DNA metabarcoding of insects and allies: an evaluation of primers and pipelines.

Metabarcoding, the coupling of DNA-based species identification and high-throughput sequencing, offers enormous promise for arthropod biodiversity studies but factors such as cost, speed and ease-of-use of bioinformatic pipelines, crucial for making the leapt from demonstration studies to a real-world application, have not yet been adequately addressed. Here, four published and one newly designed primer sets were tested across a diverse set of 80 arthropod species, representing 11 orders, to establish optimal protocols for Illumina-based metabarcoding of tropical Malaise trap samples. Two primer sets which showed the highest amplification success with individual specimen polymerase chain reaction (PCR, 98%) were used for bulk PCR and Illumina MiSeq sequencing. The sequencing outputs were subjected to both manual and simple metagenomics quality control and filtering pipelines. We obtained acceptable detection rates after bulk PCR and high-throughput sequencing (80-90% of input species) but analyses were complicated by putative heteroplasmic sequences and contamination. The manual pipeline produced similar or better outputs to the simple metagenomics pipeline (1.4 compared with 0.5 expected:unexpected Operational Taxonomic Units). Our study suggests that metabarcoding is slowly becoming as cheap, fast and easy as conventional DNA barcoding, and that Malaise trap metabarcoding may soon fulfill its potential, providing a thermometer for biodiversity.

Phylogenetics and systematics of Angiostrongylus lungworms and related taxa (Nematoda: Metastrongyloidea) inferred from the nuclear small subunit (SSU) ribosomal DNA sequences.

The Angiostrongylus lungworms are of public health and veterinary concern in many countries. At the family level, the Angiostrongylus lungworms have been included in the family Angiostrongylidae or the family Metastrongylidae. The present study was undertaken to determine the usefulness and suitability of the nuclear 18S (small subunit, SSU) rDNA sequences for differentiating various taxa of the genus Angiostrongylus, as well as to determine the systematics and phylogenetic relationship of Angiostrongylus species and other metastrongyloid taxa. This study revealed six 18S (SSU) haplotypes in A. cantonensis, indicating considerable genetic diversity. The uncorrected pairwise 'p' distances among A. cantonensis ranged from 0 to 0.86%. The 18S (SSU) rDNA sequences unequivocally distinguished the five Angiostrongylus species, confirmed the close relationship of A. cantonensis and A. malaysiensis and that of A. costaricensis and A. dujardini, and were consistent with the family status of Angiostrongylidae and Metastrongylidae. In all cases, the congeneric metastrongyloid species clustered together. There was no supporting evidence to include the genus Skrjabingylus as a member of Metastrongylidae. The genera Aelurostrongylus and Didelphostrongylus were not recovered with Angiostrongylus, indicating polyphyly of the Angiostrongylidae. Of the currently recognized families of Metastrongyloidea, only Crenosomatidae appeared to be monophyletic. In view of the unsettled questions regarding the phylogenetic relationships of various taxa of the metastrongyloid worms, further analyses using more markers and more taxa are warranted.

Mitochondrial DNA analyses reveal low genetic diversity in Culex quinquefasciatus from residential areas in Malaysia.

The present study explored the intraspecific genetic diversity, dispersal patterns and phylogeographic relationships of Culex quinquefasciatus Say (Diptera: Culicidae) in Malaysia using reference data available in GenBank in order to reveal this species' phylogenetic relationships. A statistical parsimony network of 70 taxa aligned as 624 characters of the cytochrome c oxidase subunit I (COI) gene and 685 characters of the cytochrome c oxidase subunit II (COII) gene revealed three haplotypes (A1-A3) and four haplotypes (B1-B4), respectively. The concatenated sequences of both COI and COII genes with a total of 1309 characters revealed seven haplotypes (AB1-AB7). Analysis using tcs indicated that haplotype AB1 was the common ancestor and the most widespread haplotype in Malaysia. The genetic distance based on concatenated sequences of both COI and COII genes ranged from 0.00076 to 0.00229. Sequence alignment of Cx. quinquefasciatus from Malaysia and other countries revealed four haplotypes (AA1-AA4) by the COI gene and nine haplotypes (BB1-BB9) by the COII gene. Phylogenetic analyses demonstrated that Malaysian Cx. quinquefasciatus share the same genetic lineage as East African and Asian Cx. quinquefasciatus. This study has inferred the genetic lineages, dispersal patterns and hypothetical ancestral genotypes of Cx. quinquefasciatus.

