Improved biodiversity detection using a large-volume environmental DNA sampler with in situ filtration and implications for marine eDNA sampling strategies

Metabarcoding analysis of environmental DNA samples is a promising new tool for marine biodiversity and conservation. Typically, seawater samples are obtained using Niskin bottles and filtered to collect eDNA. However, standard sample volumes are small relative to the scale of the environment, conventional collection strategies are limited, and the filtration process is time consuming. To overcome these limitations, we developed a new large-volume eDNA sampler with in situ filtration, capable of taking up to 12 samples per deployment. We conducted three deployments of our sampler on the robotic vehicle Mesobot in the Flower Garden Banks National Marine Sanctuary in the northwestern Gulf of Mexico and collected samples from 20 to 400 m depth. We compared the large volume (~40-60 liters) samples collected by Mesobot with small volume (~2 liters) samples collected using the conventional CTD-mounted Niskin bottle approach. We sequenced the V9 region of 18S rRNA, which detects a broad range of invertebrate taxa, and found that while both methods detected biodiversity changes associated with depth, our large volume samples detected approximately 66% more taxa than the CTD small volume samples. We found that the fraction of the eDNA signal originating from metazoans relative to the total eDNA signal decreased with sampling depth, indicating that larger volume samples may be especially important for detecting metazoans in mesopelagic and deep ocean environments. We also noted substantial variability in biological replicates from both the large volume Mesobot and small volume CTD sample sets. Both of the sample sets also identified taxa that the other did not; although the number of unique taxa associated with the Mesobot samples was almost four times larger than those from the CTD samples. Large volume eDNA sampling with in situ filtration, particularly when coupled with robotic platforms, has great potential for marine biodiversity surveys, and we discuss practical methodological and sampling considerations for future applications.

Bacterial and plankton communities in mariculture water sources: a case study in Nampu and Sembukan seawaters, Wonogiri, Indonesia

Seawaters in Indonesia, part of the tropical marine ecosystem, have great microbial and plankton diversity. Seawater is used as a water source for marine aquacultures, such as shrimp, milkfish, lobster, and mud crab. Sustainability of environmental resources for supporting aquaculture activities can be assessed by analyzing the actual conditions of the water source environment, including bacterial and plankton communities. However, the characteristics of bacteria and plankton communities in Indonesian seawaters have not been well documented. In this study, we investigated the bacterial and plankton communities in surface seawater from two coastal areas, i.e. Nampu and Sembukan, Wonogiri regency, Central Java, Indonesia. Bacterial diversity was analyzed using the Illumina-based high throughput sequencing with a primer set targeting the V3-V4 region of the bacterial 16S rRNA gene. Meanwhile, the plankton community (phytoplankton and zooplankton) was calculated and identified using a counting chamber method. Sequencing analysis revealed that the five dominant bacterial phyla in the two seawater samples were similar, consisting of Proteobacteria, Firmicutes, Actinobacteria, Bacteriodetes, and Cyanobacteria. Although the two sites are separated by several kilometres, the distribution of dominant bacterial phyla in both seawater samples is similar. Phytoplankton in Nampu and Sembukan were similarly dominated by Trichodesmium sp., Navicula sp., and Rhabdonema sp. Dominant zooplankton in the two sites were Euterpina, Nauplius, Oithona sp., Oncaea sp., Tigriopus sp., and Gastropoda larvae. The seawater in Nampu and Sembukan is suitable as a water source for aquaculture.

Survival and detection of bivalve transmissible neoplasia from the soft-shell clam Mya arenaria (MarBTN) in seawater

Many pathogens can cause cancer, but cancer itself does not normally act as an infectious agent. However, transmissible cancers have been found in a few cases in nature: in Tasmanian devils, dogs, and several bivalve species. The transmissible cancers in dogs and devils are known to spread through direct physical contact, but the exact route of transmission of bivalve transmissible neoplasia (BTN) has not yet been confirmed. It has been hypothesized that cancer cells could be released by diseased animals and spread through the water column to infect/engraft into other animals. To test the feasibility of this proposed mechanism of transmission, we tested the ability of BTN cells from the soft-shell clam (Mya arenaria BTN, or MarBTN) to survive in artificial seawater. We found that BTN cells are highly sensitive to salinity, with acute toxicity at salinity levels lower than those found in their environment. BTN cells also survive longer at lower temperatures, with >48% of cells surviving a week in seawater at temperatures from 4°C to 16°C, and 49% surviving for more than two weeks at 4°C. With one clam donor, living cells were observed for more than eight weeks at 4°C. We also used qPCR of environmental DNA (eDNA) to detect the presence of BTN-specific DNA in the environment. We observed release of BTN-specific DNA into the water of aquaria from tanks with highly BTN-positive clams, and we detected BTN-specific DNA in seawater samples collected from BTN-endemic areas, although the level detected was much lower. Overall, these data show that BTN cells can survive well in seawater, and they are released into the water by diseased animals, supporting the hypothesis that BTN is spread from animal-to-animal by cells through seawater.

