Species-specific Probes Research Articles

A Comparison of Peripheral Blood Smears, Autologous Cell Cultures, and Reverse Line Blot Hybridisation in Screening for Anaplasma/Ehrlichia in Roaming Dogs and Symptomatic Dogs in Trinidad.

This study compared two methods to detect cases of canine ehrlichiosis in a field setting. One method was a polymerase chain reaction for the 16S rRNA gene followed by reverse line blot hybridisation with genera and species-specific probes for Anaplasma/Ehrlichia. The second method was an autologous cell culture of peripheral leucocytes isolated from heparinised blood and maintained in a homologous canine serum in Dulbecco’s Modified Eagle medium without antibiotics. The cultures were examined under light microscopy for inclusion bodies after 48 h. Leucocytes were successfully propagated for 20 of the 34 samples submitted for autologous cell culture. Inclusion bodies were observed after cell culture in leucocytes of eight dogs. Two dogs were positive to the Anaplasma/Ehrlichia genera probe and six dogs were positive to the E. canis probe after reverse line blot hybridisation. There was acceptable agreement between reverse line blot hybridisation and cell culture results. Both reverse line blot hybridisation and autologous cell cultures can be used to detect E. canis in subclinical and clinical cases of disease. A definitive diagnosis of E. canis is best achieved by a combination of clinical signs, positive autologous cell culture, and reverse line blot hybridisation results.

Molecular quantification, a new strategy for quality control of Chinese patent medicine containing animal-derived crude drug: Qi She in Jinlong capsule as an example

Quality control for Chinese patent medicine (CPM) containing animal-derived crude drug(s) is rather difficult. The methods based on chemical composition analysis, which are commonly used in CPM consisted of plant-derived crude drugs, are often not applicable for CPM containing animal-derived crude drug, because the effective constituents of most animal-derived crude drugs remain unknown. Even if there are such methods, they are usually qualitative rather than quantitative, and the specificity is generally poor. Here we proposed a molecular quantification method for CPM containing animal-derived crude drug, based upon the hypothesis that the amount of remnant DNA fragments could reflect feeding quantity of the crude drugs and thus ensure the quality of the CPM. Take Jinlong capsule [a hepatocellular carcinoma-resisting Chinese patent medicine comprising of three fresh animal drugs, i.e. Shougong (Peking gecko, Gekko swinhonis), Qi She (sharp-snouted pitviper, Deinagkistrodon acutus), and Jinqian Baihua She (many-banded krait, Bungarus multicinctus)] as an example, we established a qPCR assay for Qi She in the capsule, which verified the feasibility of the quality control method based on molecular quantification. Species-specific primers and TaqMan probe for Qi She were designed, and the qPCR assay system was then established. The assay exhibited a good specificity; there’s a good linearity between Ct values and logarithm of the target amplicon copy numbers within the range of 8.8 × 101 to 8.8 × 106 copies/μL, and the limit of detection was 88 copies/μL. The method was validated through reproducibility, stability assessment. Recovery of spiked samples was between 91.59% and 101.69%. It was verified that the copy numbers reflected the original feeding amount of an animal-derived crude drug by self-made Jinlong capsules. The assay was successfully applied in Qi She-specific amplicon determination in 20 batches of Jinlong capsule. The study was expected to provide a new strategy for quality control of CPM containing animal-derived crude drug.

Developing, Modifying, and Validating a TaqMan Real-Time PCR Technique for Accurate Identification of Leishmania Parasites Causing Most Leishmaniasis in Iran.

