DNA Metabarcoding Approach Research Articles

Detection of Bombyx mori as a Protein Source in Feedingstuffs by Real-Time PCR with a Single-Copy Gene Target

The silkworm, Bombyx mori, is reared on a large scale, mainly for silk production. The waste from this silk production, like pupae, is underused. As an edible insect, B. mori is a good source of protein in human food and animal feed. In recent years, European legislation on the use of insects has evolved and a multitude of European companies have initiated the rearing of insects specifically for food and feed applications. Regarding animal feed, Commission Regulations (EU) 2021/1372 and 2021/1925 authorize eight insect species, including silkworm, as processed animal proteins for use in fish, pig, and poultry feed. The incorporation of edible insects into the human diet falls within Regulation (EU) No. 2015/2283 concerning novel foods. Implementation of authentication methods is imperative to ensure the conformity of the products. In the present study, we propose a specific real-time PCR method for the detection of silkworm (B. mori). The developed PCR test amplifies a 98 bp fragment of the cadherin gene. This gene is present in a single-copy per haploid genome, as demonstrated by experimental evidence. The qualitative method was successfully evaluated on the performance criteria of specificity, sensitivity, efficiency, robustness, and transferability. The applicability of the test was assessed on samples of B. mori from industry. Light microscopy and DNA metabarcoding approaches were used as a complement to genomic analysis as a means of providing authentication of the samples.

Major diet of common carp (Cyprinus carpio L.) over different developmental stages in rice-field: agroecological interactions between fishes and rice in Sichuan, China, based on DNA metabarcoding approach

Major diet of common carp (Cyprinus carpio L.) over different developmental stages in rice-field: agroecological interactions between fishes and rice in Sichuan, China, based on DNA metabarcoding approach

Bottom Trawling and Multi‐Marker eDNA Metabarcoding Surveys Reveal Highly Diverse Vertebrate and Crustacean Communities: A Case Study in an Urbanized Subtropical Estuary

ABSTRACTEstuarine habitats serve as critical feeding and nursery grounds for many aquatic species and support fisheries. However, monitoring these complex ecosystems using conventional trawling methods is destructive, costly, and labor‐intensive. This study compared trawling and a multi‐marker environmental DNA (eDNA) metabarcoding approach to monitor marine vertebrate and crustacean communities in an estuarine environment in subtropical Hong Kong. We analyzed 16 bottom trawl samples and the eDNA from 32 two‐liter water samples using primer sets specific to fishes and mammals (MiFish‐U, 12S‐V5, and Berry‐Fish) and decapod crustaceans (MiDeca). We found that the eDNA approach detected more pelagic and demersal fishes (237 vs. 106 in trawling) and elasmobranchs (6 vs. 3) than trawling. The eDNA approach was also more effective than trawling in detecting threatened vertebrates (16 vs. 4), including the Indo‐Pacific Finless Porpoise and the critically endangered Large Yellow Croaker. Among the detected fish at species level, 70 species were detected by both approaches, 32 species were detected by trawling only, and 142 species were detected by the eDNA approach only. Regarding crustaceans, the eDNA approach detected slightly fewer decapods (61 vs. 77) and stomatopods (5 vs. 8) than trawl surveys. However, the eDNA approach could be enhanced through the development of suitable decapod‐specific primers and the expansion of the local reference database. In addition, multivariate analyses of the eDNA data revealed spatial patterns of fish and crustacean assemblages that might be associated with sediment loading, oxygen, and nutrient levels. Furthermore, there was a positive correlation between eDNA read counts and trawl catch, but their correlation coefficient was low. We conclude that eDNA metabarcoding can provide high‐resolution detection of species, composition, and unravel community–environment relationships in estuarine ecosystems. Overall, integrating the non‐destructive eDNA approach can complement the conventional trawling method for better‐informed sustainable fishery management and conservation.

DNA Metabarcoding Approach as a Potential Tool for Supporting Official Food Control Programs: A Case Study.

