Source and Distribution of Emerging and Legacy Persistent Organic Pollutants in the Basins of the Eastern Indian Ocean.

Persistent organic pollutants (POPs) have received significant and ongoing attention. To establish favorable regulatory policies, it is vital to investigate the occurrence, source, and budgets of POPs worldwide. POPs including phthalic acid esters (PAEs), organophosphate esters (OPEs), brominated flame retardants (BFRs), and highly chlorinated flame retardants (HFRs) have not yet been examined in the Eastern Indian Ocean (EIO). In this study, the distribution of POPs has been investigated from surface sediments with the depth of 4369-5742 m in the Central Indian Ocean Basin (CIOB) and Wharton Basin (WB) of EIO. The average (±SD) concentrations of ∑11PAEs, ∑11OPEs, ∑4 BFRs, and ∑5HFRs were 1202.0 ± 274.36 ng g-1 dw, 15.3 ± 7.23 ng g-1 dw, 327.6 ± 211.74 pg g-1 dw, and 7.9 ± 7.45 pg g-1 dw, respectively. The high abundance of low-molecular-weight (LMW) PAEs, chlorinated OPEs, LMW BDEs, and anti-Dechlorane Plus indicated the pollution characteristics in the EIO. Correlation analysis demonstrated that LMW compounds may be derived from the high-molecular-weight compounds. The monsoon circulation, currents, and Antarctic Bottom Water may be the main drivers. POP accumulation rate, depositional flux, and mass inventory in the Indian Ocean were also estimated.

UTR introns, antisense RNA and differentially spliced transcripts between Plasmodium yoelii subspecies.

BACKGROUND: The rodent malaria parasite Plasmodium yoelii is an important animal model for studying host-parasite interaction and molecular basis of malaria pathogenesis. Although a draft genome of P. yoelii yoelii YM is available, and RNA sequencing (RNA-seq) data for several rodent malaria species (RMP) were reported recently, variations in coding regions and structure of mRNA transcript are likely present between different parasite strains or subspecies. Sequencing of cDNA libraries from additional parasite strains/subspecies will help improve the gene models and genome annotation. METHODS: Here two directional cDNA libraries from mixed blood stages of a subspecies of P. yoelii (P. y. nigeriensis NSM) with or without mefloquine (MQ) treatment were sequenced, and the sequence reads were compared to the genome and cDNA sequences of P. y. yoelii YM in public databases to investigate single nucleotide polymorphisms (SNPs) in coding regions, variations in intron-exon structure and differential splicing between P. yoelii subspecies, and variations in gene expression under MQ pressure. RESULTS: Approximately 56 million of 100 bp paired-end reads were obtained, providing an average of ~225-fold coverage for the coding regions. Comparison of the sequence reads to the YM genome revealed introns in 5' and 3' untranslated regions (UTRs), altered intron/exon boundaries, alternative splicing, overlapping sense-antisense reads, and potentially new transcripts. Interestingly, comparison of the NSM RNA-seq reads obtained here with those of YM discovered differentially spliced introns; e.g., spliced introns in one subspecies but not the other. Alignment of the NSM cDNA sequences to the YM genome sequence also identified ~84,000 SNPs between the two parasites. CONCLUSION: The discoveries of UTR introns and differentially spliced introns between P. yoelii subspecies raise interesting questions on the potential role of these introns in regulating gene expression and evolution of malaria parasites.

Linkage maps from multiple genetic crosses and loci linked to growth-related virulent phenotype in Plasmodium yoelii.

