Comparative evaluation of microscopy,OptiMAL® and 18S rRNA gene based multiplex PCR for detection of Plasmodium falciparum & Plasmodium vivax from field isolates of Bikaner,India

Objective:To evaluate microscopy,OptiMAL? and multiplex PCR for the identification of Plasmodium falciparum（P.falciparum） and Plasmodium vivax（P.vivax） from the field isolates of Bikaner,Rajasthan（Northwest India）.Methods:In this study,a multiplex PCR（P.falciparum and P.vivax） was further developed with the incorporation of Plasmodium malariae（P.malariae） specific primer and also a positive control.The performance of microscopy,plasmodium lactate dehydrogenase（pLDH） based malaria rapid diagnostic test OptiMAL? and 18S rRNA gene based multiplex PCR for the diagnosis of P.falciparum and P.vivax was compared.Results:The three species multiplex PCR if.falciparum,P.vivax and P.malariae) with an inbuilt positive control was developed and evaluated.In comparison with multiplex PCR,which showed the sensitivity and specificity of 99.36%（95%CI,98.11%-100.00%） and 100.00%（95%CI,100.00%-100.00%）,the sensitivity and specificity of microscopy was 90.44%（95%CI,88.849-95.04%） and 99.22%（95% CI,97.71%-100.00%）,and OptiMAL? was 93.58%（95%CI,89.75%-97.42%） and 97.69%（95%CI, 95.10%-100.00%）.The efficiencies were 99.65%,95.10%and 95.45%for multiplex PCK.microscopy and OptiMAL?.respectively.Conclusions:Our results raise concerns over the overall sensitivities of microscopy and OptiMAL?,when compared to the multiplex PCR and thus stress the need for new molecular interventions in the accurate detection of the malarial parasites.This further highlights the fact that further developments are needed to improve the performance of rapid diagnostic tests at field level.

Eukaryotic food sources analysis in situ of tropical common sea cucumber Holothuria leucospilota based on 18S rRNA gene high-throughput sequencing

Sea cucumber Holothuria leucospilota is one of the most widespread tropical holothurian species.In this study,eukaryotic organism composition in foregut and hindgut contents of H.leucospilota and surrounding sediments was assessed by 18S rRNA gene high-throughput sequencing.Eukaryon richness and diversity in the habitat sediments were significantly higher than those in foregut and hindgut contents of the sea cucumbers(P<0.05).The foregut content group,hindgut content group,and marine sediment group sequences were respectively assigned to 18.20±1.32,19.40±1.03,and 21.80±0.37 phyla.In the foregut contents,Nematoda(20.18%±9.59%),Mollusca(16.12%±10.49%),Chlorophyta(10.04%±4.85%),Annelida(8.72%±10.93%),Streptophyta(8.46%±4.65%),and Diatomea(5.99%±2.01%)were the predominant phyla,which showed the eukaryotic food sources of H.leucospilota were primarily belong to the above phyla.The predominant phyla in the hindgut contents were Streptophyta(45.55%±17.32%),Mollusca(4.93%±4.82%),Arthropoda(5.37%±3.08%),Diatomea(3.88%±2.34%),and Chlorophyta(3.79%±1.59%);and Annelida(37.80%±17.00%),Arthropoda(24.49%±12.53%),Platyhelminthes(7.14%±3.02%),Nematoda(4.14%±0.91%),and Diatomea(5.11%±1.35%)had large contents in the sediments.The comparatively high content of Paris genus in phylum Streptophyta in foregut contents indicated that land plants were one of the primary food sources of H.leucospilota,however the significantly higher contents of Streptophyta in hindgut contents than that in foregut contents might suggest a large part of the terrigenous detritus ingested might not be digested by H.leucospilota.UPGMA and PCoA analysis revealed that eukaryotic organism composition differed significantly between foregut contents of H.leucospilota and ambient sediments,indicating selective feeding feature of H.leucospilota.This study provided useful references for artificial feed of tropical sea cucumbers and enhanced understanding of the ecological roles of detritus-feeding macrobenthos.