Many laboratory methods are used to diagnose leishmaniasis because it is characterized by varied symptoms and caused by different Leishmania species. A quantitative real-time PCR method based on a TaqMan probe was developed and modified for accurate identification of human cutaneous leishmaniasis (caused by Leishmania major or Leishmania tropica) from endemic areas of Iran. Two gene regions of amino acid permease 3 (AAP3) and cytochrome oxidase II (COII) were considered. Six new sets of species-specific primers and probes were designed. A total of 123 samples were examined and employed to evaluate and validate real-time PCR. According to parasitic load of the genesig® Leishmania Advanced Standard Kit, a serial dilution of purified plasmid (2–2×107 copies/reaction) was prepared under the same conditions for both genes. Specific primers and probes were able to detect three and six parasite copies in AAP3 and COII genes, respectively, and were able to detect three copies of parasites for L. major and L. tropica. The sensitivities of the reference kit and our method were 98.7 and 98.1%, respectively, and specificity was 100% for detecting parasite genomes in all assays. Designed primers and probes performed well in terms of efficiency and regression coefficient. For AAP3 and COII genes, respectively, the linear log range was 7 and the correlation coefficient (R2) was 0.749 and 0.996 for the reference kit using the standard generated curve and 0.98 and 0.96 with serial dilutions of parasite DNA. This research detected L. major and L. tropica definitely and opens the horizon for the other scientists in the multiplex reactions in designing and optimization of the conditions in silico and in vivo.

TLR7 is expressed by support cells, but not sensory neurons, in ganglia

BackgroundToll-like receptor 7 (TLR7) is an innate immune receptor that detects viral single-stranded RNA and triggers the production of proinflammatory cytokines and type 1 interferons in immune cells. TLR7 agonists also modulate sensory nerve function by increasing neuronal excitability, although studies are conflicting whether sensory neurons specifically express TLR7. This uncertainty has confounded the development of a mechanistic understanding of TLR7 function in nervous tissues.MethodsTLR7 expression was tested using in situ hybridization with species-specific RNA probes in vagal and dorsal root sensory ganglia in wild-type and TLR7 knockout (KO) mice and in guinea pigs. Since TLR7 KO mice were generated by inserting an Escherichia coli lacZ gene in exon 3 of the mouse TLR7 gene, wild-type and TLR7 (KO) mouse vagal ganglia were also labeled for lacZ. In situ labeling was compared to immunohistochemistry using TLR7 antibody probes. The effects of influenza A infection on TLR7 expression in sensory ganglia and in the spleen were also assessed.ResultsIn situ probes detected TLR7 in the spleen and in small support cells adjacent to sensory neurons in the dorsal root and vagal ganglia in wild-type mice and guinea pigs, but not in TLR7 KO mice. TLR7 was co-expressed with the macrophage marker Iba1 and the satellite glial cell marker GFAP, but not with the neuronal marker PGP9.5, indicating that TLR7 is not expressed by sensory nerves in either vagal or dorsal root ganglia in mice or guinea pigs. In contrast, TLR7 antibodies labeled small- and medium-sized neurons in wild-type and TLR7 KO mice in a TLR7-independent manner. Influenza A infection caused significant weight loss and upregulation of TLR7 in the spleens, but not in vagal ganglia, in mice.ConclusionTLR7 is expressed by macrophages and satellite glial cells, but not neurons in sensory ganglia suggesting TLR7’s neuromodulatory effects are mediated indirectly via activation of neuronally-associated support cells, not through activation of neurons directly. Our data also suggest TLR7’s primary role in neuronal tissues is not related to antiviral immunity.

First report of Theileria buffeli/Theileria orientalis group and identification of piroplasms via Nested PCR-based RLB Hybridization assay in zebu cattle in the Western Highlands of Cameroon