Plasmodium yoelii is an excellent model for studying malaria pathogenesis that is often intractable to investigate using human parasites; however, genetic studies of the parasite have been hindered by lack of genome-wide linkage resources. Here, we performed 14 genetic crosses between three pairs of P. yoelii clones/subspecies, isolated 75 independent recombinant progeny from the crosses, and constructed a high-resolution linkage map for this parasite. Microsatellite genotypes from the progeny formed 14 linkage groups belonging to the 14 parasite chromosomes, allowing assignment of sequence contigs to chromosomes. Growth-related virulent phenotypes from 25 progeny of one of the crosses were significantly associated with a major locus on chromosome 13 and with two secondary loci on chromosomes 7 and 10. The chromosome 10 and 13 loci are both linked to day 5 parasitemia, and their effects on parasite growth rate are independent but additive. The locus on chromosome 7 is associated with day 10 parasitemia. The chromosome 13 locus spans ~220 kb of DNA containing 51 predicted genes, including the P. yoelii erythrocyte binding ligand, in which a C741Y substitution in the R6 domain is implicated in the change of growth rate. Similarly, the chromosome 10 locus spans ~234 kb with 71 candidate genes, containing a member of the 235-kDa rhoptry proteins (Py235) that can bind to the erythrocyte surface membrane. Atypical virulent phenotypes among the progeny were also observed. This study provides critical tools and information for genetic investigations of virulence and biology of P. yoelii.

Sources and transport of organic matter in the Ganges-Brahmaputra-Meghna River system of Bengal Basin, Bangladesh.

This study aimed to understand the sources and transport mechanism of organic matter (OM) in the Ganges-Brahmaputra-Meghna (GBM) river system in Bangladesh. We conducted analyses of total organic carbon (TOC), total nitrogen (TN), their stable isotopes (δ13C and δ15N), and sediment grain size. The results reveal a heterogeneous mixture of OM derived from terrestrial plants, aquatic environments, and anthropogenic sources. The Brahmaputra River exhibited higher concentrations of TOC and TN, with δ13C and δ15N values indicating that the OM is primarily sourced from C3 plants. Conversely, the Ganges River demonstrated lower TOC levels and higher isotopic values, reflecting significant anthropogenic inputs. The Lower Meghna showed a mixture of terrestrial and marine sources. Variations in the TOC/TN ratios across the river system underscore the complex interplay between natural and anthropogenic factors. Additionally, sediment grain size plays a crucial role, with finer sediments in the Brahmaputra River associated with increased OM concentrations, while coarser sediments in the Ganges River correlate with lower TOC and TN levels.

Size-dependent enhancement of gene expression by Plasmodium 5'UTR introns.

BACKGROUND: Eukaryotic genes contain introns that are removed by the spliceosomal machinery during mRNA maturation. Introns impose a huge energetic burden on a cell; therefore, they must play an essential role in maintaining genome stability and/or regulating gene expression. Many genes (> 50%) in Plasmodium parasites contain predicted introns, including introns in 5' and 3' untranslated regions (UTR). However, the roles of UTR introns in the gene expression of malaria parasites remain unknown. METHODS: In this study, an episomal dual-luciferase assay was developed to evaluate gene expression driven by promoters with or without a 5'UTR intron from four Plasmodium yoelii genes. To investigate the effect of the 5'UTR intron on endogenous gene expression, the pytctp gene was tagged with 3xHA at the N-terminal of the coding region, and parasites with or without the 5'UTR intron were generated using the CRISPR/Cas9 system. RESULTS: We showed that promoters with 5'UTR introns had higher activities in driving gene expression than those without 5'UTR introns. The results were confirmed in recombinant parasites expressing an HA-tagged gene (pytctp) driven by promoter with or without 5'UTR intron. The enhancement of gene expression was intron size dependent, but not the DNA sequence, e.g. the longer the intron, the higher levels of expression. Similar results were observed when a promoter from one strain of P. yoelii was introduced into different parasite strains. Finally, the 5'UTR introns were alternatively spliced in different parasite development stages, suggesting an active mechanism employed by the parasites to regulate gene expression in various developmental stages. CONCLUSIONS: Plasmodium 5'UTR introns enhance gene expression in a size-dependent manner; the presence of alternatively spliced mRNAs in different parasite developmental stages suggests that alternative slicing of 5'UTR introns is one of the key mechanisms in regulating parasite gene expression and differentiation.