Evaluation of different primers of the 18S rRNA gene to profile amoeba communities in environmental samples

Amoeboid protists,an assemblage of organisms belonging to different phylogenetic lineages,have drawn increasing attention due to their crucial ecological roles in various environments and their potential health risks.Currently,18S rRNA gene sequencing is widely applied for the detection of amoebae.However,it is not clear which is the best primer pair for 18S rRNA gene amplification in amoebae.This study compared the four most commonly used primer pairs for revealing the diversity,composition,core species,and community assembly processes of amoebae in water and sediments.We found that the choice of primers artificially influences the detection of community composition of amoebae.We also found that short-read fragments may lead to mismatches in taxonomy and were not suitable for phylogenetic analyses.In contrast,full-length primers could detect the highest number of amoeba lineages and annotate 80%of reads belonging to amoebae to known species.However,full-length primers did not detect as many amoeba species as V4 primers.Moreover,we showed that beta diversity and community assembly determination were largely unaffected by primer choice,but different primers could influence our interpretations of the ecological process underlying stochasticity and determinism.This study indicates that full-length read sequencing and V4 region Illumina sequencing are suitable for profiling amoeba diversity in the environment.

First molecular identification of Cryptosporidium by 18S rRNA in goats and association with farm management in Terengganu

Objective:To identify the prevalence of Cryptosporidium from goats in three types of farm management systems in Terengganu,Malaysia and to determine the Cryptosporidium species infecting goats by using 18 S r RNA.Methods:A total of 478 fecal samples were randomly collected from goats in three farms;199 samples were collected from intensive farm,179 samples from semi-intensive farm and 100 samples from extensive farm.The samples were processed by using formolether concentration technique and stained by using modified Ziehl–Neelsen.Positive samples were performed by using nested PCR analysis by using 18 S r RNA.Results:Out of 478 goats,207(43.3%) were found to be infected with Cryptosporidium.Goats reared under the intensive farm management system reported the highest prevalence of infection(49.7%),followed by intensive farm management system(41%) and the lowest prevalence was reported in the goats reared under semi-intensive management system(37.4%).Conclusions:The identified species found in goat was Cryptosporidium parvum.Future study on the zoonotic transmission of Cryptosporidium parvum in goats needs to be done in order to find the source of transmission of this parasite.

Morphogenesis and Molecular Phylogeny of a Marine Urostylid Ciliate Apokeronopsis wrighti(Protista, Ciliophora, Hypotrichia)

Hypotrichs are one of the highly differentiated ciliated lineages which play important roles in ecological, environmental,evolutionary and basic biological studies. In the present study, we investigated the living characteristics, infraciliature, nuclear apparatus, ontogenesis and phylogenetic position of a marine hypotrichous ciliate, Apokeronopsis wrighti Long et al., 2008, which was isolated from coastal waters in Shenzhen, China. The new isolate resembles the type population in terms of morphological characteristics, morphometrics, and SSU rRNA gene sequence that is with a 99.7% similarity. Ontogenesis of A. wrighti is characterized by oral primordium for the proter as well as marginal and dorsal kineties anlagen in both filial products formed de novo, and the cirral row arranged along the paroral and endoral arises from several anterior frontoventral-transverse cirral streaks. Phylogenetic analyses based on SSU and concatenated gene data suggest that five species of Apokeronopsis form a monophyletic clade, and the genus Apokeronopsis is closely related to Thigmokeronopsis and Metaurostylopsis.