Food authentication significantly impacts consumer health and the credibility of Food Business Operators (FBOs). As European regulations mandate the verification of food authenticity and supply chain integrity, competent authorities require access to innovative analytical methods to identify and prevent food fraud. This study utilizes the DNA metabarcoding approach on meat preparations, sampled during an official control activity. It assesses animal and plant composition by amplifying DNA fragments of the 12S rRNA and trnL (UAA) genes, respectively. The results not only confirmed the declared species but also revealed undeclared and unexpected taxa in products labelled as containing a single animal species and various unspecified plant species. Notable findings such as the presence of Murinae, Sus scrofa, Ovis aries, and Pisum sativum could raise public health concerns, compromise consumer choices made for ethical or religious reasons, and reflect the hygienic conditions of the processing plant. This study demonstrates that the DNA metabarcoding approach looks to be a promising support tool for official control authorities to ensure food authenticity and safety, and to develop risk profiles along the supply chain.

Performance of DNA metabarcoding, standard barcoding and morphological approaches in the identification of insect biodiversity.

For two decades, DNA barcoding and, more recently, DNA metabarcoding have been used for molecular species identification and estimating biodiversity. Despite their growing use, few studies have systematically evaluated these methods. This study aims to evaluate the efficacy of barcoding methods in identifying species and estimating biodiversity, by assessing their consistency with traditional morphological identification and evaluating how assignment consistency is influenced by taxonomic group, sequence similarity thresholds and geographic distance. We first analysed 951 insect specimens across three taxonomic groups: butterflies, bumblebees and parasitic wasps, using both morphological taxonomy and single-specimen COI DNA barcoding. An additional 25,047 butterfly specimens were identified by COI DNA metabarcoding. Finally, we performed a systematic review of 99 studies to assess average consistency between insect species identity assigned via morphology and COI barcoding and to examine the distribution of research effort. Species assignment consistency was influenced by taxonomic group, sequence similarity thresholds and geographic distance. An average assignment consistency of 49% was found across taxonomic groups, with parasitic wasps displaying lower consistency due to taxonomic impediment. The number of missing matches doubled with a 100% sequence similarity threshold and COI intraspecific variation increased with geographic distance. Metabarcoding results aligned well with morphological biodiversity estimates and a strong positive correlation between sequence reads and species abundance was found. The systematic review revealed an 89% average consistency and also indicated taxonomic and geographic biases in research effort. Together, our findings demonstrate that while problems persist, barcoding approaches offer robust alternatives to traditional taxonomy for biodiversity assessment.

From ctenophores to scyphozoans: parasitic spillover of a burrowing sea anemone

Most host-parasite associations are explained by phylogenetically conservative capabilities for host utilization, and therefore parasite switches between distantly related hosts are rare. Here we report the first evidence of a parasitic spillover of the burrowing sea anemone Edwardsiella carnea from the invasive ctenophore Mnemiopsis leidyi to two scyphozoan hosts: the native Mediterranean barrel jellyfish Rhizostoma pulmo and the invasive Indo-Pacific nomad jellyfish Rhopilema nomadica, collected from the Eastern Mediterranean Sea. Edwardsiella carnea planulae found in these jellyfish were identified using molecular analyses of the mitochondrial 16S and nuclear 18S rRNA genes. Overall, 93 planulae were found on tentacles, oral arms, and inside of the gastrovascular canals of the scyphomedusae, whereas no infection was observed in co-occurring ctenophores. DNA metabarcoding approach indicated seasonal presence of Edwardsiella sp. in the Eastern Mediterranean mesozooplankton, coinciding with jellyfish blooms in the region. Our findings suggest a non-specific parasitic relationship between Edwardsiella carnea and various gelatinous hosts based on shared functionality rather than evolutionary history, potentially driven by shifts in host availability due to jellyfish blooms. This spillover raises questions about the ecological impacts of parasitism on native and invasive scyphozoan hosts and the potential role of Edwardsiella in controlling their populations.