Deaggregation of mutant Plasmodium yoelii de-ubiquitinase UBP1 alters MDR1 localization to confer multidrug resistance.

Mutations in a Plasmodium de-ubiquitinase UBP1 have been linked to antimalarial drug resistance. However, the UBP1-mediated drug-resistant mechanism remains unknown. Through drug selection, genetic mapping, allelic exchange, and functional characterization, here we show that simultaneous mutations of two amino acids (I1560N and P2874T) in the Plasmodium yoelii UBP1 can mediate high-level resistance to mefloquine, lumefantrine, and piperaquine. Mechanistically, the double mutations are shown to impair UBP1 cytoplasmic aggregation and de-ubiquitinating activity, leading to increased ubiquitination levels and altered protein localization, from the parasite digestive vacuole to the plasma membrane, of the P. yoelii multidrug resistance transporter 1 (MDR1). The MDR1 on the plasma membrane enhances the efflux of substrates/drugs out of the parasite cytoplasm to confer multidrug resistance, which can be reversed by inhibition of MDR1 transport. This study reveals a previously unknown drug-resistant mechanism mediated by UBP1 through altered MDR1 localization and substrate transport direction in a mouse model, providing a new malaria treatment strategy.

Optimized protocols for improving the likelihood of cloning recombinant progeny from Plasmodium yoelii genetic crosses.

Genetic cross is a powerful tool for studying malaria genes contributing to drug resistance, parasite development, and pathogenesis. Cloning and identification of recombinant progeny (RP) is laborious and expensive, especially when a large proportion of progeny derived from self-fertilization are present in the uncloned progeny of a genetic cross. Since the frequency of cross-fertilization affects the number of recombinant progeny in a genetic cross, it is important to optimize the procedure of a genetic cross to maximize the cross-fertilization. Here we investigated the factors that might influence the chances of obtaining RP from a genetic cross and showed that different Plasmodium yoelii strains/subspecies/clones had unique abilities in producing oocysts in a mosquito midgut. When a genetic cross is performed between two parents producing different numbers of functional gametocytes, the ratio of parental parasites must be adjusted to improve the chance of obtaining RP. An optimized parental ratio could be established based on oocyst counts from single infection of each parent before crossing experiments, which may reflect the efficiency of gametocyte production and/or fertilization. The timing of progeny cloning is also important; cloning of genetic cross progeny from mice directly infected with sporozoites (vs. frozen blood after needle passage) at a time when parasitemia is low (usually <1%) could improve the chance of obtaining RP. This study provides an optimized protocol for efficiently cloning RPs from a genetic cross of malaria parasites.

Spatial distribution, source identification and flux estimation of polycyclic aromatic hydrocarbons and organochlorine pesticides in basins of the Eastern Indian Ocean.

Although polycyclic aromatic hydrocarbons (PAHs) and organochlorine pesticides (OCPs) have been recorded worldwide, information on their presence in the Eastern Indian Ocean (EIO), especially south of 10°S, remains limited. We investigated the distribution and depositional fluxes of PAHs and OCPs, and the major sources and ecological risks of PAHs in EIO surface sediments from the Central Indian Ocean (CIOB) and Wharton Basin (WB). The concentration of Σ18 PAHs and ∑10 OCPs had an average value (± SD) of 138.4 ± 52.34 and 0.8 ± 0.20 ng g-1, respectively. PAHs may mainly affected by traffic emission and biomass and wood combustion. Persistent organic pollutant accumulation rate (PAR) and depositional flux (DF) values showed that abundant PAHs might lost during top-down transport. The low trans- chordane (CHL)/cis-CHL ratio and PAR of OCPs may indicated few OCPs were inputted into the EIO recently. The results of binary isotope mixing modeling indicate the predominance of marine organic matter (MOM) in total organic carbon (TOC) of sediments. Fluoranthene (Flour) and pyrene (Py) might have potential biological effects in the EIO. The study provided background values for PAHs and OCPs in the Indian Ocean, and preliminarily revealed the fate of POPs in the open oceans.