Screening of the Gene for Chlorella Identification and Identification of oil-producing Microalgae

[Objective] The aim was to select suitable gene for Chlorella identification and to identify the oil-producing microalgae.[Method] Four candidate gene sequences,the nuclear genomic rDNA of the 18S rRNA gene,internal transcribed spacer（ITS）,internal transcribed spacer Ⅱ（ITS Ⅱ）and the chloroplast rbcL gene,were selected for Chlorella molecular identification.Through these four candidate genes,the genetic variability and distinguish ability between intra-species and inter-species was analyzed to choose the right genes for identification of the high oil-content Chlorella.On this basis,application of these gene segments were classified and identified for five fresh-water isolated Chlorella,which oil-content is more than 30%.[Result] ITS gene was a suitable gene because of its high variation and short fragment length,meanwhile its genetic distance intra-species（0.439 6±0.135 9）was larger than inter-species（0.045 7±0.084 3）.Its sequence length varied between different species whereas highly conserved in the same species.By the application of ITS sequences,respectively,five high oil-content stains were identified as one C.vulgaris,two strains of C.sorokiniana and two strains of algae Chlorella sp.[Conclusion] This study had provided reference for the establishment of identification gene pool of Chlorella.

Morphological and Phylogenetic Studies of a Copepod Species, Irodes parupenei Ho and Lin (2007), Infecting Parupeneus rubescens in Saudi Arabia

Dammam City is one of the gorgeous coastal areas in the Arabian Gulf of Saudi Arabia.The present study aimed to ex-amine one of the copepod species infecting the rosy goatfish that represents a highly consumed fish species by the local population in the Arabian Gulf.The copepod species isolated from the infected fish specimens belong to the family Taeniacanthidae and was iden-tified as Irodes parupenei Ho and Lin(2007),primarily based on its morphological,morphometric,and ultrastructural characteris-tics,especially the structures of the dorsal cephalic area,segmentation of the first antenna,the absence of the maxilliped claw in the fe-male specimens,and the setation and spinulation of the legs 2-4 for the adult females are of great significance in the taxonomic iden-tification.The 18S rRNA gene sequence was analyzed to ensure the precise identity and exact taxonomic status of the copepod species.The result showed that this copepod species belong to Taenicanthidae and closely related to Irodes sauridi(gb|JF781550.1)in the same taxon.More details on the specificity of the goatfish for Irodes species and identifying these parasitic taxa using molecular analysis are given in the present study.

The first record of Pavlova pinguis (Pavlovophyceae, Haptophyta) in China seas

One strain of unicellular flagellated yellow-green algae was successlully isolated lrom the coastal area near Tianjin in the Bohai Sea in May 2015. The strain ranged from round to elongated in shape. Most of the cells possessed active motility, and some cells formed non-motile aggregation. Based on evidences from morphology, ultrastructure and molecular analysis, we identified the strain as Pavlova pinguis which belonged to Pavlovophyceae, Haptophyta. For Pavlovophyceae, only Pavlova viridis （Diacronema viridis） was reported in China seas prior to this study and it played an important role in aquaculture. This is the first record of Pavlova pinguis in Chinese waters.

<i>Chroococcidiorella tianjinensis</i>gen. et sp. nov. (Trebouxiophyceae, Chlorophyta), a Green Alga Arises from the Cyanobacterium TDX16

All algae documented so far are of unknown origin. Here, we provide a taxonomic description of the first origin-known alga TDX16-DE that arises from the Chroococcidiopsis-like endosymbiotic cyanobacterium TDX16 by de novo organelle biogenesis after acquiring its green algal host Haematococcus pluvialis’s DNA. TDX16-DE is spherical or oval, with a diameter of 2.0-3.6 μm, containing typical chlorophyte pigments of chlorophyll a, chlorophyll b and lutein and reproducing by autosporulation, whose 18S rRNA gene sequence shows the highest similarity of 99.7% to that of Chlorella vulgaris. However, TDX16-DE is only about half the size of C. vulgaris and structurally similar to C. vulgaris only in having a chloroplast-localized pyrenoid, but differs from C. vulgaris in that 1) it possesses a double-membraned cytoplasmic envelope but lacks endoplasmic reticulum and Golgi apparatus;and 2) its nucleus is enclosed by two sets of envelopes (four unit membranes). Therefore, based on these characters and the cyanobacterial origin, we describe TDX16-DE as a new genus and species, Chroococcidiorella tianjinensis gen. et sp. nov., which sets the basis for multidisciplinary research.