Structural variability of protist assemblages in surface sediments across Italian Mediterranean marine subregions

Marine sediments host heterogeneous protist communities consisting of both living benthic microorganisms and planktonic resting stages. Despite their key functions in marine ecosystem processes and biogeochemical cycles, their structure and dynamics are largely unknown. In the present study, with a spatially intensive sampling design we investigated benthic protist diversity and function of surface sediment samples from three subregions of the Mediterranean Sea, through an environmental DNA metabarcoding approach targeting the 18S V4 region of rRNA gene. Protists were characterized at the taxonomic level and trophic function, both in terms of alpha diversity and community composition, testing for potential differences among marine subregions and bathymetric groups. Overall, Alveolata and Stramenopiles were the two divisions that dominated the communities. These dominant groups exhibited significant differences among the three Mediterranean subregions in the alpha diversity estimates based on the detected ASVs, for all computed indices (ASV richness, Shannon and Simpson indices). Protist communities were also found to be significantly different in terms of composition at the order rank in the three subregions p-value < 0.01). These differences were mainly driven by Anoecales, Peridiniales, Borokales, Paraliales and Gonyaulacales, which together contributed almost 80% of the average dissimilarity. Anoecales was the dominant order in the Ionian – Central Mediterranean and Adriatic Sea, but with considerably different relative abundances (52% and 36%, respectively), while Borokales was the dominant order in the Western Mediterranean Sea (33%). Similarly, significant differences among the three marine subregions were also highlighted when protist assemblages were examined in terms of trophic function, both in terms of alpha diversity (calculated on the ASVs for each trophic group) and community composition p-value < 0.01. In particular, the Adriatic Sea stood out for having the highest relative abundance of autotrophic/mixotrophic components in the surface sediments analyzed. Conversely, no significant differences in protist assemblages were found among depth groups. This study provided new insights into the taxonomic and trophic composition of benthic protist communities found in Mediterranean surface sediments, revealing geographical differences among regional seas. The results were discussed in relation to the Mediterranean environmental features that could generate the differences among benthic protist communities.

First DNA metabarcoding diet assessment on the critically endangered Tricolour Langur, Presbytischrysomelascruciger.

Presbytischrysomelascruciger or also known as the Tricolour langur-is rare, endemic to Sarawak and Kalimantan in Borneo and classified as a critically endangered subspecies. The current P.c.cruciger population size is uncertain because the numbers are continuously decreasing. At present, there is no comprehensive scientific report on P.c.cruciger in Sarawak, although this subspecies is known to inhabit Maludam area. Recent first sighting of P.c.cruciger in Jemoreng Sarawak presents a research opportunity to study its feeding from a molecular ecology perspective. Herein, we report the first findings on the dietary intake of P.c.cruciger using a high-throughput DNA metabarcoding approach. We emphasise the diet intake of P.c.cruciger from Jemoreng Protected Forest in Sarawak using DNA metabarcoding of the trnL region. Preliminary findings revealed 11 amplicon sequence variants (ASV) classified into one phylum, four classes, four orders, four families, three genera and three plant species. Fibraureatinctoria (akar kuning; Family Menispermaceae), Poikilospermumsuaveolens (akar jangkang; Family Urticaceae) and Litchichinensis (lychee; Family Sapindaceae) were the three main plant species that were consumed by P.c.cruciger. Understanding the dietary intake of P.c.cruciger is of paramount importance for their conservation and management of the habitat areas where their population resides.

Disentangling eukaryotic biodiversity patterns from man‐made environments (port and marina) and nearby coral reefs in the Red Sea: A focus on the surveillance of non‐indigenous species

AbstractMan‐made environments such as ports and marinas are gateways for many marine non‐indigenous species (mNIS) transported via shipping worldwide. These habitats are often the focus of biosecurity programs for the surveillance of mNIS, but there have been few studies investigating the distribution of biofouling communities in natural habitats and nearby artificial habitats. Here, following a DNA metabarcoding approach, we tested for differences in spatio‐temporal trends of biological pioneer communities established after one‐week colonization between a port, a marina, and coral reefs using two different matrices (PVC panels and water samples) in the central Red Sea. In addition, we aimed to investigate differences in the prevalence of mNIS between man‐made and natural habitats during the summer and winter seasons, and whether the number and patterns of mNIS differed between the port (an active historic port subjected to intense shipping traffic) and the marina (a small recently developed marina utilized by small and medium research vessels). The community structure from samples collected at the two coral reefs showed greater similarity to each other compared to that obtained from the port and the marina. A total of 29 mNIS were detected across all sites, of which 16 were reported in the Red Sea for the first time, with Jeddah port hosting 28 mNIS. By applying standardized methodological approaches, this study provides comparable data regarding the monitoring of introduced and invasive species, particularly within the data‐poor Red Sea, a major shipping corridor. This study serves as the foundation for implementing a robust and rapid baseline monitoring system to assess native biodiversity and facilitate the early detection of NIS.