Morphology, Chemical Characterization and Sources of Microplastics in a Coastal City in The Equatorial Zone With Diverse Anthropogenic Activities (Fortaleza City, Brazil)

The aim of the present study was to perform morphological and chemical characterizations of microplastics (MPs) found in seawater samples from the coast of the city of Fortaleza (CE) using Fourier-transform infrared (FTIR) spectroscopy, Raman spectroscopy and differential scanning calorimetry (DSC). Sampling was performed using a neuston sampler. MPs were separated based on the difference in density. MPs with varied morphologies were found. Fibers and fragments were the most abundant (57% and 36.2%, respectively). FTIR, Raman spectroscopy and DSC confirmed the presence of polyurethane and alkyd resin, polyethylene, polypropylene, polystyrene, polyamide blends, thermoplastic rubber and polyester fibers. Many studies have chemically characterized plastic materials using FTIR, Raman spectroscopy and DSC and common MPs, such as polyethylene, polypropylene, polyamide and polyester, have been characterized. However, characterization becomes more complex when MPs are less common particles, weathered particles and blends of polymers and additives. There is also less information on these types of MPs in commercial polymer databases. Therefore, the MP spectra obtained in this study can serve as a database to compare and characterize common as well as less common, more complex MPs.

Environmental DNA (eDNA) reveals endangered narrow sawfish across Indonesian Reefs

Environmental DNA or eDNA is a powerful method to uncover marine organisms in the seawaters. Furthermore, many marine species are difficult to determine in the sea waters because of their rare existence based on the visual census. In this study, we implemented environmental DNA to investigate the presence of the endangered species of narrow sawfish Anoxypristis cuspidata in Indonesia. Four liters of seawater samples were collected at six locations near the coral reefs ecosystem of Indonesia and filtered at 0.45 μm filter paper. DNA was extracted from the filter paper then Polymerase Chain Reaction (PCR) amplification was performed using the cytochrome c oxidase subunit I (COI) primer and analyzed by Next Generation Sequencing (NGS). The findings revealed that narrow sawfish exist in Indonesian waters, and it also simultaneously showed that environmental DNA could detect rare species. The environmental DNA approach to identifying narrow sawfish can provide reliable results and be used as a survey tool to protect endangered threatened and protected (ETP) species.

D-alanine metabolism via D-Ala aminotransferase by marine Gammaproteobacteria , Pseudoalteromonas sp. CF6-2

As the most abundant D-amino acid (DAA) in the ocean, D-alanine (D-Ala) is a key component of peptidoglycan in bacterial cell wall. However, the underlying mechanisms of bacterial metabolization of D-Ala through microbial food web remain largely unknown. In this study, the metabolism of D-Ala by marine bacterium Pseudoalteromonas sp. CF6-2 was investigated. Based on genomic, transcriptional and biochemical analyses combined with gene knockout, D-Ala aminotransferase was found to be indispensable for the catabolism of D-Ala in strain CF6-2. Investigation on other marine bacteria also showed that D-Ala aminotransferase gene is a reliable indicator for their ability to utilize D-Ala. Bioinformatic investigation revealed that D-Ala aminotransferase sequences are prevalent in genomes of marine bacteria and metagenomes, especially in seawater samples, and Gammaproteobacteria represents the predominant group containing D-Ala aminotransferase. Thus, Gammaproteobacteria is likely the dominant group to utilize D-Ala via D-Ala aminotransferase to drive the recycling and mineralization of D-Ala in the ocean. IMPORTANCE As the most abundant D-amino acid in the ocean, D-Ala is a component of marine DON (Dissolved organic nitrogen) pool. However, the underlying mechanism of bacterial metabolization of D-Ala to drive the recycling and mineralization of D-Ala in the ocean is still largely unknown. The results in this study showed that D-Ala aminotransferase is specific and indispensable for D-Ala catabolism in marine bacteria, and that marine bacteria containing D-Ala aminotransferase genes are predominantly Gammaproteobacteria widely distributed in global oceans. This study reveals marine D-Ala utilizing bacteria and the mechanism of their metabolization of D-Ala. The results shed light on the mechanisms of recycling and mineralization of D-Ala driven by bacteria in the ocean, which are helpful in understanding oceanic microbial-mediated nitrogen cycle.