Current susceptibility status of Malaysian Culex quinquefasciatus (Diptera: Culicidae) against DDT, propoxur, malathion, and permethrin.

A nationwide investigation was carried out to determine the current susceptibility status of Culex quinquefasciatus Say populations against four active ingredients representing four major insecticide classes: DDT, propoxur, malathion, and permethrin. Across 14 study sites, both larval and adult bioassays exhibited dissimilar trends in susceptibility. A correlation between propoxur and malathion resistance and between propoxur and permethrin resistance in larval bioassays was found. The results obtained from this study provide baseline information for vector control programs conducted by local authorities. The susceptibility status of this mosquito should be monitored from time to time to ensure the effectiveness of current vector control operations in Malaysia.

First molecular genotyping of voltage gated sodium channel alleles in Culex quinquefasciatus populations in Malaysia.

A nationwide investigation was performed to detect the presence of 1014 mutation(s) in voltage gated sodium channel (kdr) gene of Culex quinquefasciatus from 14 residential areas across 13 states and a federal territory in Malaysia. Molecular genotyping of kdr mutation was performed via a modified three tubes allele-specific-polymerase chain reaction (AS-PCR) and direct sequencing of kdr gene. Based on the results of AS-PCR, homozygous susceptible (SS) genotype was found in nine out of 14 populations with 38 individuals from a total sample size of 140. Heterozygous (RS) genotype was most predominant (99 individuals) and distributed across all study sites. Homozygous resistance (RR) genotype was detected in Perak (one individual) and Selangor (two individuals). The resistance kdr allele frequencies ranged from 0.1 to 0.55, with the highest being detected in Cx. quinquefasciatus population from Selangor. This study has documented the first field-evolved instance of 1014F mutation in Malaysian mosquitoes and the findings of this study could be utilized in the implementation of strategic measures in vector control programs in Malaysia.

The effects of nickel(II) and chromium(VI) on oxygen demand, nitrogen and metal removal in a sequencing batch reactor.

The objective of this study was to evaluate the effects ofNi(II) and Cr(VI) individually and in combination on the simultaneous removal of chemical oxygen demand (COD), nitrogen and metals under a sequencing batch reactor (SBR) operation. Three identical laboratory-scale SBRs were operated with FILL, REACT, SETTLE, DRAW and IDLE periods in a ratio of 1:12:1:2:8 for a cycle time of 24 h until the steady state was achieved. Nickel(II) at increasing concentrations up to 35 mg/L was added to one of the reactors; Cr(VI) at increasing concentrations up to 25 mg/L was added to a second reactor; while a combination of Ni(II) and Cr(VI) in equal concentrations up to 10 mg/L was added to a third reactor. The results demonstrate that both Ni(II) and Cr(VI) exerted a more pronounced inhibitory effect on the removal of ammonia nitrogen (AN) than on COD removal. Synergistic and antagonistic inhibitory effects on the rates of COD and AN removal, respectively, were observed for the 50% Ni(II) and 50% Cr(VI) (w/w) mixture in the concentration range between 10 and 20 mg/L. The simultaneous presence of 50% Ni(II) and 50% Cr(VI) at a concentration of 20 mg/L resulted in system failure.

Treatment of p-nitrophenol in an adsorbent-supplemented sequencing batch reactor.