Piroplasms infections are tick-borne diseases caused by haemoparasite of the genus Theileria or Babesia. They have a great impact on livestock production, especially cattle in sub-Saharan countries. However, data on the prevalence of bovine piroplasms and their genetic diversity are scanty in Cameroon. This study was aimed at highlight the species composition and determine the prevalence of piroplasms infecting cattle in the Western Highlands of Cameroon. To achieve this aim, blood samples from a total of 162 cattle were collected and examined using Reverse Line Blot hybridization (RLB) assay. The amplified hypervariable V4 region of the 18S rRNA gene of bovine piroplasms species, including Theileria parva, T. annulata, T mutans, T. velifera, T. buffeli/T. orientalis, T. taurotragi, Theileria sp (buffalo), Babesia bovis, B. bigemina, B. divergens, B. major and B. occultans was hybridized against species-specific probes. RLB hybridization assay revealed the presence of four piroplasms species with the overall prevalence of infection of 82.1%. Theileria velifera (71.6%) was the most prevalent species followed by Theileria mutans (43.21%), Theileria buffeli/T. orientalis (5.55%) and Babesia bigemina (3.7%). However, the study provided the first molecular evidence for the presence of T. buffeli/T. orientalis group species in cattle in Cameroon. Higher overall prevalence of infection of tick-borne pathogens was observed in this study area as well as the increase in prevalence and widespread of T. velifera and the observance of a new species of piroplasms. These results are an indication that special attention should be given to epizootiological investigations alongside well-adopted control programs.

Infection of oomycetes and bacteria associated with their specific colocalization in chum salmon eggs

To elucidate the localization of oomycetes (water molds) and bacteria in a single egg of chum salmon, Oncorhynchus keta , eggs infected with oomycetes were examined by bright-field microscopy, scanning electron microscopy (SEM), and fluorescent in situ hybridization (FISH) using oomycete universal and bacterial species-specific probes. Furthermore, DNA barcoding for oomycetes and bacteria was performed on the chorion and cytoplasm of the eggs using genomic DNA extracted from the sections of eggs used for FISH analyses. Using bright-field microscopy and SEM, it was shown that oomycete hyphae penetrated the chorion and invaded the cytoplasm of the eggs. Based on DNA barcoding, two oomycetes ( Pythium monospermum and Pythium sp. SE-OP1), and three bacterial species ( Flavobacterium plurextorum , Flavobacterium sp. SE-BF1 and Undibacterium pigrum ) were predominantly detected in the infected eggs. FISH analyses indicated that localization of the oomycetes was not noticeable different between the chorion and cytoplasm of the eggs. In contrast, FISH analyses using bacterial species-specific probes demonstrated clear differences in their localization. Flavobacterium sp. SE-BF1 and U. pigrum were predominantly found inside the chorion and around the oomycete hyphae invading the cytoplasm, respectively. These results are the first to indicate the possibility that U. pigrum has a specific relationship with oomycetes and coordinately invades eggs with oomycetes. However, it seems that Flavobacterium sp. SE-BF1 invades by a mechanism different from that of U. pigrum . Although this study is preliminary and the interaction between oomycetes and U. pigrum associated with chum salmon egg infection needs to be elucidated in detail, this finding may provide new insights into the infection mechanism of bacteria in salmon eggs, which is a longstanding problem in salmon hatcheries. • Oomycete hyphae penetrate the chorion and invad the cytoplasm of chum salmon eggs • Specific distribution of a bacterial species around the oomycete hyphae in the eggs • Possibility of a specific relationship between oomycetes and the bacterial species

Molecular diagnostics of the pigeon pea cyst nematode, Heterodera cajani Koshy, 1967, using real-time PCR

Summary The pigeon pea cyst nematode, Heterodera cajani, is an important nematode pest of pigeon pea that is present in all major growing regions of this crop in India and reported from Pakistan, Egypt and Myanmar. In this study, a new real-time PCR assay for detection of H. cajani using a species-specific primer and a TaqMan probe was developed. The primers and a probe were designed to amplify the COI gene fragment. The specificity of the primer-probe set was tested in singleplex or multiplex reactions against target and non-target nematodes. In multiplex real-time PCR experiments with the specific and universal primer-probe sets, the signals were simultaneously observed for COI and D3 of 28S rRNA target genes. The results showed that the real-time PCR assay with species-specific primer and probe was sensitive enough to detect H. cajani DNA extracted from 0.003 egg or second-stage juvenile.