Seasonal Variations of Picoeukaryote Community Structure in Zhoushan Fishing Ground,East China Sea

Picoeukaryotes(<2-3μm)are major oceanic primary producers and play a crucial role for the functioning of marine ecosystems.However,the community structure of such communities remains poorly understood,especially in the East China Sea(ECS).We investigated the seasonal variations of abundance and diversity of picoeukaryotes,and recorded environmental variables,along a transect in Zhoushan Fishing Ground.High-throughput sequencing was used for sequencing the V4-V5 variable region within the 18S rRNA gene to analyze genetic diversity and relative abundance of picoeukaryotes.A total of 40 phyla,68 classes,99 orders,126 families,and 140 genera were observed.The estimated richness and diversity indices were both higher in each season.The composition and dominant picoeukaryotes changed significantly at the class level with the seasons alternating.Basidiomycota,Ciliophora,Ascomycota and Cryptomonadales were observed throughout the year.The Linear Discriminant Analysis(LDA)revealed the predominant discriminant taxa of four seasonal groups.Redundancy analysis(RDA)showed that NO2^−and salinity played very important roles to picoeukaryotes for all the spring samples and DS5 sample in winter,PO4^3−and pH affected mostly picoeukaryotes for winter samples and autumn samples.

Dynamics of the Eukaryotic Microbial Community at the Breeding Sites of the Large Yellow Croaker Pseudosciaena crocea in the Southern East China Sea

Clarifying eukaryotic microbial spatial distribution patterns and their determinants is an important idea in ecological research.However,information on the distribution patterns of eukaryotic microbial community structures(EMCSs)within oceans remains unclear.In this study,surface water samples from the southern East China Sea(SECS)were collected to investigate the spatiotemporal variation in EMCSs by using 18S rRNA high-throughput sequencing technology and the impact of this variation on Pseudosciaena crocea during the breeding season.The results indicated that the distribution patterns of the eukaryotic microbial community structure were different among the Sansha Bay,Mindong and Wentai reserves and the offshore East China Sea.In addition,there were notable potential effects of EMCSs on fishery activities.The variation partitioning analysis showed the environmental and spatial factors caused 53.4%of the variation in the EMCSs,indicating that spatially structured environmental factors were the key determinants of the EMCSs spatial heterogeneity in the SECS and may have contributed to the general distribution of P.crocea.In addition,all the environmental factors were the main factors driving the distribution of eukaryotic microbes except for total phosphorus.Furthermore,it was noted some phytoplankton such as Poterioochromonas and Rhizophydium of fungi in Sansha Bay can effectively prevent Cyanobacteria blooms.Chrysophyceae are natural high-quality baits for juvenile fish distributed in Sansha Bay,Mindong and Wentai reserves.This study provides a part of the insight into potential eukaryotic community distributions in large water bodies and how they are affected by environmental factors.

Identifying the ‘unidentified’ fungi: a global-scale long-read third-generation sequencing approach

Molecular identification methods,in particular high-throughput sequencing tools,have greatly improved our knowledge about fungal diversity and biogeography,but many of the recovered taxa from natural environments cannot be identified to species or even higher taxonomic levels.This study addresses the phylogenetic placement of previously unrecognized fungal groups by using two complementary approaches:(i)third-generation amplicon sequencing analysis of DNA from global soil samples,screening out ITS reads of<90%similarity to other available Sanger sequences,and(ii)analysis of common fungal taxa that were previously indicated to be enigmatic in terms of taxonomic placement based on the ITS sequences alone(so-called top50 sequences).For the global soil samples,we chose to amplify the full rRNA gene operon using four partly overlapping amplicons and multiple newly developed primers or primer combinations that cover nearly all fungi and a vast majority of non-fungal eukaryotes.We extracted the rRNA 18S(SSU)and 28S(LSU)genes and performed phylogenetic analyses against carefully selected reference material.Both SSU and LSU analyses placed most soil sequences and top50 sequences to known orders and classes,but tens of monophyletic groups and single sequences remained outside described taxa.Furthermore,the LSU analyses recovered a few small groups of sequences that may potentially represent novel phyla.We conclude that rRNA genes-based phylogenetic analyses are efficient tools for determining phylogenetic relationships of fungal taxa that cannot be placed to any order or class using ITS sequences alone.However,in many instances,longer rRNA gene sequences and availability of both SSU and LSU reads are needed to improve taxonomic resolution.By leveraging third-generation sequencing from global soil samples,we successfully provided phylogenetic placement for many previously unidentified sequences and broadened our view on the fungal tree of life,with 10-20%new order-level taxa.In addition,the PacBio sequence data greatly extends fungal class-level information in reference databases.