Human ApoE2 protects mice against Plasmodium berghei ANKA experimental cerebral malaria.

Cerebral malaria (CM) is a severe neurological complication of Plasmodium falciparum infection with acute brain lesions. Genetic variations in both host and parasite have been associated with susceptibility to CM, but the underlying molecular mechanism remains unclear. Here, we demonstrate that variants of human apolipoprotein E (hApoE) impact the outcome of Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (ECM). Mice carrying the hApoE2 isoform have fewer intracerebral hemorrhages and are more resistant to ECM than mice bearing the hApoE3, hApoE4, or endogenous murine ApoE (mApoE). hApoE2 mice infected with PbA showed increased splenomegaly and IFN-γ levels in serum but reduced cerebral cell apoptosis that correlated with the survival advantage against ECM. In addition, upregulated expression of genes associated with lipid metabolism and downregulated expression of genes linked to immune responses were observed in the brain tissue of hApoE2 mice relative to ECM-susceptible mice after PbA infection. Notably, serum cholesterol and the cholesterol content of brain-infiltrating CD8+ T cells are significantly higher in infected hApoE2 mice, which might contribute to a significant reduction in the sequestration of brain CD8+ T cells. Consistent with the finding that fewer brain lesions occurred in infected hApoE2 mice, fewer behavioral deficits were observed in the hApoE2 mice. Finally, a meta-analysis of publicly available data also showed an increased hApoE2 allele in the malaria-endemic African population, suggesting malaria selection. This study shows that hApoE2 protects mice from ECM through suppression of CD8+ T cell activation and migration to the brain and enhanced cholesterol metabolism.IMPORTANCECerebral malaria (CM) is the deadliest complication of malaria infection with an estimated 15%-25% mortality. Even with timely and effective treatment with antimalarial drugs such as quinine and artemisinin derivatives, survivors of CM may suffer long-term cognitive and neurological impairment. Here, we show that human apolipoprotein E variant 2 (hApoE2) protects mice from experimental CM (ECM) via suppression of CD8+ T cell activation and infiltration to the brain, enhanced cholesterol metabolism, and increased IFN-γ production, leading to reduced endothelial cell apoptosis, BBB disruption, and ECM symptoms. Our results suggest that hApoE can be an important factor for risk assessment and treatment of CM in humans.

Selenium-dependent glutathione peroxidase gene expression during gonad development and its response to LPS and H2O2 challenge in Scylla paramamosain.

A selenium-dependent glutathione peroxidase cDNA was obtained from green mud crab Scylla paramamosain (SpGPx) by homology PCR technique and rapid amplification of cDNA ends (RACE) methods. The 1135 bp full-length cDNA contains a 9 bp 5'-untranslated region (UTR), an open reading frame (ORF) of 564 bp encoded a deduced protein of 187 amino acids (aa), and a 562 bp 3'-UTR with a 100 bp conserved eukaryotic selenocysteine insertion sequence (SECIS). It involves a putative selenocysteine (Sec4°, or U4°) residue which is encoded by an opal codon, ¹²7TGA¹²9, and forms an active site with residues Q74 and W¹4². Sequence characterization revealed that SpGPx contain a characteristic GPx signature motif 2 (64LAFPCNQF7¹), an active site motif (¹5²WNFEKF¹57), a potential N-glycosylation site (76NTT78), and two residues (R9° and R¹68) which contribute to the electrostatic architecture by directing the glutathione donor substrate. Multiple sequence alignment and phylogenetic analysis showed that SpGPx share a high level of identities and closer relationship with other selected invertebrate GPxs and vertebrate GPx1 and GPx2. Molecular modelling analysis results also supported these observations. Real time quantitative PCR analysis revealed that SpGPx was constitutively expressed in 10 selected tissues, and its expression level in gill and testis was higher than that in the other tissues (p < 0.05). The SpGPx expression increased and then declined during ovarian and testicular development implying thatnscrpits yowed that SpGPx might play an important role in gonad development by protecting them from oxidative stress. The expression of SpGPx mRNA was induced by lipopolysaccharide (LPS) and hydrogen peroxide (H2O2) in hepatopancreas and haemocytes. The results suggested that SpGPx was implicated in the immune response induced by LPS and H2O2.

