Deep sea lobsters are highly valued for seafood and provide the basis of important commercial fisheries in many parts of the world. Despite their economic significance, relatively little is known about their natural diets. Microscopic analyses of foregut content in some species have suffered from low taxonomic resolution, with many of the dietary items difficult to reliably identify as their tissue is easily digested. DNA metabarcoding has the potential to provide greater taxonomic resolution of the diet of the New Zealand scampi (Metanephrops challengeri) through the identification of gut contents, but a number of methodological concerns need to be overcome first to ensure optimum DNA metabarcoding results. In this study, a range of methodological parameters were tested to determine the optimum protocols for DNA metabarcoding, and provide a first view of M. challengeri diet. Several PCR protocols were tested, using two universal primer pairs targeting the 18S rRNA and COI genes, on DNA extracted from both frozen and ethanol preserved samples for both foregut and hindgut digesta. The selection of appropriate DNA polymerases, buffers and methods for reducing PCR inhibitors (including the use of BSA) were found to be critical. Amplification from frozen or ethanol preserved gut contents appeared similarly dependable. The COI gene was found to be more effective than 18S rRNA gene for identifying large eukaryotic taxa from the digesta; however, it was less successfully amplified. The 18S rRNA gene was more easily amplified, but identified mostly smaller marine organisms such as plankton and parasites. This preliminary analysis of the diet of M. challengeri identified a range of species (13,541 reads identified as diet), which included the ghost shark (Hydrolagus novaezealandiae), silver warehou (Seriolella punctata), tall sea pen (Funiculina quadrangularis) and the salp (Ihlea racovitzai), suggesting that they have a varied diet, with a high reliance on scavenging a diverse range of pelagic and benthic species from the seafloor.

Plankton are central to planetary ecology, generating 50% of Earth’s atmospheric oxygen and forming the largest system of interconnected life at the base of the marine food chain. Yet, current oceanographic models aimed at predicting global climate change lack high-resolution biological data, emphasizing the need for innovative approaches to collect plankton biodiversity and distribution data over larger spatial, temporal, and taxonomic scales. The significant number of boats, ranging from small sailing yachts to large commercial vessels, that ply the world’s oceans every day could help scientists collect thousands of valuable plankton samples. Traditional Plankton Nets (TPN) are not suited to the speed of a recreational craft cruising in the high seas (i.e., at speeds >2 knots). We developed and validated the efficiency of a lightweight, easily deployable Cruising Speed Net (CSN) that enables the collection of ocean surface micro- and mesoplankton at speeds up to 5 knots. Field testing was conducted during two distinct research cruises along coastal and oceanic latitudinal gradients (SSV Robert C. Seamans in New Zealand and RV Investigator in the south-east Indian Ocean). DNA metabarcoding performed on the collected plankton samples showed the TPN and CSN yielded identical sequence-based diversity at low speed, with the CSN also effective at higher speed for characterizing latitudinal distribution of plankton communities. The CSN represents a valuable new tool for expanding the global collection of plankton data.

Deep-sea benthic ecosystems are difficult to study, particularly when trying to clarify diet and trophic relationships. New Zealand scampi Metanephrops challengeri are endemic, commercially prized deep-sea lobsters that are bottom trawled. These lobsters are typically the dominant mobile megafaunal species in the deep-sea benthic habitat, and their burrowing behaviour plays an important role in bioturbation of seafloor habitats. DNA metabarcoding was undertaken on the gut contents of 66 scampi from 4 fishery management areas using COI and 18S rRNA markers to better understand their feeding habits and trophic role. Scampi were confirmed to be opportunistic benthic scavengers, with the gut samples containing over 150 species, ranging from small (e.g. alveolates) to large eukaryotes (e.g. fish). The main dietary components consisted of crabs and prawns, but also included macroalgae and fish. Significant differences were found among scampi gut contents when comparing season and geographic region, but not when comparing sex and size. Due to their generalist scavenging nature, scampi play an important role in the deep-sea benthic ecosystems and are natural benthic samplers that are well suited to being used as deep-sea ecosystem/biodiversity monitors.

Dietary and predator-prey studies are more frequently relying on DNA metabarcoding methods, typically achieving results that have a better taxonomic resolution (e.g., species-level) than previous methods. With the continuous advancement in sequencing technology, what was previously accessible only as a large, fixed structure in a laboratory, which had a limited number of users, has now advanced to a small and readily usable device. In this study, we used the gut (content and lining) from juvenile lanternfish (Hygophum) specimens to compare the short-read sequencing capability of the portable Nanopore MinION with the Illumina MiSeq. Primers common in dietary DNA metabarcoding work (COI "Leray primers" and 18S rRNA V4 "Zhan primers") were used, with an additional comparison of cost-effective COI "Lobo primers" (targeting the same COI fragment) for the proficiency in species detection of a broad range of taxa. Our results indicate high congruency between sequencing machines for, not only taxonomic assignments, but also relative read abundance of the main dietary items. We also identified that Nanopore sequencing is more cost-effective.The Lobo primers are comparable to that of Leray, but substantially reduce the primer set price without compromising detection of taxa. Using both COI and 18S broadened the taxonomic scope, providing greater prey detection. Overall, this preliminary study was successful in creating a foundation for future dietary work involving larvae and transformation stage fishes whereby the content of the gut need not be separated from the gut lining to detect prey. The Hygophum diet detected here aligns with previous research that suggests the main dietary items to be calanoid copepods, but using molecular methods, soft prey was more readily identified compared to studies using visual methods of identification of dietary items. Overall, this study found that Nanopore sequencing is suitable for short-read DNA metabarcoding and can provide rapid access to sequencing results.

Background: Probiotics are effective to rectify the imbalanced gut microbiota in the diseased cohorts. Two Bifidobacterium strains (LI09 and LI10) were found to alleviate D-galactosamine-induced liver damage (LD) in rats in our previous work. A series of bioinformatic and statistical analyses were performed to determine the vital bacteria in the gut microbiotas altered by the LI09 or LI10 in rats. Results: Two groups of representative phylotypes could distinguish the gut microbiotas of LI09 or LI10 groups from the other groups. Among them, OTU170_Porphyromonadaceae acted as a gatekeeper in LI09 group, while OTU12_Bacteroides was determined with multiple correlations in the gut network of LI10 group. Multiple reduced OTUs associated with LC and increased OTUs associated with health were determined in LI09 or LI10 groups, among which, increased OTU51_Barnesiella and reduced OTU99_Barnesiella could be associated with the protective effects of both the two probiotics. The gut microbiotas in LI09, LI10 and positive control groups were clustered into three clusters, i.e., Cluster_1_Microbiota, Cluster_2_Microbiota and Cluster_3_Microbiota, by Partition Around Medoids clustering analysis. Cluster_2_Microbiota was determined at least dysbiotic status due to its greatest LD dysbiosis ratio, lowest levels of liver function variables and plasma cytokines compared with the two other clustered microbiotas, suggesting the treated rats in Cluster_2 were at better health status. Conclusion: Our findings suggest that OTU170_Porphyromonadaceae and OTU12_Bacteroides are vital in the gut microbiotas altered by LI09 and LI10. Characteristics of the LD cohorts treated by LI09 or LI10 at different gut microbial colonization states could help monitor the cohorts' health status.