Ontogenesis and systematic position of a new hypotrichous ciliate,Chaetospira sinica sp.nov.,with an improved diagnosis of the poorly defined family Chaetospiridae Jankowski,1985(Protozoa,Ciliophora,Hypotrichia)

Ciliates are unique single-celled organisms that play important roles in ecological,environmental,evolutionary,and ontogenetic research.In the present study,phylogenetic analyses based on 18S rRNA gene sequence data reveal that Chaetospira sinica sp.nov.clusters with Stichotricha aculeata with strong to full support(97%ML,1.00 BI),but is not closely related to members of Spirofilidae Gelei,1929 to which Chaetospira and Stichotricha have previously been assigned.Phylogenetic analyses,together with morphological and morphogenetic data from Chaetospira sinica sp.nov.,support the validity of family Chaetospiridae Jankowski,1985.Chaetospira and Stichotricha are here assigned to the family Chaetospiridae,the improved diagnosis of which is as follows:non-dorsomarginalian Hypotrichia with flask-shaped body;oral region extending along narrow anterior neck region;lorica usually present;two ventral and two marginal cirral rows,all distinctly spiraled or obliquely curved;pretransverse and transverse cirri absent.The basic morphogenetic features in C.sinica sp.nov.can be summarized as:(1)the oral primordium for the opisthe develops de novo and the parental adoral zone is completely retained by the proter;(2)all ventral cirral anlagen and marginal anlagen developed intrakinetally;(3)three dorsal kineties anlagen formed intrakinetally in each daughter cell;and(4)macronuclear nodules fuse into a single mass.Exconjugant cells were also isolated and their morphologic and molecular data are provided.

Morphological and molecular examination of the ciliate family Lagynusidae(Protista,Ciliophora,Prostomatea)with descriptions of two new genera and two new species from China

Ciliates in the class Prostomatea play an important role in the global microbial loop due to their signifcant abundances and broad feeding strategies at the foundation of food webs.Despite their importance in ecosystems,the taxonomy and systematics of this group of ciliates has long been poorly understood,with this being especially true for members of the family Lagynusidae.Here we examine four lagynusids collected from sandy beaches in China,using silver-staining and 18S rRNA gene sequencing techniques.These investigations revealed two new genera and two new species and provided details for two little known forms:Penardella marina gen.nov.,sp.nov.,Apolagynus cucumis(as reported by Penard.Études sur les infusoires d’eau douce.Georg and Cie,Genève,1922)gen.nov.,comb.nov.,Lagynus minutus sp.nov.,and Lagynus elegans(Engelmann in Z Wiss Zool 11:347-393,1862)Quennerstedt(Acta Univ Lund 4:1-48,1867).Penardella gen.nov.can be morphologically distinguished by having more than three dikinetidal perioral kineties.Apolagynus gen.nov.difers from the closely related genus Lagynus in the absence of a conspicuous neck-like region.The ciliature of Apolagynus cucumis is revealed here for the frst time,which demonstrates the classifcation of this species within Lagynusidae.Furthermore,Apolagynus binucleatus(Jiang et al.,2021)comb.nov.is established according to the new fnding.The results of our phylogenetic analyses based on the 18S rRNA gene support the establishment of two new genera and indicate that Lagynusidae is monophyletic,which further strengthens its valid taxonomic status.