A DNA microarray for the authentication of giant tiger prawn (Penaeus monodon) and whiteleg shrimp (Penaeus (Litopenaeus) vannamei): a proof-of-principle

This proof-of-principle study describes the development of a rapid and easy-to-use DNA microarray assay for the authentication of giant tiger prawns and whiteleg shrimp. Following DNA extraction and conventional end-point PCR of a 16S rDNA segment, the PCR products are hybridised to species-specific oligonucleotide probes on DNA microarrays located at the bottom of centrifuge tubes (ArrayTubes) and the resulting signal patterns are compared to those of reference specimens. A total of 21 species-specific probes were designed and signal patterns were recorded for 47 crustacean specimens belonging to 16 species of seven families. A hierarchical clustering of the signal patterns demonstrated the specificity of the DNA microarray for the two target species. The DNA microarray can easily be expanded to other important crustaceans. As the complete assay can be performed within half a day and does not require taxonomic expertise, it represents a rapid and simple alternative to tedious DNA barcoding and could be used by crustacean trading companies as well as food control authorities for authentication of crustacean commodities.Graphical abstract

A novel duplex ddPCR assay for detection and differential diagnosis of Ascaridia galli and Heterakis gallinarum eggs from chickens feces

Since the EU ban on battery cages, many studies have listed Ascaridia galli and Heterakis gallinarum as the most common roundworms in the European laying hen population. A complicating factor is that the eggs of these parasites are almost identical. Thus, lack of molecular diagnostic approaches has driven epidemiological studies to take on necropsy for species discrimination, which is labor and cost intensive. Here, we describe a novel diagnostic tool based on droplet digital PCR for simultaneous identification and absolute quantification of the eggs of both of these ascarids in chickens’ droppings using two different genus-specific primer-probe sets targeting the second internal transcribed spacer region (ITS-2) in the nuclear ribosomal (rRNA) gene array. No cross-reaction was observed when different combinations of DNA and species-specific primers and probes were tested. The lowest obtained frequency threshold for the detection of H. gallinarum in the presence of a constant A. galli DNA concentration was determined to be 0.8 %. After validation, we used the assay to analyze field samples collected from several Swedish laying hen farms. Out of 134 samples, 86 (64 %) were positive for A. galli while 11 (8.3 %) samples were positive for H. gallinarum. These samples were initially analyzed with flotation technique for detection of ascarid eggs. The results of the Cohen’s kappa indicated substantial agreement (85.8 %) between the two tests. In conclusion, we have validated a novel molecular-based diagnostic tool for quantification and differentiation between intestinal parasites of major importance in chickens with high precision. Although this study focuses on identification of parasites of laying hens, the findings may well have a bearing on all types of chicken production systems. The present study lays the groundwork for future research into epidemiology of these two important chicken parasite species.

Novel rRNA-depletion methods for total RNA sequencing and ribosome profiling developed for avian species

Deep sequencing of RNAs has greatly aided the study of the transcriptome, enabling comprehensive gene expression profiling and the identification of novel transcripts. While messenger RNAs (mRNAs) are of the greatest interest in gene expression studies as they encode for proteins, mRNAs make up only 3 to 5% of total RNAs, with the majority comprising ribosomal RNAs (rRNAs). Therefore, applications of deep sequencing to RNA face the challenge of how to efficiently enrich mRNA species prior to library construction. Traditional methods extract mRNAs using oligo-dT primers targeting the poly-A tail on mRNAs; however, this approach is not comprehensive as it does not capture mRNAs lacking the poly-A tail or other long non-coding RNAs that we may be interested in. Alternative mRNA enrichment methods deplete rRNAs, but such approaches require species-specific probes and the commercially available kits are costly and have only been developed for a limited number of model organisms. Here, we describe a quick, cost-effective method for depleting rRNAs using custom-designed oligos, using chickens as an example species for probe design. With this optimized protocol, we have not only removed the rRNAs from total RNAs for RNA-seq library construction but also depleted rRNA fragments from ribosome-protected fragments for ribosome profiling. Currently, this is the only rRNA depletion-based method for avian species; this method thus provides a valuable resource for both the scientific community and the poultry industry.