Endolithic Fungal Diversity in Antarctic Oligocene Rock Samples Explored Using DNA Metabarcoding.

In this study, we evaluated the fungal diversity present associated with cores of Oligocene rocks using a DNA metabarcoding approach. We detected 940,969 DNA reads grouped into 198 amplicon sequence variants (ASVs) representing the phyla Ascomycota, Basidiomycota, Mortierellomycota, Chytridiomycota, Mucoromycota, Rozellomycota, Blastocladiomycota, Monoblepharomycota, Zoopagomycota, Aphelidiomycota (Fungi) and the fungal-like Oomycota (Stramenopila), in rank abundance order. Pseudogymnoascus pannorum, Penicillium sp., Aspergillus sp., Cladosporium sp., Aspergillaceae sp. and Diaporthaceae sp. were assessed to be dominant taxa, with 22 fungal ASVs displaying intermediate abundance and 170 being minor components of the assigned fungal diversity. The data obtained displayed high diversity indices, while rarefaction indicated that the majority of the diversity was detected. However, the diversity indices varied between the cores analysed. The endolithic fungal community detected using a metabarcoding approach in the Oligocene rock samples examined contains a rich and complex mycobiome comprising taxa with different lifestyles, comparable with the diversity reported in recent studies of a range of Antarctic habitats. Due to the high fungal diversity detected, our results suggest the necessity of further research to develop strategies to isolate these fungi in culture for evolutionary, physiological, and biogeochemical studies, and to assess their potential role in biotechnological applications.

DNA metabarcoding as a tool for characterising the spatio-temporal distribution of planktonic larvae in the phylum Echinodermata

AbstractMetabarcoding is revolutionising the analysis of biodiversity in marine ecosystems, especially as it provides a means of detecting and identifying cryptic life stages in field samples. The planktonic larval stage of many species underpins the abundance and distribution of adult populations but is challenging to characterise given the small size of larvae and diffuse distributions in pelagic waters. Yet, planktonic larval dynamics are key to understanding phenomena observed in adult populations, such as the boom-and-bust dynamics exhibited by some echinoderms. Rapid changes in echinoderm population density can have significant effects on local benthic ecosystems. For example, outbreaks of the crown-of-thorns sea star (CoTS) on the Great Barrier Reef (GBR) have led to considerable declines in coral cover. Here, we used a DNA metabarcoding approach to investigate the spatio-temporal distribution and diversity of echinoderm larvae on the GBR, including CoTS. Generalised linear mixed models revealed that echinoderm larval richness, was significantly correlated with temporal variables (i.e. season and year) which is consistent with expected fluctuations in larval output based on adult spawning periodicity. However, neither site-specific differences in echinoderm larval richness, nor correlations between larval composition and environmental, temporal, or spatial variables were found. This study validates the utility of metabarcoding approaches for detecting and characterising echinoderm larvae, including CoTS, which could prove useful to future monitoring efforts. Our findings suggest that metabarcoding can be used to better understand the life history of planktonic larvae, and analyses combining environmental (e.g., temperature, nutrients) and oceanographic (e.g., currents) data could deliver valuable information on the factors influencing their spatio-temporal distributions.

