Pinctada Research Articles

Genomic and transcriptomic analysis to investigate the growth heterosis mechanisms of pearl oysters (Pinctada fucata martensii)

Pinctada fucata martensii, a bivalve mollusk highly valued for seawater pearl production, exhibits growth traits crucial for pearl yield and quality. Although hybridization promotes growth, the mechanisms underlying growth heterosis in pearl oysters remain unclear. This study utilized two inbred populations (PB and PY) and one hybrid population (F) constructed from pearl oysters. Comparative analysis of growth traits, along with whole-genome sequencing (WGS) and RNA sequencing (RNA-seq), revealed that hybrid F showed superior growth traits. Compared with the parents, hybrid F exhibited significant heterosis for shell height (10.422%), shell length (11.361%), shell width (11.411%), and total weight (36.254%). Analysis of population genetic structure indicated increased heterozygosity in hybrid F through hybridization. Expression pattern analysis revealed that approximately 93.79% of the DEGs displayed dominant expression, with approximately 6.21% exhibited overdominant, indicating pivotal roles of dominant genes in heterosis development. There were more paternal dominant genes than maternal genes, suggesting that paternal genetic information significantly impacts heterosis in hybrid F. Furthermore, selective sweep and expression pattern analyses revealed that immune defense, metabolism, and regulatory processes are correlated with hybrid phenotypic growth traits in pearl oysters. In conclusion, this study explored the molecular mechanisms that influence the hybrid growth advantage of pearl oysters, with the aim of providing valuable insights and ideas for breeding research in aquaculture.

Biomineral photonic materials with a gradient structure: The ligament of the pearl oyster Pinctada margaritifera

The bivalve fibrous ligaments (FLs) are unusual biomineralized materials evolved to open two shell valves as needed. For a long time, the inside beautiful structural colors of the FLs have received little attention. Here, using optical microscopy, SEM, and fiber optic spectrometry combined with the image processing method and pair correlation function (Pcf) analysis, we investigate the structure and optical property of the FL of the pearl oyster Pinctada margaritifera. In this FL, we find that: (1) the aragonite fibers are uniform in diameter with an average of 98 ± 10 nm; (2) they are spatially organized with short-range order; and (3) they vary gradiently in their nearest-neighbor distance (d) from 148 nm to 103 nm along the lateral-inner direction of the FL. Therefore, this FL possesses a 2D amorphous gradient photonic structure, which results in that the FL displays extremely variable structural colors spanning from blue to yellow. In short, this work provides a new model of biological photonic structure, which may inspire the design and synthesis of novel photonic materials.

Inhibitors of LAMP used to detect Tenacibaculum sp. strain Pbs-1 associated with black-spot shell disease in Akoya pearl oysters, and additives to reduce the effect of the inhibitors

Black-spot shell disease is an unresolved disease that decreases pearl quality and threatens pearl oyster survival. In previous studies, the bacterium Tenacibaculum sp. strain Pbs-1 was isolated from diseased Akoya pearl oysters Pinctada fucata, and a rapid, specific, and sensitive loop-mediated isothermal amplification (LAMP) assay for detecting this pathogen was established. This technology has considerable potential for routine diagnosis of strain Pbs-1 in oyster hatcheries and/or pearl farms; therefore, it is vital to identify substances in environmental samples that might inhibit LAMP and to find additives that can reduce the inhibition. In this study, we investigated the effects of six chemicals or proteins, otherwise known as conventional PCR inhibitors, on LAMP, using the DNA of strain Pbs-1 as template: humic acid, urea, iron (III) chloride hexahydrate, melanin, myoglobin, and Ethylenediamine-N,N,N′,N′-tetraacetic acid, disodium salt, dihydrate (EDTA; pH 6.5). Next, to reduce the effects of identified inhibitors, we tested the addition of bovine serum albumin (BSA) or T4 gene 32 protein (gp32) to the LAMP assay. When 50 ng of DNA template was used, 4 ng/μL of humic acid, 0.05% melanin, and 10 mM of EDTA (pH 6.5) inhibited the LAMP reaction, whereas myoglobin, urea, and FeCl3 had no effect. When 50 pg of DNA template was used, 4 ng/μL of humic acid, 0.05% melanin, 4 μg/μL of myoglobin, 10 μg/μL of urea, and 10 mM of EDTA inhibited the LAMP reaction. Thus, it was shown that the gene-amplification inhibitory effect of melanin, humic acid, and urea could be reduced by adding BSA or gp32 to the LAMP reaction mixture. This technique could be applied as part of a protocol to prevent mass mortalities of pearl oysters; moreover, the results enhance our knowledge about substances that inhibit LAMP and methods to reduce the inhibition, which have rarely been reported.