Semi-quantitative evaluation of Babesia bovis and B. bigemina infection levels estimated by HRM analysis

Bovine babesiosis is economically the most important arthropod-borne disease of cattle worldwide. The most significant damage caused by bovine babesiosis is attributed to Babesia bovis due to its higher pathogenicity. This study aimed to develop a real-time PCR method followed by HRM (high-resolution melting) analysis for the simultaneous detection of B. bovis and B. bigemina, enabling a semi-quantitative analysis of Babesia levels using a single-tube reaction. The HRM was compared with real-time PCR using species-specific hydrolysis probes. The HRM analysis allowed to differentiate both Babesia species and was sensitive in the detection and differentiation of 10% for each Babesia species in the sample. Our results suggest the use of this method to estimate the prevalence of infections by B. bovis or B. bigemina as an alternative to the methods of absolute quantification by real-time PCR since it neither requires precise estimates of the number of DNA loads nor the construction of calibration curves. The simultaneous detection of the two Babesia species can be used to characterise the infection levels in cattle populations from different geographical regions, allowing a better control of these diseases.

Quantification of Actaea racemosa L. (black cohosh) from some of its potential adulterants using qPCR and dPCR methods

The demand for popular natural health products (NHPs) such as Black Cohosh is increasing considerably, which in turn challenges quality assurance (QA) throughout the supply chain. To detect and quantify the target species present in a given NHP, DNA-based molecular techniques such as Real-time quantitative PCR (qPCR) and digital PCR (dPCR) are standard tools in the food and pathogen testing industries. There is a gap in the literature concerning validated quantitative PCR methods for botanicals that can be utilized for QA and good manufacturing practices. The objective of this study is to develop an efficient quantification method using qPCR and dPCR techniques for the detection and quantification of Actaea racemosa (Black cohosh) NHPs from its potential adulterants. These developed methods are validated for applicability on commercial NHPs. Species-specific hydrolysis probe assays were designed to analyze the black cohosh NHPs using qPCR and dPCR techniques. The results confirmed that the developed qPCR and dPCR methods are highly precise for identifying and quantifying black cohosh NHPs, indicating their potential applicability in future routine industrial and laboratory testing. This enables a single qPCR test to determine not only the presence of a specific botanical, but also the amount when mixed with an adulterant.

Character-based identification key for commercially important fishes of Pulicat lake: tool for conservation and management

Fishes are an important group of vertebrates in the animal world and make a significant contribution to global biodiversity. Fish is used as a source of food and contains many essential vitamins and fatty acids. The study of fish and their stability is important because, from year to year, fish stocks are often very important. For the conservation and management of these dwindling resources, correct identification of species is a prerequisite. Character-based methods of identification are of considerable use in this context, which classify specimens into species using classification rules that compactly describe species in terms of key diagnostic nucleotides in the gene sequences chosen. In this study, a total of 56 species of fishes distributed in Pulicat lake waters is taken as the target group. Mitochondrial CO1 sequences of each species were downloaded and modified. The species-specific diagnostic nucleotides for the selected group of species were identified using the BLOG version 2.0 software. Species-specific probes with a length range of 18–37 bp were designed on the basis of identified diagnostic nucleotide sites. The method is an effective tool for quickly and easily obtaining a significant amount of reliable information and could be used for forensic applications and conservation of fishes in Pulicat Lake.