The objective of this research was to evaluate the treatment ofp-nitrophenol (PNP) as a sole organic carbon source using a sequencing batch reactor (SBR) with the addition of adsorbent. Two types of adsorbents, namely powdered activated carbon (PAC) and pyrolysed rice husk (PRH) were used in this study. Two identical SBRs, each with a working volume of 10 L, were operated with fill, react, settle, draw and idle periods in the ratio of 2:8:1:0.75:0.25 for a cycle time of 12 h. The results showed that, without the addition of adsorbent, increasing the influent PNP concentration to 200 mg/L resulted in the deterioration of chemical oxygen demand (COD) removal efficiency and PNP removal efficiency in the SBRs. Improvement in the performance of the SBR was observed with the addition of PAC. When the dosage of 1.0 g PAC/cycle was applied, COD removal of 95% and almost complete removal of PNP were achieved at the influent PNP concentration of 300 mg/L. The kinetic study showed that the rates of COD and PNP removal can be described by the first-order kinetics. The enhancement of performance in the PAC-supplemented SBR was postulated to be due to the initial adsorption of PNP by the freshly added and the bioregenerated PAC, thus reducing the inhibition on the microorganisms. The PRH was found to be ineffective because of its relatively low adsorption capacity for PNP, compared with that of PAC.

Bioregeneration of powdered activated carbon in the treatment of alkyl-substituted phenolic compounds in simultaneous adsorption and biodegradation processes.

The role of bioregeneration process in renewing the adsorbent surface for further adsorption of organics during simultaneous adsorption and biodegradation processes has been well recognized. The extent of bioregeneration of powdered activated carbon (PAC) as an adsorbent loaded with phenol, p-methylphenol, p-ethylphenol and p-isopropylphenol, respectively, in the simultaneous adsorption and biodegradation processes were quantitatively determined using oxygen uptake as a measure of substrate consumption. Bioregeneration phenomenon was also evaluated in the simultaneous adsorption and biodegradation processes under sequencing batch reactor (SBR) operation to treat synthetic wastewater containing 1200 mg l(-1) phenol and p-methylphenol, respectively. The SBR systems were operated with FILL, REACT, SETTLE, DRAW and IDLE periods in the ratio of 4:6:1:0.75:0.25 for a cycle time of 12 h. The results show that the percentage of desorption from loaded PAC decreased in the order phenol>p-methylphenol>p-ethylphenol>p-isopropylphenol. For the treatment of phenol and p-methylphenol in the SBR reactors, respectively, the simultaneous adsorption and biodegradation processes were able to produce a consistent effluent quality of COD < or = 100 mg l(-1) when the applied PAC dosage was 0.115 and 0.143 g PAC per cycle, respectively. When no further PAC was added, the treatment performance deteriorated to that of the case without PAC addition after 68 and 48 cycles of SBR operation, respectively, for phenol and p-methylphenol. This observation is consistent with the greater extent of bioregeneration for phenol-loaded PAC as compared to p-methylphenol-loaded PAC.

Oxygen demand, nitrogen and copper removal by free-water-surface and subsurface-flow constructed wetlands under tropical conditions.

This study was conducted to: (1) assess the role of wetland vegetation in the removal of oxygen demand and nitrogen under tropical conditions, (2) estimate the uptake of nitrogen and copper by wetland plants and (3) investigate the speciation of Cu in wetland media among four operationally defined host fractions, namely exchangeable, carbonate, reducible and organically bound. Four laboratory-scale wetland units, two free-water-surface (FWS) and two subsurface-flow (SF) with one of each planted with cattails (Typha augustifolia), were fed with primary-treated sewage and operated at nominal retention times of 0.6-7 days. The influent and effluent BOD/COD and nitrogen concentrations were monitored to assess the performance of the wetland units for various mass loading rates. At the end of the study, all cattail plants were harvested and analyzed for total Kjeldahl nitrogen (TKN). Four other wetland units, which were identical to the first four, were fed with domestic wastewater spiked with copper in increasing concentrations. Copper speciation patterns in the sand layer were determined at the end of the study. The results showed that wetland vegetation did not play an important role in oxygen demand removal but were capable of removing about 22% and 26% of the nitrogen input in the FWS and SF wetland units, respectively. Mass balance analysis indicated that less than 1% of copper introduced was taken up by the cattails. Copper speciation patterns in the sand media showed that the exchangeable fraction contributed 30-57% and 63-80% of the nonresidual copper in the planted and unplanted FWS wetlands, respectively. For SF units, the percentages were 52-62% and 59-67%, respectively. This indicates that large amount of copper in the media were potentially remobilizable.