Metabolic fingerprints and immune response of silverlip pearl oysters (Pinctada maxima) under low salinity event

Marine ecosystems are prone to hyposalinity events due to rainfall and freshwater inflow, which has led marine bivalves to develop mechanisms to adapt to salinity fluctuations over a long period of evolution. This study conducted a comprehensive assessment to examine the physiological impact of a low salinity event on Pinctada maxima, which included measure- ments of haemolymph electrolytes, haemocytes functions, and non-targeted metabolomics analyses. A total of 92 metabolites were detected in the gill tissue of P. maxima. Six hours after exposure, low salinity stress impacted five metabolomic pathways, with succinic acid and alanine being highly upregulated. However, the electrolyte and the measured immune response were unaffected at that sampling time. After 24 h of exposure to low salinity conditions, a significant decrease in the concentrations of key electrolytes in the haemolymph was observed, including Na+, Cl−, K+, and Ca2+. Furthermore, the immune responses of the pearl oysters were affected by hypo-salinity stress. Additionally, the total haemocyte count decreased significantly, and some important immune functions, such as the phagocytosis capacity and the intracellular oxidative activity were suppressed. After 72 h of exposure to low salinity, an increase in alanine and succinic acid levels indicated the onset of anaerobic metabolism. The prolonged hyposalinity exposure (72 h) impacted nine metabolomic pathways. The downregulation of adenosine suggested the impairment of the cyclic adenosine monophosphate (cAMP) pathway, which inhibits ambient inorganic ions entering the gill cells. This study identified potential metabolic biomarkers such as succinic acid in oysters exposed to hyposalinity stress.

Antioxidant Activity and Oxidative Damage Associated with Seeding Surgery for Pearl Culture in the Winged Pearl Oyster Pteria sterna.

To evaluate the antioxidant activity and oxidative damage by relaxing, wounding, and seeding of a saibo of different origin on Pteria sterna hosts, five oyster treatments were included: (1) relaxed (REL) but neither wounded nor seeded; (2) relaxed and wounded (WOU) but not seeded; (3) relaxed, wounded, and seeded with an allograft (ALL); (4) relaxed, wounded, and seeded with an autograft (AUT); and (5) unrelaxed, unwounded, and unseeded as control (CTR). Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and thiobarbituric acid (TBARS) activity were quantified between 3 and 24 h post-seeding. Compared to the CTR oysters, which did not suffer oxidative stress, SOD activity significantly decreased in the gonad and digestive gland in all treatments and decreased in mantle tissue in AUT oysters; this indicates that the entire process of preparing oysters for pearl culture (relaxing, wounding, and seeding) generates oxidative stress in the host. CAT was not a sensitive enzyme for measuring the short-term response of oysters to the wounding-seeding processes but rather a more prolonged or chronic stress. Similar to SOD, the lowest GPx and TBARS activity in seeded oysters evidenced their susceptibility to oxidative stress and damage, particularly in the WOU treatment. Evidence from this study indicates that SOD is a more sensitive enzyme for measuring the short-term response of the host oyster to the wounding and seeding of a saibo. It is also clear that the host undergoes stress at all stages of the pearl culture process, mostly during gonad wounding and regardless of the origin of saibo.

The effects of pearl oyster shell-derived bone grafts on TNF-alfa levels: An in vivo study in Cavia porcellus

Abstract Objective: This study aims to assess the effect of bone graft materials derived from pearl oyster shells on the tumor necrosis factor-alpha (TNF-α) level. Methods: This research utilized a bone graft containing hydroxyapatite Pinctada maxima (HPM) powder obtained from pearl oyster shells. Material testing was conducted on 30 male guinea pigs aged 8-10 weeks, divided into three groups: the negative control group, the group treated solely with HPM, and the group treated with HPM combined with platelet-rich fibrin (PRF). Each treatment group was observed for 7 and 14 days. The results were analyzed using One-way ANOVA with a significance level of p<0.05. Results: The findings demonstrated a decrease in the average TNF-α levels across all groups. On observation days 7 and 14, the HPM group exhibited a significant difference compared to the negative control group, indicating reduced TNF-α levels. However, no significant difference was observed when comparing the HPM group with the HPM and PRF groups. Conclusions: Bone grafts derived from pearl oyster shells possess high levels of hydroxyapatite, and the addition of PRF into the bone graft effectively reduces TNF-α.