Simultaneous quantification of the most common and proteolytic Pseudomonas species in raw milk by multiplex qPCR

The heat-stable peptidase AprX, secreted by psychrotolerant Pseudomonas species in raw milk, is a major cause of destabilization and premature spoilage of ultra-high temperature (UHT) milk and milk products. To enable rapid detection and quantification of seven frequent and proteolytic Pseudomonas species (P. proteolytica, P. gessardii, P. lactis, P. fluorescens, P. protegens, P. lundensis, and P. fragi) in raw milk, we developed two triplex qPCR assays taking into account species-dependent differences in AprX activity. Besides five species-specific hydrolysis probes, targeting the aprX gene, a universal rpoB probe was included in the assay to determine the total Pseudomonas counts. For all six probes, linear regression lines between Cq value and target DNA concentration were obtained in singleplex as well as in multiplex approaches, yielding R2 values of > 0.975 and amplification efficiencies of 85–97%. Moreover, high specificity was determined using genomic DNA of 75 Pseudomonas strains, assigned to 57 species, and 40 other bacterial species as templates in the qPCR. Quantification of the target species and total Pseudomonas counts resulted in linear detection ranges of approx. 103–107 cfu/ml, which correspond well to common Pseudomonas counts in raw milk. Application of the assay using 60 raw milk samples from different dairies showed good agreement of total Pseudomonas counts calculated by qPCR with cell counts derived from cultivation. Furthermore, a remarkably high variability regarding the species composition was observed for each milk sample, whereby P. lundensis and P. proteolytica/P. gessardii were the predominant species detected.Key points• Multiplex qPCR for quantification of seven proteolytic Pseudomonas species and total Pseudomonas counts in raw milk• High specificity and sensitivity via hydrolysis probes against aprX and rpoB• Rapid method to determine Pseudomonas contamination in raw milk and predict spoilage potential

A DNA microarray assay for authenticating five important marine mammal species in food and feed

Material identification in processed and unprocessed food and feed is crucial for ensuring the safety and hygiene of food and feed products. Therefore, to identify possible marine mammal components in feed, we study developed a DNA microarray with species-specific oligonucleotide probes that enable the rapid identification of five important marine mammal species (dolphins, seals, sea lions, white whales, and finless porpoises). The assay was tested using five target marine mammal species, and the probe patterns were compared with those of three fish meals (for feed) to see if they contained traces of marine mammals. All five marine mammal species could be distinguished by the microarray, and no marine mammal-derived ingredients were detected in the three fish meals. This study indicates that DNA microarray-based detection is relatively easy and effective for identification of non-compliant marine mammal ingredients in seafood or feed.

Development of fluorescence in situ hybridization (FISH) probes to detect and enumerate Gambierdiscus species

Ciguatera poisoning (CP) is a syndrome caused by the bioaccumulation of lipophilic ciguatoxins in coral reef fish and invertebrates, and their subsequent consumption by humans. These phycotoxins are produced by Gambierdiscus spp., tropical epiphytic dinoflagellates that live on a variety of macrophytes, as well as on dead corals and sand. Recent taxonomic studies have identified novel diversity within the Gambierdiscus genus, with at least 18 species and several sub-groups now identified, many of which co-occur and differ significantly in toxicity. The ability to accurately and quickly distinguish Gambierdiscus species in field samples and determine community composition and abundance is central to assessing CP risk, yet most Gambierdiscus species are indistinguishable using light microscopy, and other enumeration methods are semi-quantitative. In order to investigate the spatial and temporal dynamics of Gambierdiscus species and community toxicity, new tools for species identification and enumeration in field samples are needed. Here, fluorescence in situ hybridization (FISH) probes were designed for seven species commonly found in the Caribbean Sea and Pacific Ocean, permitting their enumeration in field samples using epifluorescence microscopy. This technique enables the assessment of community composition and accurate determination of cell abundances of individual species. Molecular probes detecting G. australes, G. belizeanus, G. caribaeus, G. carolinianus, G. carpenteri, and the G. silvae/G. polynesiensis clade were designed using alignments of large subunit ribosomal RNA (rRNA) sequences. These probes were tested for specificity and cross-reactivity through experiments in which field samples were spiked with known concentrations of Gambierdiscus cultures, and analyzed to confirm that Gambierdiscus can be successfully detected and enumerated by FISH in the presence of detritus and other organisms. These probes were then used to characterize Gambierdiscus community structure in field samples collected from the Florida Keys and Hawai‘i, USA. The probes revealed the co-occurrence of multiple species at each location. Time-series FISH analyses of samples collected from the Florida Keys quantified seasonal shifts in community composition as well as fluctuations in overall Gambierdiscus cell abundance. Application of species-specific FISH probes provides a powerful new tool to those seeking to target individual Gambierdiscus species, including significant toxin-producers, in field populations. Moving forward, analysis of Gambierdiscus community composition across multiple environments and over time will also allow species dynamics to be linked to environmental parameters, improving our ability to understand and manage the current and changing risks of CP worldwide.