Speciation patterns of heavy metals in tropical estuarine anoxic and oxidized sediments by different sequential extraction schemes.

The speciation patterns of Cu, Cd, Zn, Pb, Fe and Mn in sediment samples under anoxic and oxidized conditions were investigated using three-stage, four-stage and five-stage sequential extraction schemes. All the extraction schemes identify the non-residual metal among three basic operationally-defined host fractions, namely, exchangeable, reducible and organic/sulfide bound. The anoxic sediment samples were found to have been oxidized during the extraction stage for the reducible fraction under the three-stage and four-stage schemes and the moderately reducible fraction under the five-stage scheme despite the maintenance of an oxygen-free environment. This artifact has resulted in an over-representation of the reducible fraction and an under-representation of the organic/sulfide fraction in the heavy metal speciation patterns of anoxic sediment samples. For Cd, Zn and Pb which had > 70% associated with the acid volatile sulfide in the organic/sulfide fraction, this artifact has resulted in the observation of a decrease in the reducible fraction and, in some cases, an increase in the organic/sulfide fraction upon oxidation of the anoxic sediment samples.

The kinetics of bioaccumulation of zinc, copper, lead and cadmium by oysters (Crassostrea iredalei and C. belcheri) under tropical field conditions.

A study on the kinetics of accumulation and depuration of Zn, Cu, Pb and Cd by the oysters (Crassostrea iredalei and Crassostrea belcheri) cultured at two locations in the Merbok Estuary, Malaysia was conducted. A first-order kinetic model was employed to fit the experimental data in order to estimate the rate constants for uptake and elimination processes and to predict the bioconcentration factors (BCF). Among the four metals studied, only the Zn accumulation process could not be modelled using first-order kinetics. The elimination rate constants estimated from depuration data for C. iredalei are found to be much greater than those from accumulation data. The results suggest that the values of kinetic parameters and BCFs derived under conditions of both aqueous and dietary exposure are probably more site- than species-specific.

The effect of heavy metals on nitrogen and oxygen demand removal in constructed wetlands.

The objective of this study is to investigate the respective effects of Zn, Pb and Cd as well as the combined effect of Zn, Pb, Cd and Cu on the removal of nitrogen and oxygen demand in constructed wetlands. Four laboratory-scale gravel-filled subsurface-flow constructed wetland units planted with cattails (Typha latifolia) were operated outdoors and fed with primary-treated domestic wastewater at a constant flow rate of 25 ml/min. After 6 months, three of the wetland units were fed with the same type of wastewater spiked with Zn(II), Pb(II) and Cd(II), respectively, at 20, 5 and 1 mg/l for a further 9 months. The remaining unit was fed with the same type of wastewater spiked with a combination of Zn(II), Pb(II), Cd(II) and Cu(II) at concentrations of 10, 2.5, 0.5 and 5 mg/l, respectively, over the same period. The chemical oxygen demand (COD) and ammoniacal nitrogen (AN) concentrations were monitored at the inlet, outlet and three additional locations along the length of the wetland units to assess the performance of the wetland units at various metal loadings. At the end of the study, all cattail plants were harvested for the determination of total Kjeldahl nitrogen and metal concentrations. The results showed that the COD removal efficiency was practically independent of increasing metal loading or a combination of metal loadings during the duration of the study. In contrast, the AN removal efficiency deteriorated progressively with increasing metal loading. The relative effect of the heavy metals was found to increase in the order: Zn

Effects of adsorbents and copper(II) on activated sludge microorganisms and sequencing batch reactor treatment process.