Oral Administration of Nacre Extract from Pearl Oyster Shells Has Anti-Aging Effects on Skin and Muscle, and Extends the Lifespan in SAMP8 Mice.

Pearl oysters have been extensively utilized in pearl production; however, most pearl oyster shells are discarded as industrial waste. In a previous study, we demonstrated that the intraperitoneal administration of pearl oyster shell-derived nacre extract (NE) prevented d-galactose-induced brain and skin aging. In this study, we examined the anti-aging effects of orally administered NE in senescence-accelerated mice (SAMP8). Feeding SAMP8 mice NE prevented the development of aging-related characteristics, such as coarse and dull hair, which are commonly observed in aged mice. Additionally, the NE mitigated muscle aging in SAMP8 mice, such as a decline in grip strength. Histological analysis of skeletal muscle revealed that the NE suppressed the expression of aging markers, cyclin-dependent kinase inhibitor 2A (p16) and cyclin-dependent kinase inhibitor 1 (p21), and increased the expression of sirtuin1 and peroxisome proliferator-activated receptor gamma coactivator 1 (PGC1)- α, which are involved in muscle synthesis. These findings suggest that the oral administration of NE suppresses skeletal muscle aging. Moreover, NE administration suppressed skin aging, including a decline in water content. Interestingly, oral administration of NE significantly extended the lifespan of SAMP8 mice, suggesting that its effectiveness as an anti-aging agent of various tissues including skeletal muscle, skin, and adipose tissue.

The role of Pm-miR-184-3p in regulating the immune response in the pearl oyster Pinctada fucata martensii

microRNAs are a class of non-coding RNAs with post-transcriptional regulatory functions in eukaryotes. In our previous study, miR-184-3p was identified in the hemocyte transcriptome of Pinctada fucata martensii (Pm-miR-184-3p), and its expression was shown to be up-regulated following transplantation surgery; however, its role in regulating transplantation immunity has not yet been clarified. Here, the role of Pm-miR-184-3p in regulating the immune response of P. f. martensii was studied. The expression of Pm-miR-184-3p increased following the stimulation of pathogen-associated molecular patterns, and Pm-miR-184-3p overexpression increased the activity of antioxidant-related enzymes, such as superoxide dismutase and catalase. Transcriptome analysis obtained 1096 differentially expressed genes (DEGs) after overexpression of Pm-miR-184-3p, and these DEGs were significantly enriched in conserved pathways such as the Cell cycle pathway and NF-kappa B signaling pathway, as well as GO terms including base excision repair, cell cycle, and DNA replication, suggesting that Pm-miR-184-3p could enhance the inflammation process. Target prediction and dual luciferase analysis revealed that pro-inflammatory related genes Pm-TLR3 and Pm-FN were the potential target of Pm-miR-184-3p. We speculate that Pm-miR-184-3p may utilize negative regulation of target genes to delay the activation of corresponding immune pathways, potentially preventing excessive inflammatory responses and achieving a delicate balance within the organism. Overall, Pm-miR-184-3p play a key role in regulating cellular responses to transplantation. Our findings provide new insights into the immune response of P. f. martensii to transplantation.

Pearl Fisheries in South Asia: Archaeological Evidence from Pre-Colonial and Colonial Shell Middens around the Gulf of Mannar in Sri Lanka

AbstractThis study investigates pre-colonial and colonial-era pearl fisheries in Sri Lanka using archaeological data from the Gulf of Mannar (GoM). Following surface surveys, test pits were excavated in three pearl oyster middens and one coastal settlement. The archaeomalacological study of the molluscan remains from the middens confirms that the pearl oyster (Pinctada fucata) was the predominant species exploited, providing a clear picture of the pearl oyster bank ecosystems used at the industrial level. Morphometric analysis of the pearl oysters allows for an assessment of exploitation pressure. Ceramic and radiocarbon analyses date the high impact of the pearl industry from the tenth to the nineteenth centuries. The study of by-catch fauna provides insight into pearl bank ecosystems over time. These archaeological findings highlight the impact of industrial extractions on a single species.