Sedimentary DNA reveals phytoplankton diversity loss in a deep maar lake during the Anthropocene

AbstractAnthropogenic‐driven environmental change, including current climate warming, has influenced lake ecosystems globally during the Anthropocene. Phytoplankton are important indicators of environmental changes in lakes and play a fundamental role in maintaining the functioning and stability of these ecosystems. However, the extent to which lake phytoplankton were affected by anthropogenic or climatic forces during the Anthropocene remains unclear. Here, we investigated the 160‐yr‐long dynamics of the phytoplankton community (cyanobacteria and eukaryotic microalgae) in response to anthropogenic forcing in Sihailongwan Maar Lake—a candidate for a Global boundary Stratotype Section and Point for demarcation of the Anthropocene—using DNA metabarcoding and traditional paleolimnological approaches. Our results show a significant decline in phytoplankton diversity and an abrupt shift in community composition around the 1950s, corresponding to the beginning of the “Great Acceleration” period. Specifically, phytoplankton taxa coexistence patterns, niche differentiation, and assembly mechanisms changed significantly after the 1950s. Overall, increases in air temperature and anthropogenic forcing appear to be the dominant controls for community reorganization and diversity decline of the phytoplankton from this deep maar lake. A neutral community model suggests that phytoplankton community composition was mainly controlled by stochastic processes before the 1950s; however, as time progressed, deterministic effects driven by anthropogenic global warming increased. The results of this study imply that anthropogenic perturbations have led to a loss of phytoplankton diversity and a further decline in ecological resilience in deep lakes, with likely knock‐on effects on the productivity and function of lake ecosystems.

DNA metabarcoding reveals the seasonal variation of dietary composition of Taihangshan macaque (Macaca mulatta tcheliensis), Jiyuan, north China.

Dietary analysis in wildlife is fundamental for understanding their flexible response to seasonal changes and developing effective conservation management measures. Taihangshan macaque (Macaca mulatta tcheliensis) is the northernmost population of rhesus macaque, currently only distributed in the southern Mt. Taihangshan area. This area belongs to a semi-arid region resulting in limited plant food availability for Taihangshan macaques, with seasonal variation. Herein, we used a chloroplast trnL DNA metabarcoding approach to identify the plant diet diversity and composition from 100 fecal samples of Taihangshan macaque in four seasons (spring, summer, autumn, and winter) from 2020 to 2021. The results revealed that (1) a total of 48 distinct families, 88 genera, and 52 species within the 105 food items that were consumed by Taihangshan macaques throughout the year; (2) the diversity of food items exhibited significant differences across the four seasons; (3) Rosaceae, Rhamnaceae, Fagaceae, and Poaceae are the preferential food items for Taihangshan macaques and have different relative abundances, fluctuating with seasonal variation. DNA metabarcoding can expand our understanding of the repertoire of food items consumed by Taihangshan macaques by detecting some consumed food items in this population that were not yet discovered using traditional methods. Therefore, the integrative results from traditional methods and DNA metabarcoding can provide a fundamental understanding of dietary composition to guide the conservation management of Taihangshan macaques.

A full-length 18S ribosomal DNA metabarcoding approach for determining protist community diversity using Nanopore sequencing.

Protist diversity studies are frequently conducted using DNA metabarcoding methods. Currently, most studies have utilized short read sequences to assess protist diversity. One limitation of using short read sequences is the low resolution of the markers. For better taxonomic resolution longer sequences of the 18S rDNA are required because the full-length has both conserved and hypervariable regions. In this study, a new primer pair combination was used to amplify the full-length 18S rDNA and its efficacy was validated with a test community and then validated with field samples. Full-length sequences obtained with the Nanopore MinION for protist diversity from field samples were compared with Illumina MiSeq V4 and V8-V9 short reads. Sequences generated from the high-throughput sequencers are Amplicon Sequence Variants (ASVs). Metabarcoding results show high congruency among the long reads and short reads in taxonomic annotation at the major taxonomic group level; however, not all taxa could be successfully detected from sequences. Based on the criteria of ≥95% similarity and ≥1000 bp query length, 298 genera were identified by all markers in the field samples, 250 (84%) were detected by 18S, while only 226 (76%) by V4 and 213 (71%) by V8-V9. Of the total 85 dinoflagellate genera observed, 19 genera were not defined by 18S dinoflagellate ASVs compared to only three among the total 52 diatom genera. The discrepancy in this resolution is due to the lack of taxonomically available 18S reference sequences in particular for dinoflagellates. Overall, this preliminary investigation demonstrates that application of the full-length 18S rDNA approach can be successful in field studies.