A new malaria antigen produces partial protection against Plasmodium yoelii challenge.

Of all the parasitic diseases, malaria is the number one killer. Despite tremendous efforts in disease control and research, nearly a million people, primarily children, still die from the disease each year, partly due to drug resistance and the lack of an effective vaccine. Many parasite antigens have been identified and evaluated for vaccine development; however, none has been approved for human use. Antigenic variation, complex life cycle, and inadequate understanding of the mechanisms of parasite-host interaction and of host immune response all contribute to the lack of an effective vaccine for malaria control. In a recent search of genome-wide polymorphism in Plasmodium falciparum, several molecules were found to be recognized by sera from patients infected with the P. falciparum parasite. Here, we have expressed a 350-amino acid N terminus from one of the homologous candidate antigen genes from the rodent malaria parasite Plasmodium yoelii (Py01157, a putative dentin phosphorin) in bacteria and evaluated the immune response and protection generated after immunization with the recombinant protein. We showed that the recombinant protein was recognized by sera from both mice and humans infected with malaria parasites. Partial protection was observed after challenge with non-lethal P. yoelii 17XNL but not with the lethal P. yoelii 17XL parasite. Further tests using a full-length protein or the conserved C terminus may provide additional information on whether this protein has the potential for being a malaria vaccine.

Molecular cloning and characterization of glutamine synthetase, a tegumental protein from Schistosoma japonicum.

Glutamine synthetase catalyzes the synthesis of glutamine, providing nitrogen for the production of purines, pyrimidines, amino acids, and other compounds required in many pivotal cellular events. Herein, a full-length cDNA encoding Schistosoma japonicum glutamine synthetase (SjGS) was isolated from 21-day schistosomes. The entire open reading frame of SjGS contains a 1,095-bp coding region corresponding to 364 amino acids with a calculated molecular weight of 40.7 kDa. NCBIP blast shows that the putative amino acid of SjGS contains a classic ß-grasp domain and a catalytic domain of glutamine synthetase. The relative mRNA expression of SjGS was evaluated in 7-, 13-, 21-, 28-, 35-, and 42-day worms of S. japonicum in the final host and higher expression at day 21, and 42 worms were observed. This protein was also detected in worm extracts using Western blot. Immunofluorescence studies indicated that the SjGS protein was mainly distributed on tegument and parenchyma in 28-day adult worms. The recombinant glutamine synthetase with a molecular weight of 45 kDa was expressed in Escherichia coli and purified in its active form. The enzyme activity of the recombinant protein was 3.30 ± 0.67 U.µg-1. The enzyme activity was highly stable over a wide range of pH (6-9) and temperature (25-40 °C) under physiological conditions. The transcription of SjGS was upregulated in praziquantel-treated worms at 2-, 4-, and 24-h posttreatment compared with the untreated control. As a first step towards the clarification of the role of glutamine synthetase in schistosome species, we have cloned and characterized cDNAs encoding SjGS in S. japonicum, and the data presented suggest that SjGS is an important molecule in the development of the schistosome.

Distribution and assessment of heavy metal in sediments of Malacca Strait.