Probiotic Potential of Lactobacillus Strains Isolated from the Pearl Oyster (Pinctada radiata)

Probiotic Potential of Lactobacillus Strains Isolated from the Pearl Oyster (Pinctada radiata)

A novel birnavirus identified as the causative agent of summer atrophy of pearl oyster (Pinctada fucata (Gould)).

The Akoya pearl oyster (Pinctada fucata (Gould)) is the most important species for pearl cultivation in Japan. Mass mortality of 0-year-old juvenile oysters and anomalies in adults, known as summer atrophy, have been observed in major pearl farming areas during the season when seawater temperatures exceed about 20 °C since 2019. In this study, we identified a novel birnavirus as the pathogen of summer atrophy and named it Pinctada birnavirus (PiBV). PiBV was first presumed to be the causative agent when it was detected specifically and frequently in the infected oysters in a comparative metatranscriptomics of experimentally infected and healthy pearl oysters. Subsequently, the symptoms of summer atrophy were reproduced by infection tests using purified PiBV. Infection of juvenile oysters with PiBV resulted in an increase in the PiBV genome followed by the atrophy of soft body and subsequent mortality. Immunostaining with a mouse antiserum against a recombinant PiBV protein showed that the virus antigen was localized mainly in the epithelial cells on the outer surface of the mantle. Although the phylogenetic analysis using maximum likelihood method placed PiBV at the root of the genus Entomobirnavirus, the identity of the bi-segmented, genomic RNA to that of known birnaviruses at the full-length amino acid level was low, suggesting that PiBV forms a new genus. The discovery of PiBV will be the basis for research to control this emerging disease.

Effect of nacre extract from pearl oyster shells against behavioral and psychological symptoms of dementia in senescence-accelerated mouse P8 (SAMP8)

Pearls have been used in traditional Chinese medicine for the treatment of myopia, epilepsy, and palpitations. We previously demonstrated that the intraperitoneal administration of nacre extract from pearl oyster shells prevents lipopolysaccharide-induced anxiety and depression-like behavior. In this study, nacre extract was orally administered to senescence-accelerated mice-prone 8 (SAMP8) mice at 125 ppm and 250 ppm for 26 weeks. Behavioral tests were performed at 28 weeks of age, followed by molecular analysis at 31 weeks. Administration of nacre extract suppressed anxiety and depressive behavior in SAMP8 mice. In addition, nacre extract ameliorated aggressive behavior and disruption of circadian rhythms in SAMP8 mice. Histological analysis showed that nacre extract prevented neurodegeneration in the hippocampus. RNA sequencing analysis of the brain revealed that nacre extract suppressed endoplasmic reticulum (ER) stress. In addition, nacre extract also suppressed increase in expression of inflammation-related proteins, such as tumor necrosis factor-alpha and interleukin-1. These results suggest that the oral administration of nacre extract suppresses behavioral alterations by suppressing ER stress and inflammation associated with aging in SAMP8 mice.

Characterization, functional analysis, and expression of TNFR16 from Pinctada fucata martensii in response to exogenous stimuli and RNA interference

Marine pearls from Pinctada fucata martensii are valuable for decoration, skin care, and health care, but their quality and yield can be threatened by diseases, pearl nuclei transplantation rejection, and other external factors. The present study investigated the immune function of the tumor necrosis factor receptor (TNFR) gene in P. f. martensii to better understand immune responses related to nuclei rejection. The PmTNFR16 cDNA sequence obtained from cloning was 1675 bp in length and encoded 406 amino acid residues. Sequence analysis revealed that PmTNFR16 comprises one signal peptide domain, four TNFR domains, one transmembrane domain, and one death domain. Expression analysis revealed PmTNFR16 was highly expressed in the mantle and significantly up-regulated after exogenous stimuli. The effects of PmTNFR16 on immune effector molecules within the NF-κB signaling pathway were further explored using RNA interference probes for PmTNFR16. Pre-treatment of pearl oysters with dsRNA-PmTNFR before nucleus-insertion resulted in decreased expression of PmTNFR16 by 53 % at 24 h (p<0.01). Similarly, the expression of the NF-κB signaling pathway immune-related genes TRAF2, TRADD, IKK, NIK, and NF-κB significantly decreased at 24 h. The results indicated that PmTNFR16 was successfully silenced as were the expression of immune-related genes in the downstream NF-κB signaling pathway. Overall, these results indicate that PmTNFR16 may be involved in the immune response of the host organism by mediating the NF-κB signaling pathway. These observations provide a framework for future studies of the immune mechanisms of pearl oysters.