Assessment of fishery resources using environmental DNA: Small yellow croaker (Larimichthys polyactis) in East China Sea.

Species distribution monitoring and biomass assessment are crucial for fishery management and resource conservation. However, traditional methods such as motor trawling are costly and less effective than the novel environmental DNA (eDNA) approach. This study employs eDNA approach to investigate horizontal and vertical distributions of small yellow croaker (Larimichthys polyactis), an economically important species, in the East China Sea. The analysis of 171 eDNA samples collected from 44 stations using the species-specific primers and Taqman probe suggests a presence of small yellow croaker at 28 sampling layers in 44 stations. Significant differences in croaker eDNA concentrations were revealed among sampling stations and layers, consistent with previous findings through motor-trawl capture offshore and nearshore ichthyoplakton surveys, indicating small yellow croaker exhibits strong regional distribution and layer preference. In addition, we found a high eDNA concentration of small yellow croaker in the surface waters beyond the motor-trawl prohibition line, which confirms spawning grounds have been expanded from nearshore to offshore areas. Such expansion of spawning grounds could be a response by small yellow croaker to stressors such as overfishing, climate change, and nearshore environment contamination. To identify environmental variables potentially associated with small yellow croaker presence and absence, we conducted a correlation analysis between eDNA concentration and environmental variables, and the results provide a guideline for further investigation of fishery resources in the future. In conclusion, this study demonstrates the power of the eDNA approach in monitoring small yellow croaker at extensive geographic scales. The developed protocols and the findings are expected to assist in long-term monitoring and protection programs and benefit sustainable fishery in small yellow croaker.

Validation and Optimization of qPCR Method for Identification of Actaea racemosa (Black Cohosh) NHPs.

Actaea racemosa (black cohosh) herbal dietary supplements are commonly used to treat menopausal symptoms in women. However, there is a considerable risk of contamination of A. racemosa herbal products in the natural health product (NHP) industry, impacting potential efficacy. Authentication of A. racemosa products is challenging because of the standard, multi-part analytical chemistry methods that may be too costly and not appropriate for both raw and finished products. In this paper, we discuss developing and validating quick alternative biotechnology methods to authenticate A. racemosa herbal dietary supplements, based on the use of a species-specific hydrolysis PCR probe assay. A qPCR-based species-specific hydrolysis probe assay was designed, validated, and optimized for precisely identifying the species of interest using the following analytical validation criteria: (1) specificity (accuracy) in determining the target species ingredient, while not identifying other non-target species; (2) sensitivity in detecting the smallest amount of the target material; and (3) reliability (repeatability and reproducibility) in detecting the target species in raw materials on a real-time PCR platform. The results show that the species-specific hydrolysis probe assay was successfully developed for the raw materials and powders of A. racemosa. The specificity of the test was 100% to the target species. The efficiency of the assay was observed to be 99%, and the reliability of the assay was 100% for the raw/starting and powder materials. The method developed in this study can be used to authenticate and perform qualitative analysis of A. racemosa supplements.

