Microbial community diversity of an integrated constructed wetland used for treatment of sewage.

The microbial community diversity in Constructed Wetland System (CWS) plays a key role in the removal of pollutants from waste water. An integrated functional CWS developed at Neela Hauz Biodiversity Park, Delhi was selected to assess the diversity in composition and structure of microbial community diversity of sludge and sediment of CWS, based on metagenomic approach using 16S rRNA genes. The sediment showed higher diversity than sludge and both formed distinct clusters. The taxonomic structure of the microbial community of CWS is represented by 6,731 OTUs distributed among 2 kingdoms, 103 phyla, 227 classes, 337 orders, 320 families, 295 identified genera, and 84 identified species. The relative abundance of top 5 dominant phyla of sludge and sediment varied from 3.77% (Acidobacteria) to 35.33% (Proteobacteria) and 4.07% (Firmicutes) to 28.20% (Proteobacteria), respectively. The range of variation in relative abundance of top 5 dominant genera of sludge and sediment was 2.58% (Hyphomicrobium) to 6.61% (Planctomyces) and 2.47% (Clostridium) to 4.22% (Syntrophobacter), respectively. The rich microbial diversity of CWS makes it perform better in pollutants removal (59.91-95.76%) than other CWs. Based on the abundance values of taxa, the taxa are grouped under four frequency distribution classes-abundant (>20), common (10-19), rare (5-9), and very rare (1-4). The unique structure of microbial communities of integrated CWS is that the number of abundant taxa decreases in descending order of taxonomic hierarchy, while the number of rare and very rare taxa increases. For example, the number of abundant phyla was 14 and 21 in sludge and sediment, respectively and both communities have only 3 abundant genera each. This is in contrast to 4 and 17 very rare phyla in sludge and sediment, respectively and both the communities have 114 and 91 very rare genera, respectively. The outcomes of the study is that the integrated CWS has much higher microbial community diversity than the diversity reported for other CWs, and the rich diversity can be used for optimizing the performance efficiency of CWS in the removal of pollutants from waste water. Such structural diversity might be an adaptation to heterogeneous environment of CWS.

Expression Analysis Reveals Differentially Expressed Genes in BPH and WBPH Associated with Resistance in Rice RILs Derived from a Cross between RP2068 and TN1.

BPH (brown planthopper) and WBPH (white backed planthopper) are significant rice pests that often co-occur as sympatric species and cause substantial yield loss. Despite their genetic similarities, different host-resistance genes confer resistance against these two hoppers. The defense mechanisms in rice against these pests are complex, and the molecular processes regulating their responses remain largely unknown. This study used specific recombinant inbred lines (RILs) derived from a cross between rice varieties RP2068-18-3-5 (BPH- and WBPH-resistant) and TN1 (BPH- and WBPH-susceptible) to investigate the mechanisms of interaction between these planthoppers and their rice hosts. WBPH and BPH were allowed to feed on specific RILs, and RNA-Seq was carried out on WBPH insects. Transcriptome profiling and qRT-PCR results revealed differential expression of genes involved in detoxification, digestion, transportation, cuticle formation, splicing, and RNA processing. A higher expression of sugar transporters was observed in both hoppers feeding on rice with resistance against either hopper. This is the first comparative analysis of gene expressions in these insects fed on genetically similar hosts but with differential resistance to BPH and WBPH. These results complement our earlier findings on the differential gene expression of the same RILs (BPH- or WBPH-infested) utilized in this study. Moreover, identifying insect genes and pathways responsible for countering host defense would augment our understanding of BPH and WBPH interaction with their rice hosts and enable us to develop lasting strategies to control these significant pests.

Midgut serine proteinases participate in dietary adaptations of the castor (Eri) silkworm Samia ricini Anderson transferred from Ricinus communis to an ancestral host, Ailanthus excelsa Roxb.

Dietary change influenced the life-history traits, nutritional utilization, and midgut serine proteinases in the larvae of the domesticated polyphagous S. ricini, transferred from R. communis (common name: castor; family Euphorbiaceae; the host plant implicated in its domestication) to A. excelsa (common name: Indian tree of heaven; family Simaroubaceae; an ancestral host of wild Samia species). Significantly higher values for fecundity and body weight were observed in larvae feeding on R. communis (Scr diet), and they took less time to reach pupation than insects feeding on A. excelsa (Scai diet). Nevertheless, the nutritional index for efficiency of conversion of digested matter (ECD) was similar for larvae feeding on the two plant species, suggesting the physiological adaptation of S. ricini (especially older instars) to an A. excelsa diet. In vitro protease assays and gelatinolytic zymograms using diagnostic substrates and protease inhibitors revealed significantly elevated levels (p ≤ 0.05) of digestive trypsins, which may be associated with the metabolic costs influencing slow growth in larvae feeding on A. excelsa. RT-PCR with semidegenerate serine proteinase gene-specific primers, and cloning and sequencing of 3' cDNA ends identified a large gene family comprising at least two groups of putative chymotrypsins (i.e., Sr I and Sr II) resembling invertebrate brachyurins/collagenases with wide substrate specificities, and five groups of putative trypsins (i.e., Sr III, Sr IV, Sr V, Sr VII, and Sr VIII). Quantitative RT-PCR indicated that transcripts belonging to the Sr I, Sr III, Sr IV, and Sr V groups, especially the Sr IV group (resembling achelase I from Lonomia achelous), were expressed differentially in the midguts of fourth instars reared on the two plant species. Sequence similarity indicated shared lineages with lepidopteran orthologs associated with expression in the gut, protein digestion, and phytophagy. The results obtained are discussed in the context of larval serine proteinases in dietary adaptations, domestication, and exploration of new host plant species for commercial rearing of S. ricini.