Electrical and thermal properties of bio-inspired laminated Cu/Ti3SiC2/C composites reinforced by graphene nanoplatelets

Taking inspiration from the unique hierarchical structure of bio-materials like pearl oyster, this study employs bio-inspired composites technological route using flake powder metallurgy to prepare laminated Cu/Ti3SiC2/C composites. The prepared composites have clear laminated structure, uniform dispersion of reinforcing phase and clean interface. At a Cu-plated GNPs content of 0.5 wt%, the composites displayed tensile strength of 300.65 MPa, compressive strength of 575.32 MPa, electrical conductivity of 26.31 MS/m, and thermal conductivity of 285.87 W·m−1·K−1. The reinforcement of composites is caused by the load transfer effect of the laminated structure. During the loading process, the laminated structure creates multiple paths for deflecting cracks, which leads to the redistribution of the applied load. Moreover, the homogeneous distribution of GNPs throughout the laminated structure extends the mean free path of both free electrons and phonons within the material, consequently bolstering the electrical and thermal conductivity of the composites.

Structural and Functional Analysis of the Amorphous Calcium Carbonate-Binding Protein Paramyosin in the Shell of the Pearl Oyster, Pinctada fucata.

Amorphous calcium carbonate (ACC) is an important precursor phase for the formation of aragonite crystals in the shells of Pinctada fucata. To identify the ACC-binding protein in the inner aragonite layer of the shell, extracts from the shell were used in the ACC-binding experiments. Semiquantitative analyses using liquid chromatography-mass spectrometry revealed that paramyosin was strongly associated with ACC in the shell. We discovered that paramyosin, a major component of the adductor muscle, was included in the myostracum, which is the microstructure of the shell attached to the adductor muscle. Purified paramyosin accumulates calcium carbonate and induces the prism structure of aragonite crystals, which is related to the morphology of prism aragonite crystals in the myostracum. Nuclear magnetic resonance measurements revealed that the Glu-rich region was bound to ACC. Activity of the Glu-rich region was stronger than that of the Asp-rich region. These results suggest that paramyosin in the adductor muscle is involved in the formation of aragonite prisms in the myostracum.

Liquid Motion: Reimaging Maritime History through an African Lens

“Liquid Motion” examines how African women and men perceived, understood, and interacted with oceans and rivers through swimming, underwater diving, surfing, canoe-making, and canoeing. Africans inspire us to rethink assumptions about maritime history, by considering maritime traditions absent in the Western lexicon, like harnessing wave energy to transport goods through the surf or swimming into the depths to salvage goods from shipwrecks or harvest pearl oysters. Enslaved Africans carried these traditions to the Americas, where they used them to benefit their exploited lives and enslavers exploited them to generate wealth.

The Influence of Transportation Infrastructure on Community Economic Improvement in Makassar Island, Baubau City

Makassar Island is an island area in Baubau City, Southeast Sulawesi Province which is included in the Kokalukuna District. It consists of two villages, namely Liwuto Village and Sukanayo Village with a land area of 1.04 km2. In general, 70% of the livelihood of residents on Makassar Island is fishing, seaweed farming, pearl oysters, fish marketing businesses, and crossing services (sea taxis). As an island area, the movement of both goods and people within this region is closely related to the infrastructure of docks and highways. This study aims to analyze the influence of transportation infrastructure in improving the economy of the people on Makassar Island. The analysis methods used in this study are Likert analysis, correlation analysis and simple linear regression analysis. Based on the results of the analysis, it was found that transportation infrastructure has a strong relationship with the community's economy. Furthermore, the results of the study are presented in the form of a crosstab between each indicator of the transportation infrastructure variable (X) with the economic variable of the community (Y) on the island.