Wastewater treatment systems employing simultaneous adsorption and biodegradation processes have proven to be effective in treating toxic pollutants present in industrial wastewater. The objective of this study is to evaluate the effect of Cu(II) and the efficacy of the powdered activated carbon (PAC) and activated rice husk (ARH) in reducing the toxic effect of Cu(II) on the activated sludge microorganisms. The ARH was prepared by treatment with concentrated nitric acid for 15 h at 60-65 degrees C. The sequencing batch reactor (SBR) systems were operated with FILL, REACT, SETTLE, DRAW and IDLE modes in the ratio of 0.5:3.5:1:0.75:0.25 for a cycle time of 6 h. The Cu(II) and COD removal efficiency were 90 and 85%, respectively, in the SBR system containing 10 mg/l Cu(II) with the addition of 143 mg/l PAC or 1.0 g PAC per cycle. In the case of 715 mg/l ARH or 5.0 g ARH per cycle addition, the Cu(II) and COD removal efficiency were 85 and 92%, respectively. ARH can be used as an alternate adsorbent to PAC in the simultaneous adsorption and biodegradation wastewater treatment process for the removal of Cu(II). The specific oxygen uptake rate (SOUR) and kinetic studies show that the addition of PAC and ARH reduce the toxic effect of Cu(II) on the activated sludge microorganisms.

Treatment of phenolic wastewater using agricultural wastes as an adsorbent in a sequencing batch reactor.

The objective of this study is to investigate the potential of the activated rice husk to be used as an alternative adsorbent to powdered activated carbon (PAC) in the simultaneous adsorption and biodegradation processes under sequencing batch reactor (SBR) operation to treat synthetic wastewater containing phenol, p-methylphenol, p-ethylphenol and p-isopropylphenol. The rice husk (PRH) was activated by pyrolysis at 600 degrees C for 5 hours in a nitrogen atmosphere. Using the Langmuir model, the limiting adsorption capacities of PRH for the phenols were found to vary from 0.015-0.05 of those of PAC. The SBR reactors with and without adsorbent addition were operated with fill, react, settle, draw and idle periods in the ratio of 4:6:1:0.76:0.25 for a cycle time of 12 hours. For phenolic wastewater containing, 1,200 mg/L phenol, 1,200 mg/L p-methylphenol, 800 mg/L p-ethylphenol and 660 mg/L p-isopropylphenol, it was found that the biodegradation process alone was unable to produce effluent of quality which would satisfy the discharge standards of COD < or = 100 mg/L and phenol concentration < or = 1 mg/L. The addition of PAC in the ratio of PAC/phenolic compound at 0.095 g/g for phenol, 0.119 g/g for p-methylpheol, 0.179 g/g for p-ethylphenol and 0.220 g/g for p-isopropylphenol, can improve the effluent quality to satisfy the discharge standards. Equivalent treatment performance was achieved with the use of PRH at dosages of 2-3 times higher than those of PAC for all the phenolic wastewater studied. The increased adsorption capacity of PRH shown in the treatment indicates bioregeneration of the adsorbed surface during the treatment process.

Removal and speciation of heavy metals along the treatment path of wastewater in subsurface-flow constructed wetlands.