Cyclospora cayetanensis infections among diarrheal outpatients in Shanghai： a retrospective case study

Cyclospora cayetanensis is a foodborne and waterborne pathogen that causes endemic and epidemic human diarrhea worldwide. A few epidemiological studies regarding C. cayetanensis infections in China have been conducted. During 2013, a total of 291 stool specimens were collected from patients with diarrhea at a hospital in urban Shanghai. C. cayetanensis was not detected in any of the stool specimens by traditional microscopy, whereas five stool specimens （1.72%, 5/291） were positive by PCR. These positive cases confirmed by molecular technology were all in the adult group （mean age 27.8 years; 2.94%, 5/170） with watery diarrhea. Marked infection occurred in the rainy season of May and July. Sequence and phylogenetic analyses of the partial 18S rRNA genes of C. cayetanensis isolated showed intra-species diversity of this parasite. This study showed, for the first time, that C. cayetanensis is a pathogen in outpatients with diarrhea in Shanghai, albeit at a low level. However, the transmission dynamics of this parasite in these patients remain uncertain.

Hypotrichidium tisiae(Gelei,1929)Gelei,1954:a unique hypotrichid ciliate having a highly specialized developmental pattern during binary division

In sharp contrast to their pelagic relatives,the oligotrichs,the overwhelming majority of hypotrich ciliates inhabit the benthos.Only a few species,including those of the genus Hypotrichidium Ilowaisky,1921,have adapted to a planktonic lifestyle.The ontogenetic mode of the highly differentiated ciliate,Hypotrichidium tisiae(Gelei,1929)Gelei,1954,is unknown.In this study,the interphase morphology and the ontogenetic process of this species are investigated.Accordingly,the previously unidentified ciliary pattern of Hypotrichidium is redefined.The main morphogenetic features are as follows:(1)The parental adoral zone of membranelles is inherited completely by the proter and the oral primordium of the opisthe arises in a deep pouch.(2)Five frontoventral cirral anlagen(FVA)are formed:FVA I contributes to the single frontal cirrus,FVA II–IV generate three frontoventral cirral rows,FVA V migrates and forms postoral ventral cirri.(3)All marginal cirral row anlagen develop de novo:each of the two left anlagen forms a single cirral row,while the single right anlage fragments into anterior and posterior parts.(4)Two dorsal kinety anlagen occur de novo,with the right one fragmenting to form kineties 2 and 3.(5)Two long caudal cirral rows are formed at the ends of dorsal kineties 1 and 3.On the basis of the morphogenetic features and phylogenetic analyses,the assignment of Hypotrichidium to the family Spirofilidae Gelei,1929 within Postoralida is supported.The establishment of separate families for the slender“tubicolous”spirofilids and the highly helical spirofilids is also validated.

Amazonian Dark Earth and Its Black Carbon Particles Harbor Different Fungal Abundance and Diversity

Amazonian Dark Earth(ADE) is a highly fertile soil of anthropogenic origin characterized by high levels of charred black carbon(BC). It is considered a model of fertility; however, knowledge on the fungal community structure and diversity inhabiting ADE and its BC particles is scarce. Fungal community structure and diversity of ADE and its BC from four sites under different land uses(three agricultural systems and a secondary pristine forest) in the Brazilian Central Amazon were evaluated by 18S rRNA gene pyrosequencing. Fungal communities in ADE and BC were dissimilar and showed differential abundances of fungal operational taxonomic units(OTUs). Estimated fungal species richness(abundance-based coverage estimate and Chao-1 index) and diversity estimators(Shannon and Simpson's reciprocal indices) were higher in ADE than in BC in all agricultural areas. No differences were observed in those parameters in ADE and BC samples from the secondary forest. Pezizomycotina fungi and OTUs assigned to Cordyceps confragosa,Acremonium vitellinum, Camarops microspora, and Hirsutella rhossiliensis were more abundant in BC particles than in ADE. These findings represent a breakthrough in our understanding of fungal communities in BC particles from ADE, and will be valuable in future studies considering biochar application in soil.