Biodiversity of autotrophic euglenids based on the group specific DNA metabarcoding approach

This study reports a comprehensive analysis of photoautotrophic euglenids’ distribution and biodiversity in 16 small water bodies of various types (including fish ponds, field ponds, rural ponds and park ponds) located in three regions of Poland: Masovia, Masuria and Pomerania during a period of three years. By employing a euglenid specific barcode marker and a curated database of V2 18S rDNA sequences it was possible to identify 97.7 % of euglenid reads at species level. A total of 152 species classified in 13 genera were identified. The number of euglenid species found in one pond varied from 40 to 102. The most common species were Euglena agilis and Euglenaria caudata, found in every analysed waterbody. The highest number of observed species belonged to Trachelomonas and Phacus. Certain species exhibited a tendency to coexist, suggesting the presence of distinct species assemblages. Among them, the most distinctive cluster was associated with water bodies located in the Masuria region, characterized also by the greatest species richness, including many very rare species: Euglenaformis chlorophoenicea, Lepocinclis autumnalis, L. marssonii, Trachelomonas eurystoma, T. manschurica, T. mucosa, T. zuberi, T. zuberi var. nepos.

DNA-metabarcoding supports trophic flexibility and reveals new prey species for the Galapagos sea lion.

Tropical ecosystems are challenging for pinnipeds due to fluctuating food availability. According to previous research, the Galapagos sea lion (GSL, Zalophus wollebaeki) adopts trophic flexibility to face such conditions. However, this hypothesis comes from studies using traditional methods (hard-parts analysis of scat and isotopic analysis from tissue). We studied the diet of five rookeries in the southeastern Galapagos bioregion (which harbors the highest GSL density), via DNA-metabarcoding of scat samples. The DNA-metabarcoding approach may identify consumed prey with a higher taxonomic resolution than isotopic analysis, while not depending on hard-parts remaining through digestion. Our study included five different rookeries to look for evidence of trophic flexibility at the bioregional level. We detected 98 prey OTUs (124 scats), mostly assigned to bony-fish taxa; we identified novel prey items, including a shark, rays, and several deep-sea fish. Our data supported the trophic flexibility of GSL throughout the studied bioregion since different individuals from the same rookery consumed prey coming from different habitats and trophic levels. Significant diet differentiations were found among rookeries, particularly between Punta Pitt and Santa Fe. Punta Pitt rookery, with a more pronounced bathymetry and lower productivity, was distinguished by a high trophic level and consumption of a high proportion of deep-sea prey; meanwhile, Santa Fe, located in more productive, shallow waters over the shelf, consumed a high proportion of epipelagic planktivorous fish. Geographic location and heterogeneous bathymetry of El Malecon, Española, and Floreana rookeries would allow the animals therein to access both, epipelagic prey over the shelf, and deep-sea prey out of the shelf; this would lead to a higher prey richness and diet variability there. These findings provide evidence of GSL adopting a trophic flexibility to tune their diets to different ecological contexts. This strategy would be crucial for this endangered species to overcome the challenges faced in a habitat with fluctuating foraging conditions.

Soil Fertilization with Palm Oil Mill Effluent Has a Short-Term Effect on the Bacterial Diversity of an Amazonian Agricultural Land Area.

Palm oil derived from the fruits of Elaeis guineensis Jacq. has global economic importance and is largely produced in tropical regions. The palm oil production process leads to a highly polluting waste called palm oil mill effluent (POME). A strategy commonly used by producers to overcome environmental issues and to improve soil fertility is the reuse of POME as a fertilizer due to the chemical and biological characteristics of the effluent. In this research, three groups were analyzed: soil without POME application (control group) and soil samples after 4 and 9 days of POME application. An environmental DNA metabarcoding approach was used. eDNA was extracted, and the V4 region of the 16S rRNA gene was amplified and sequenced in the Illumina MiSeq platform. The abundance of Proteobacteria (48.1%) and Firmicutes (9.0%) was higher in fertilized soil, while Bacteroidetes (20.3%) and Verrucomicrobia (7.8%) were more abundant in control soil. Additionally, the effluent seemed to modify soil characteristics favoring taxa responsible for the mineralization of organic compounds and nitrogen fixation such as species of Gammaproteobacteria class. Our study highlights the influence of POME on soil biological components and contributes to the sustainable production of palm oil in the Amazon.