This study was conducted to: (1) evaluate the performance of constructed wetlands in removing Zn, Pb and Cd, respectively, and Zn, Pb, Cd and Cu in combination and (2) investigate the speciation patterns of the dissolved metals differentiated according to their detectability by anodic stripping voltammetry (ASV) and their lability towards Chelex resin along the treatment path of metal-containing wastewater in horizontal subsurface-flow constructed wetlands. Four laboratory scale wetland units planted with cattails (Typha latifolia) were operated outdoors for six months. Three of the units were, respectively, fed with primary-treated domestic wastewater spiked with Zn(II), Pb(II) and Cd(II) whilst the fourth was spiked with a combination of Zn(II), Pb(II), Cd(II) and Cu(II). The results demonstrate that a metal removal efficiency of over 99% was achievable for wetland units treating the metals singly or in combination provided the sorption capacity of the media was not exceeded. When treating the metals in combination, an antagonistic effect, more significantly for Pb and Cd, on the sorptive metal uptake by media was observed. Based on the metal speciation patterns, the wetland system seemed to be capable of maintaining the ASV-labile metal species at relatively low level (< 10%) before media exhaustion.

Heavy metals and adsorbents effects on activated sludge microorganisms.

The sorption of Cu(II) and Cd(II) from synthetic solution by powdered activated carbon (PAC), biomass, rice husk (RH) and activated rice husk (ARH) were investigate under batch conditions. After activated by concentrated nitric acid for 15 hours at 60-65 degrees C, the adsorption capacity for RH was increased. The adsorbents arranged in the increasing order of adsorption capacities to the Langmuir Q degree parameter were biomass > PAC > ARH > RH. The addition of adsorbents in base mix solution had increased the specific oxygen uptake rate (SOUR) activated sludge microorganisms with and without the presence of metals. The increased of SOUR were due to the ability of PAC and RH in reducing the inhibitory effect of metals on microorganisms and provide a reaction site between activated sludge microorganisms and substrates.

Effects of co-substrate and biomass acclimation concentration on the bioregeneration of azo dye-loaded mono-amine modified silica.

Bioregeneration of mono-amine modified silica gel (MAMS) adsorbent loaded with Acid Orange 7 (AO7), Acid Yellow 9 (AY9) and Acid Red 14 (AR14), respectively, was investigated under two different operational conditions, namely absence/presence of sucrose/bacto-peptone as the co-substrate and different biomass acclimation concentrations. The results revealed that the AY9- and AR14-loaded MAMS adsorbents could almost be completely bioregenerated but only in the presence of co-substrate whereas the bioregeneration of AO7-loaded MAMS could achieve up to 71% in the absence of the co-substrate. These differences could be related to the structural properties of the investigated azo dyes. In addition, the results showed that the bioregeneration duration of AO7-loaded MAMS could be progressively shortened by using biomass acclimated to increasingly higher AO7 concentration. However, the bioregeneration efficiencies were found to be relatively unchanged under different biomass acclimation concentrations.

Bioregeneration of activated carbon and activated rice husk loaded with phenolic compounds: Kinetic modeling.

A kinetic model consisting of first-order desorption and biodegradation processes was developed to describe the bioregeneration of phenol- and p-nitrophenol-loaded powdered activated carbon (PAC) and pyrolyzed rice husk (PRH), respectively. Different dosages of PAC and PRH were loaded with phenol or p-nitrophenol by contacting with the respective phenolic compound at various concentrations. The kinetic model was used to fit the phenol or p-nitrophenol concentration data in the bulk solution during the bioregeneration process to determine the rate constants of desorption, k(d), and biodegradation, k. The results showed that the kinetic model fitted relatively well (R(2)>0.9) to the experimental data for the phenol- and p-nitrophenol-loaded PAC as well as p-nitrophenol-loaded PRH. Comparison of the values of k(d) and k shows that k is much greater than k(d). This indicates clearly that the desorption process is the rate-determining step in bioregeneration and k(d) can be used to characterize the rate of bioregeneration. The trend of the variation of the k(d) values with the dosages of PAC or PRH used suggests that higher rate of bioregeneration can be achieved under non-excess adsorbent dosage condition.