In this study, we explored the heavy metal elements in 42 surface sediments from the Malacca Strait in terms of distribution, controlling factors, environmental quality, and primary sources. An analysis of grain size revealed finer sediments near the coast of Malaysia, which gradually thickened toward offshore. In addition, heavy metal elements were abundantly distributed near the coastal area of Malaysia, with a gradual decrease toward the sea; their content increased within waters close to the Perak estuary. Source analysis of heavy metals showed that Cr, Hg, Zn, Cd, and Cu were mostly derived from natural weathering, and their distribution was significantly influenced by sediment grain size. As and Pb were affected by human activities. The environmental quality assessment results showed that Cu, Cr, Cd, and Zn in our study regions were pollution-free. Pb elements showed low-to-moderate pollution, and Hg showed a certain degree of ecological risk due to its high toxicity coefficient. The content of As elements in surface sediments increased significantly when compared to the background value, with several evaluation methods indicating a high-risk index. According to these findings, the area near the mouth of the Perak River is the most polluted, followed by the surrounding coastal areas.

A checklist of parasitic nematodes from marine fishes of China.

A checklist of the parasitic nematodes of Chinese marine fishes is presented. This fauna comprises 90 species, representing 31 genera, 13 families, nine superfamilies, three orders and two subclasses. Additional details for each species include the hosts, localities and references which represent the source of these data.

Distribution and assessment of heavy metal contents in surface sediments of the western Sunda Shelf.

The heavy metal contents (Cr, Cu, Zn, Cd, Pb, Hg, and As) of 88 surface sediment samples from the western Sunda Shelf were analyzed to determine their spatial distribution patterns and contamination status. The results demonstrated that high enrichment regions of heavy metals were focused in the Kelantan, Pahang, and Ambat river estuaries, and deep water regions of the study area. These high enrichment regions were mainly controlled by riverine inputs and their hydrodynamic conditions. The enrichment factor (EF), geoaccumulation index (Igeo), and potential ecological risk index (PERI) were used to assess heavy metal accumulation. The results indicated that the study area was not significantly contaminated overall at the time of the study; however, Cd, As, and Hg were at levels corresponding to moderate contamination at many stations located in the Pahang River estuary, Kelantan River estuary, and north-eastern region of the study area, primarily because of anthropogenic activities.

Lead isotopes in the Central Yellow Sea Mud: Evidence of atmospheric deposition and its implication for regional energy consumption shift.

Anthropogenic activities have increased lead (Pb) emissions and impacted their spatiotemporal distributions in coastal seas. To quantify the increasing variability of Pb and identify the specific origins and their corresponding magnitudes, Pb and Pb isotopes are investigated in a well-placed sediment core covering the period of 1928-2008 in the Central Yellow Sea Mud (CYSM). The concentration of Pb varied from 27.17 µg/g to 37.30 µg/g upwardly along the core, with pronounced anthropogenic disturbance since the late 1960s. The Pb input history of the CYSM experienced five stages according to industrialization levels and Pb contamination, with relative pristine stages from 1928 to 1969 and human activity-impacted stages from 1969 to 2008. The 206Pb/207Pb ratio demonstrated an overall decreasing profile while the 208Pb/206Pb ratio displayed the reverse trend upwardly along the core, possibly due to the atmospheric delivery of anthropogenic Pb emissions from northern China. Furthermore, 208Pb/206Pb vs. 206Pb/207Pb shows certain linearity between natural sediment sources and anthropogenic emissions of Pb (atmospheric deposition); thus, atmospheric inputs account for 34-43% of the Pb in the sediment since Pb enrichment using the two-endmember mixing model. Moreover, the steep decrease in 206Pb/207Pb and rapid increase in 208Pb/206Pb since the 1970s suggest the introduction of leaded gasoline and the increasing proportionate consumption of gasoline relative to total energy consumption. The continuously decreasing 206Pb/207Pb ratio and increasing 208Pb/206Pb ratio since 2000 are the combined results of coal consumption, nonferrous smelting, and residual Pb contamination from leaded gasoline, which is quite distinctive from cases in North America and Europe. The relatively high 206Pb/207Pb and low 208Pb/206Pb ratios before 1969 represent the natural Pb isotopic signatures. Hence, Pb input is significantly affected by regional energy consumption and restructuring, and the Pb isotopic ratios may be a potential proxy for the shift in energy consumption.