Influence of geographical origin and shell positions on nacre color and thickness in mabé pearl oysters Pteria penguin

To examine variations in nacre color and thickness in Pteria penguin oysters obtained from Tonga and Japan, a novel approach with a focus on their potential for mabé pearl production was used. Detailed analyses of color and microstructure revealed that the middle position of the right-valve nacre (position 1) primarily showed a reduction in yellow pigmentation. This was indicated by a decrease in the yellowness index (YI) and an increase in the whiteness index (WI). In contrast, the anterior and middle positions of the left-valve nacre (positions 2 and 3) consistently displayed a higher level of yellow pigmentation. A comparison of oysters from different geographical origin found that Tonga wild-caught oysters had significantly thicker nacre than Tonga culture-site oysters, suggesting the influence of probable genetic diversity and environmental factors on nacre formation. Notably, nacre at position 1 was generally thinner, while at positions 2 and 3 it was thicker, which would influence the resultant pearl quality and attributes. We demonstrate the significance of the nacre position on the valve for mabé pearl production: position 1 tended to produce white pearls with pronounced interference colors, whereas position 2 tended to yield gold pearls. Furthermore, our findings highlight the significant impact of the oysters' geographic origin on mabé pearl color and quality. This study emphasizes the importance of considering the position of shell nacre and the potential influences of the oysters' genetic origins and environmental factors on mabé pearl formation.

Effects of titanium dioxide nanoparticle exposure on the gut microbiota of pearl oyster (Pinctada fucata martensii)

With the advancement of nanotechnology and the growing utilization of nanomaterials, titanium dioxide (TiO2) has been released into aquatic environments, posing potential ecotoxicological risks to aquatic organisms. In this study, the toxicological effects of TiO2 nanoparticles were investigated on the intestinal health of pearl oyster (Pinctada fucata martensii). The pearl oysters were subjected to a 14-day exposure to 5-mg/L TiO2 nanoparticle, followed by a 7-day recovery period. Subsequently, the intestinal tissues were analyzed using 16S rDNA high-throughput sequencing. The results from LEfSe analysis revealed that TiO2 nanoparticle increased the susceptibility of pearl oysters to potential pathogenic bacteria infections. Additionally, the TiO2 nanoparticles led to alterations in the abundance of microbial communities in the gut of pearl oysters. Notable changes included a decrease in the relative abundance of Phaeobacter and Nautella, and an increase in the Actinobacteria, which could potentially impact the immune function of pearl oysters. The abundance of Firmicutes and Bacteroidetes, as well as the expression of genes related to energy metabolism (AMPK, PK, SCS-1, SCS-2, SCS-3), were down-regulated, suggesting that TiO2 nanoparticles exposure may affect the digestive and energy metabolic functions of pearl oysters. Furthermore, the short-term recovery of seven days did not fully restore these levels to normal. These findings provide crucial insights and serve as an important reference for understanding the toxic effects of TiO2 nanoparticles on bivalves.

Environmental DNA metabarcoding for the detection of the silverlip pearl oyster (Pinctada maxima) offshore of Eighty Mile Beach in northwest Australia

Effective fishery management relies on accurate knowledge of the spatial distribution of stocks of target species to efficiently focus fishing efforts. However, the distribution and abundance of species such as the silverlip pearl oyster (Pinctada maxima) can be difficult to determine with traditional survey methods since populations are patchy, often occur in low densities, and individuals can be quite cryptic, living partially buried in sediment. Environmental DNA is increasingly used to survey marine species and has proven to be particularly useful in detecting rare or elusive organisms. Here, we compared the presence of silverlip pearl oysters detected via eDNA metabarcoding to traditional towed video surveys offshore of Eighty Mile Beach, northwest Australia. Seawater samples were collected at the end of towed video transects and sites were categorised into shallow (<40 m depth) or deep (40 m +) groups. We applied an oyster-specific 16S metabarcoding assay as well as a universal 18S assay to identify potential indicator taxa associated with the presence of pearl oysters. Using the 16S assay, we detected silverlip pearl oysters at three of the 12 sites sampled, and found that eDNA records generally matched towed video transect data, with both survey methods detecting the target species predominantly in inshore waters at depths <40 m. Our results indicate that metabarcoding of seawater eDNA can be applied to detect cryptic species of commercial importance but highlight limitations around detecting organisms at very low abundances.