Insights into freshwater ciliate diversity through high throughput DNA metabarcoding.

The freshwater bodies of India are highly biodiverse but still understudied, especially concerning ciliates. Ciliates constitute a significant portion of eukaryotic diversity and play crucial roles in microbial loops, nutrient recycling, and ecosystem maintenance. The present study aimed to elucidate ciliate diversity in three freshwater sites in the Delhi region of India: Okhla Bird Sanctuary (OBS), Sanjay Lake (SL), and Raj Ghat pond (RJ). This study represents the first investigation into the taxonomic diversity and richness of freshwater ciliates in India using a high-throughput DNA metabarcoding approach. For the analysis, total environmental DNA was extracted from the three freshwater samples, followed by sequencing of the 18S V4 barcode region and subsequent phylogenetic analyses. Operational taxonomic units (OTU) analyses revealed maximum species diversity in OBS (106), followed by SL (104) and RJ (99) sites. Ciliates from the classes Oligohymenophorea, Prostomatea, and Spirotrichea were dominant in the three sites. The study discusses the ability of the metabarcoding approach to uncover unknown and rare species. The study highlights the need for refined reference databases and cautious interpretation of the high-throughput sequencing-generated data while emphasizing the complementary nature of molecular and morphological approaches in studying ciliate diversity.

Uncovering floral composition of paper wasp nests (Hymenoptera: Vespidae: Polistes) through DNA metabarcoding

As the social organism, Polistes wasps build a communal nest using woody fibers with saliva for sustaining brood and adult population throughout the season. Limited information exists regarding the identification specific plant materials employed in wasp nest building. Thus, we firstly tested if the DNA metabarcoding approach utilizing rbcL and trnL molecular markers could identify the plant species quantitatively and qualitatively inform the mixed-origin woody samples. A threshold of 0.01 proportion of reads was applied for rbcL and trnL molecular markers, while this threshold for median proportion was 0.0025. In assessing taxa richness, the median proportion demonstrated superior performance, exhibiting higher taxa detection power, however, rbcL marker outperformed in quantitative analysis. Subsequently, we applied DNA metabarcoding to identify the plant materials from the nests of two Polistes species, P. mandarinus and P. rothneyi. The results showed that higher preference of Quercus and Robinia as the major nest building materials regardless of the surrounding plant communities, by two wasp species. Material diversity was higher for P. rothneyi than P. mandarinus, which may explain the abundance of this species possibly with heightened adaptive capacities in their nesting behavior. This study demonstrated that DNA metabarcoding could identify the complex nest-building plant materials of paper wasps and provide insights into their ecological interactions in the natural ecosystem.

Interspecific variation in the diet of Symphalangussyndactylus and Macacanemestrina at Genting Highlands, Pahang, Peninsular Malaysia.

Primate communities in the Genting Highlands consist of a single species of Hylobatidae and four species of Cercopithecidae, which are known to exhibit social interaction behaviour. Thus, a study on the diets of Symphalangussyndactylus (siamang; family Hylobatidae) and Macacanemestrina (pig-tailed macaque; family Cercopithecidae) was carried out at Genting Highlands, in order to compare the dietary preferences and interspecific competition between the two primate families. A DNA metabarcoding approach was used to analyse diet intake using non-invasive samples based on the trnL region. Based on the 140 amplicon sequence variants (ASVs) generated, 26 plant orders, 46 different families, 60 genera and 49 species were identified from 23 different plant classes. Fabaceae and Moraceae were classified as the most preferred plants at the family level for S.syndactylus; meanwhile, Piperaceae and Arecaceae were classified as the most preferred for M.nemestrina. Only six out of the 60 different plant genera classified in this study, were found to be consumed by both species. Therefore, the low similarity of preferred plants in the diets between the two families suggests that there is little interspecific competition. These findings are important for future conservation management of highland primates, especially in the Genting Highlands.