Digenean parasites of Chinese marine fishes: a list of species, hosts and geographical distribution.

In the literature, 630 species of Digenea (Trematoda) have been reported from Chinese marine fishes. These belong to 209 genera and 35 families. The names of these species, along with their hosts, geographical distribution and records, are listed in this paper.

Anthropogenic effect on heavy metal contents in surface sediments of the Bengal Basin river system, Bangladesh.

Heavy metals (Sc, Cr, Co, Ni, Cu, Ga, Ge, Nb, Mo, Cd, In, Hf, Ta, W, Tl, Pb, Bi, Th, and U) in the surface sediments of the Ganges-Brahmaputra-Meghna (GBM) river system of the Bengal Basin (BB) were measured to evaluate the heavy metal contamination and anthropogenic influence. The average concentration levels of most of the heavy metals (except Mo and Tl) were above the average crustal and shale values. Contamination indices, including the contamination factor, pollution load index, enrichment factor, and geo-accumulation index, and multivariate statistical analyses indicated that the GBM is slightly polluted by heavy metals with some considerable pollution from Bi, Th, Ta, Cd, Nb, Pb, In, and U. Among the four individual rivers, the Brahmaputra River and Ganges-Brahmaputra (GB) confluence river sediments contain higher heavy metal concentrations than do the Ganges and Meghna Rivers, which may be caused by the effects of local municipal discharge, industrial or urban wastes, and ferry crossing activities.

Distribution, sources and chemical screening-level assessment of toxic metals in the northern Bay of Bengal, Bangladesh.

The present study aims to examine the distribution, sources and potential risks of toxic metals in the northern Bay of Bengal, Bangladesh. We found Cu, Pb, Zn and Hg exhibited similar spatial distribution pattern. Influenced by the Ganges-Brahmaputra River and the Karnafuli River, there were higher concentrations of these metals associated with the finer sediment and higher TOC in the northeastern portion of the study area. Moreover, coal transportation was assumed to account for the distinctive spatial distribution of As with higher concentration down the Port of Chittagong in the eastern boundary. Chemical-screening level assessment demonstrated the majority of the metals exceeded the threshold effect values, indicating certain possibility of adverse effect. The concentrations of Ni were higher than the possible hazardous values, suggesting high possibility of harmful consequences. The uncontaminated sediments mainly distributed in northwestern and the central portions affected by the delta erosion and marine transported sediments.

Generation and functional characterisation of Plasmodium yoelii csp deletion mutants using a microhomology-based CRISPR/Cas9 method.

CRISPR/Cas9 is a powerful genome editing method that has greatly facilitated functional studies in many eukaryotic organisms including malaria parasites. Due to the lack of genes encoding enzymes necessary for the non-homologous end joining DNA repair pathway, genetic manipulation of malaria parasite genomes is generally accomplished through homologous recombination requiring the presence of DNA templates. Recently, an alternative double-strand break repair pathway, microhomology-mediated end joining, was found in the Plasmodium falciparum parasite. Taking advantage of the MMEJ pathway, we developed a MMEJ-based CRISPR/Cas9 (mCRISPR) strategy to efficiently generate multiple mutant parasites simultaneously in genes with repetitive sequences. As a proof of principle, we successfully produced various size mutants in the central repeat region of the Plasmodium yoelii circumsporozoite surface protein without the use of template DNA. Monitoring mixed parasite populations and individual parasites with different sizes of CSP-CRR showed that the CSP-CRR plays a role in the development of mosquito stages, with severe developmental defects in parasites with large deletions in the repeat region. However, the majority of the csp mutant parasite clones grew similarly to the wild type P. yoelii 17XL parasite in mice. This study develops a useful technique to efficiently generate mutant parasites with deletions or insertions, and shows that the CSP-CRR plays a role in parasite development in mosquito.