Keanekaragaman Jenis dan Kepadatan Zooplankton di Perairan Pulau Makassar Kota Baubau

The purpose of the study was to determine the type and amount of zooplankton in the waters of Makassar Island, Baubau City. From the results of this study, it is expected that significant information regarding the types and zooplankton found in the sea around Makassar Island, Baubau City, whose waters are suitable for aquaculture. Furthermore, it is hoped that the results of this study can help improve and optimize the use of aquatic biological resources in the future. This research was conducted in the waters of Makassar Island, Baubau City, from August to October 2021. Sample identification was carried out at the Biology Laboratory of the Muhammadiyah University of Buton. The tool in this research field is plankton net no. 25 to filter zooplankton, GPS determines the sampling location, thermometer measures temperature, handrefactometer measures salinity, DO (Dissolved Oxygen) meter measures sea air oxygen, plastic bottles store seawater samples, small plastic bottles store zooplankton samples, and cool boxes for storing samples. Materials in the field are label paper and markers to label the sample, 70% alcohol to preserve the sample, and aqua water to sterilize the instrument. 1) From the three highest locations found 18 genera of zooplankton belonging to the Crustacea group. 2) With 33 ind/l, the genus OIthona dominates the waters of Makasar Island, Baubau City. 3) In the three locations, there was no variation in zooplankton density.

Occurrence of thraustochytrids: the fungoid protists vis-a-vis marine macroalgae (seaweeds) along the coast of Goa, India

Thraustochytrids are fungoid protists ubiquitous in the marine environment and found to be associated with decaying macroalgae. Not much is known about their association with living macroalgae. Hence in the present study, different macroalgal samples were collected from various beaches of Goa to examine the presence of thraustochytrids during a four-year-long study. Brown, red and green algae were found to be substrata of thraustochytrids. Thraustochytrids were isolated on pine pollen baiting from 17 to 58% of the specimens. Thraustochytrids isolated from various macroalgae belonged to the genera Oblongichytrium, Schizochytrium, Ulkenia, and Thraustochytrium. Labyrinthula sp. was also found once on the green alga Bryopsis hypnoides. These were generally found during dry seasons rather than in monsoons. The seasonal occurrence of thraustochytrids was found to be associated with temporal variation in macroalgal diversity. The statistical analysis supported individual or interactive effects of both factors viz, seasons and macroalgal diversity, on the occurrence of thraustochytrids. Thraustochytrids were also isolated from seawater adjoining macroalgae and from estuarine water at all times of the year. Oblongichytrium sp. was isolated from the green alga Ulva compressa and Anjuna seawater samples at the same time, thus indicating that thraustochytrids from seawater could inhabit the macroalgae.

Enzyme Method-Based Microfluidic Chip for the Rapid Detection of Copper Ions

Metal ions in high concentrations can pollute the marine environment. Human activities and industrial pollution are the causes of Cu2+ contamination. Here, we report our discovery of an enzyme method-based microfluidic that can be used to rapidly detect Cu2+ in seawater. In this method, Cu2+ is reduced to Cu+ to inhibit horseradish peroxidase (HRP) activity, which then results in the color distortion of the reaction solution. The chip provides both naked eye and spectrophotometer modalities. Cu2+ concentrations have an ideal linear relationship, with absorbance values ranging from 3.91 nM to 256 μM. The proposed enzyme method-based microfluidic chip detects Cu2+ with a limit of detection (LOD) of 0.87 nM. Other common metal ions do not affect the operation of the chip. The successful detection of Cu2+ was achieved using three real seawater samples, verifying the ability of the chip in practical applications. Furthermore, the chip realizes the functions of two AND gates in series and has potential practical implementations in biochemical detection and biological computing.

210Po/210Pb Disequilibria in the Eastern Tropical North Pacific

The 210Po/210Pb disequilibrium was attempted to reveal the small-scale particle dynamics in the eastern tropical North Pacific. Seawater samples in the full water column were collected from three sites in the Tehuantepec bowl near the East Pacific Ridge for determination of dissolved and particulate 210Po and 210Pb. Our results show that TPo/TPb activity ratios in the full water column at the three sites are less than 1, with an average of 0.56, indicating that the total 210Po in the oligotrophic sea is significantly deficient. The activity ratios of DPo/DPb in the dissolved phase are less than 1, while those in the particulate phase are greater than 1 (except for the bottom 300 m), indicating fractionation between 210Po and 210Pb in the scavenging process. A negative linear relationship between 210Po deficit and silicate proves that biological activities are responsible for 210Po deficiency in the upper 200 m. However, the deficit of 210Po in the bottom 300 m may be caused by the horizontal transport of the hydrothermal plume. After correcting the horizontal contribution, the removal rates of 210Po for the 200–1,500 m and the bottom 300 m layers increased by 7.5–21 and 26.1–29.5%, respectively. Correspondingly, the variation range of the residence time of a total 210Po became smaller. Our calculations suggest that horizontal transport is acting as a stabilizer for small-scale variation in the 210Po deficit in the eastern tropical North Pacific. Our study highlights the need to pay more attention to the small-scale variation of 210Po deficit when applying 210Po/210Pb disequilibria to trace biogeochemical processes, and the mechanism responsible for this variation deserves further study.