near Field Communication. Our technique allows tetherless retrieval from small sea vessels at the sea surface. This innovation simplifies aquatic monitoring logistics by eliminating the need for surface buoys or equipment retrieval from the seabed during each deployment. Our approach also advances benthic and deep-sea marine biodiversity assessments by enabling easy systems deployment and recapture without pingers. We validated the system through 10 seawater tests, reaching depths of 278 m, accumulating 6 h of submerged data collection, and 17 d during continuous water immersion. We provide a detailed guide for building this robust, reusable, user-friendly tool for diverse aquatic monitoring assessments. Additionally, we share key lessons learned, paving the way toward more democratized, customizable, and widely accessible applications capable of reaching the deepest seas.

Measuring dissolved nitrous oxide (N₂O), a potent greenhouse gas and contributor to ozone depletion, is essential for understanding its aquatic dynamics and informing climate mitigation and emission estimates. Dissolved N₂O concentration measurements typically involve headspace equilibration of water samples in sealed containers, followed by gas chromatography analysis. This manual method is labor-intensive and often requires toxic preservatives. Alternatively, air-water exchangers coupled with laser analyzers provide high-precision continuous measurements but lack sample storage capabilities and require frequent relocation and setup to capture spatiotemporal variations. We developed an automated gas bag (AGB) collection system for collecting N₂O samples (AGB-N₂O) from discrete water samples, which could then be analyzed for concentration using laser analyzers. This method combines the field-friendly sample collection and storage of the manual method with the precision of exchangers and laser analyzers. Field experiments tested four setups of exchangers with varying internal volumes (2 L vs. 1 L) and water flow rates (small nozzle: 0.75 L min⁻¹ vs. medium nozzle: 3 L min⁻¹) at sites with low vs. high N₂O concentrations (13 nM vs. 95 nM). The 2-L exchanger with a medium nozzle achieved the fastest equilibration times of 2.25 and 0.08 min for high and low N₂O sites, respectively. The AGB-N₂O showed comparable results to the manual method for measuring dissolved N₂O concentrations (p > 0.05). However, the AGB-N₂O demonstrated significantly lower standard deviations, indicating higher precision and consistency. These findings demonstrate the suitability of the AGB-N₂O for diverse aquatic environments, offering reliable and efficient N₂O measurements.

This study evaluates the impact of sampling protocols on the measurement of particulate inorganic carbon (PIC) in ocean waters, an essential component for understanding the global carbon cycle and climate regulation. The study compares four protocols for estimating PIC in discrete water column samples, focusing on the effects of filter pore size (0.4 vs. 0.8 μm) and rinsing agents (pH-adjusted MilliQ water with NH₄OH vs. potassium tetraborate buffer). Five coccolithophore strains were selected to represent variations in PIC content resulting from species-specific differences in coccolith mass, coccolith number per cell, and life cycle phase. Discrete samples were analyzed using inductively coupled plasma mass spectrometry. Statistical analyses show no significant differences in PIC concentrations between protocols, filter types, or rinsing agents, confirming the robustness and precision of the measurement method. In addition, the non-calcifying strain provided insights into the measurement uncertainty and enabled us to quantify the precision of the sampling method. These results suggest that researchers can use any tested protocol without compromising data quality. This will improve the reliability and comparability of PIC measurements and contribute to a more precise understanding of ocean carbon dynamics and climate regulation.

Water clarity regulates irradiance penetration in aquatic environments, influencing physical and biological dynamics: irradiance penetration affects heat transfer in the water column and provides energy through photosynthetically active radiation (PAR) in the euphotic zone, which is vital for light-dependent organisms. The ability to accurately assess water clarity is therefore important in several aquatic science contexts, from data analysis and process interpretation to modeling. Common metrics used to quantify water clarity include the vertical irradiance attenuation coefficient $��K�$, a measure of irradiance penetration, and the Secchi disk depth ($��z_{\mathrm{SD}}�$), a measure of water visibility. The enduring simplicity and low cost of the Secchi disk has made it a global standard for measuring water clarity for almost two centuries. In contrast, $��K�$ is typically determined using expensive instruments that measure underwater irradiance profiles. This highlights the need for innovative, cost-effective methods that integrate both types of measurements. Here we present DISCO, a low-cost, easy-to-build instrument that retains the traditional appearance of a Secchi disk, and is equipped with photoresistors (also known as light-dependent resistors, LDRs) both looking upwards and downwards for planar irradiance measurements. DISCO is also equipped with low-cost temperature and pressure sensors, all connected to an ArduinoUNO board. DISCO was tested in two mountain lakes together with high resolution PAR, temperature and pressure sensors to calibrate the LDRs and validate its performance. The results show that the proposed instrument is able to measure the irradiance attenuation coefficients with an error of less than 10$��\%�$ compared to the reference PAR sensor.

Experimental streams (ESs) are enclosures of fluvial ecosystems designed as flume microcosms to simulate flowing waters and assess environmental disturbances on aquatic communities' structure and function under controlled conditions. This study presents an ES system that combines ecological relevance with reliability in a compact design, offering rapid responses to iron ore tailings. The system used periphyton biofilm as a bioindicator in a recirculating flume microcosm over 65 d. Periphyton is a key bioindicator, representing critical aquatic metabolic processes such as primary production. The system includes artificial glass channels, microscope slide substrates, and recirculating pumps. It was developed with replicates, three controls, and three treatments, each consisting of a main channel and a water reservoir, for a total volume of 10.0 L. Six sampling campaigns evaluated periphyton biofilm and water quality indicators under acute, short, and long-term exposures. The system's reliability is ensured by standardizing setup conditions, maintaining river water properties, ensuring uniform flow, and standardizing slide sampling. Realism is achieved by renewing large water volumes, regular inoculation of periphyton propagules, and the appropriate sampling frequency for periphyton and water quality. The results showed that the ES, a simple and low-cost system (US$569), was sensitive to indicators throughout the exposure period. This approach is practical for assessing various aquatic stressors.