A Robust Symbiotic Relationship Between the Ciliate Paramecium multimicronucleatum and the Bacterium Ca. Trichorickettsia Mobilis.

Close reciprocal interactions in symbiotic systems have suggested the holobiont concept, in which the host and its microbiota are considered as a single entity. Ciliates are known for their ability to form symbiotic associations with prokaryotes. Relationships between the partners in such systems vary from mutualism to parasitism and differ significantly in their robustness. We assessed the viability of the ciliate Paramecium multimicronucleatum and its ability to maintain its intranuclear endosymbiont Ca. Trichorickettsia mobilis (Rickettsiaceae) after treatment with antibiotics characterized by different mode of action, such as ampicillin, streptomycin, chloramphenicol, tetracycline. The presence of endosymbionts in the host cell was determined by means of living cell observations made using differential interference contrast or fluorescence in situ hybridization with the species-specific oligonucleotide probe (FISH). Administration of antibiotics traditionally used in treatments of rickettsioses, tetracycline and chloramphenicol, depending on the concentration used and the ciliate strain treated, either caused death of both, infected and control cells, or did not affect the ability of the host to maintain the intranuclear endosymbiont. The surviving cells always manifested motile bacteria in the macronucleus. Streptomycin treatment never led to the loss of endosymbionts in any of the four infected strains, and nearly all ciliates remained viable. Ampicillin treatment never caused host cell death, but resulted in formation of filamentous and immobile oval bacterial forms. Under repeated ampicillin treatments, a part of endosymbionts was registered in the host cytoplasm, as evidenced both by FISH and transmission electron microscopy. Endosymbionts located in the host cytoplasm were enclosed in vacuoles, apparently, corresponding to autophagosomes. Nevertheless, the bacteria seemed to persist in this compartment and might cause relapse of the infection. Although the antibiotic sensitivity profile of Trichorickettsia seems to resemble that of other representatives of Rickettsiaceae, causative agents of severe diseases in humans, neither of the antibiotic treatments used in this study resulted in an aposymbiotic cell line, apparently, due to the protists’ sensitivity to tetracyclines, the drugs of preference in rickettsiosis treatment. The observed robustness of this symbiotic system makes it a good model for further elaboration of the holobiont concept.

Assessment of fishery resources using environmental DNA: The large yellow croaker (Larimichthys crocea) in the East China Sea

Fisheries management and resource conservation rely upon accurate information on the distribution and abundance of major species. The large yellow croaker (Larimichthys crocea) was once one of the most economically important marine fish in East Asia; however, its wild fishery resource has collapsed since the 1980s. Until recently, the natural resources of L. crocea started to increase due to the practice of stock enhancement and summer fishing moratorium. In this study, we developed the species-specific primers and probe and investigated the distribution and abundance of L. crocea using the environmental DNA (eDNA) approach. We collected 171 samples from 44 stations (190,467 km2 sea areas) in the East China Sea and detected 96 positive eDNA samples of L. crocea (56 %) at 29 stations (66 %). We found a significant difference in eDNA concentrations among different stations, indicating a regional distribution in L. crocea. We observed vertical variations in the eDNA concentrations of L. crocea among different water layers and detected the eDNA signals in water depths around 40 m and the bottom at multiple stations. We discovered eDNA hotspots with high concentrations in the Yushan fishing ground, which suggesting an offshore spawning ground might have been established possibly in response to nearshore environmental pollution and overfishing. We evaluated the effect of environmental variables on the distribution of L. crocea, found total depth and temperature were significantly correlated with the eDNA presence and concentration, and built a general linear model for the presence or absence prediction of L. crocea with environmental parameters. This study demonstrates the robustness of the eDNA approach in investigating and monitoring natural resources of a crucial marine fish and thus contributes to the long-term assessment of stocking and other strategies in fisheries enhancement.