Polymorphisms in the hypervariable control region of the mitochondrial DNA differentiate BPH populations.

The brown planthopper (BPH; Nilaparvata lugens) is one of India's most destructive pests of rice. BPH, a monophagous migratory insect, reported from all major rice-growing ecosystems of the country, is capable of traversing large distances and causing massive crop loss. A crucial step for developing viable management strategies is understanding its population dynamics. Very few reliable markers are currently available to screen BPH populations for their diversity. In the current investigation, we developed a combinatorial approach using the polymorphism present within the mitochondrial Control Region of BPH and in the nuclear genome (genomic simple sequence repeats; gSSRs) to unravel the diversity present in BPH populations collected from various rice-growing regions of India. Using two specific primer pairs, the complete Control Region (1112 to 2612 bp) was PCR amplified as two overlapping fragments, cloned and sequenced from BPH individuals representing nine different populations. Results revealed extensive polymorphism within this region due to a variable number of tandem repeats. The three selected gSSR markers also exhibited population-specific amplification patterns. Overall genetic diversity between the nine populations was high (>5%). Further, in silico double-digestion of the consensus sequences of the Control Region, with HpyCH4IV and Tsp45I restriction enzymes, revealed unique restriction fragment length polymorphisms (digital-RFLPs; dRFLPs) that differentiated all the nine BPH populations. To the best of our knowledge, this is the first report of markers developed from the Control Region of the BPH mitogenome that can differentiate populations. Eventually, such reliable and rapid marker-based identification of BPH populations will pave the way for an efficient pest management strategy.

Consequences of 'no-choice, fixed time' reciprocal host plant switches on nutrition and gut serine protease gene expression in Pieris brassicae L. (Lepidoptera: Pieridae).

Rapid adaptive responses were evident from reciprocal host-plant switches on performance, digestive physiology and relative gene expression of gut serine proteases in larvae of crucifer pest P. brassicae transferred from cauliflower (CF, Brassica oleracea var. botrytis, family Brassicaceae) to an alternate host, garden nasturtium, (GN, Tropaeolum majus L., family Tropaeolaceae) and vice-versa under laboratory conditions. Estimation of nutritional indices indicated that larvae of all instars tested consumed the least food and gained less weight on CF-GN diet (significant at p≤0.05) as compared to larvae feeding on CF-CF, GN-GN and GN-CF diets suggesting that the switch to GN was nutritionally less favorable for larval growth. Nevertheless, these larvae, especially fourth instars, were adroit in utilizing and digesting GN as a new host plant type. In vitro protease assays conducted to understand associated physiological responses within twelve hours indicated that levels and properties of gut proteases were significantly influenced by type of natal host-plant consumed, change in diet as well as larval age. Activities of gut trypsins and chymotrypsins in larvae feeding on CF-GN and GN-CF diets were distinct, and represented shifts toward profiles observed in larvae feeding continuously on GN-GN and CF-CF diets respectively. Results with diagnostic protease inhibitors like TLCK, STI and SBBI in these assays and gelatinolytic zymograms indicated complex and contrasting trends in gut serine protease activities in different instars from CF-GN diet versus GN-CF diet, likely due to ingestion of plant protease inhibitors present in the new diet. Cloning and sequencing of serine protease gene fragments expressed in gut tissues of fourth instar P. brassicae revealed diverse transcripts encoding putative trypsins and chymotrypsins belonging to at least ten lineages. Sequences of members of each lineage closely resembled lepidopteran serine protease orthologs including uncharacterized transcripts from Pieris rapae. Differential regulation of serine protease genes (Pbr1-Pbr5) was observed in larval guts of P. brassicae from CF-CF and GN-GN diets while expression of transcripts encoding two putative trypsins (Pbr3 and Pbr5) were significantly different in larvae from CF-GN and GN-CF diets. These results suggested that some gut serine proteases that were differentially expressed in larvae feeding on different species of host plants were also involved in rapid adaptations to dietary switches. A gene encoding nitrile-specifier protein (nsp) likely involved in detoxification of toxic products from interactions of ingested host plant glucosinolates with myrosinases was expressed to similar levels in these larvae. Taken together, these snapshots reflected contrasts in physiological and developmental plasticity of P. brassicae larvae to nutritional challenges from wide dietary switches in the short term and the prominent role of gut serine proteases in rapid dietary adaptations. This study may be useful in designing novel management strategies targeting candidate gut serine proteases of P. brassicae using RNA interference, gene editing or crops with transgenes encoding protease inhibitors from taxonomically-distant host plants.

Midgut serine proteases and alternative host plant utilization in Pieris brassicae L.

Pieris brassicae L. is a serious pest of cultivated crucifers in several parts of the world. Larvae of P. brassicae also feed prolifically on garden nasturtium (Tropaeolum majus L., of the family Tropaeolaceae). Proteolytic digestion was studied in larvae feeding on multiple hosts. Fourth instars were collected from cauliflower fields before transfer onto detached, aerial tissues of selected host plants in the lab. Variable levels of midgut proteases were detected in larvae fed on different hosts using protein substrates (casein and recombinant RBCL cloned from cauliflower) and diagnostic, synthetic substrates. Qualitative changes in midgut trypsin activities and quantitative changes in midgut chymotrypsin activities were implicated in physiological adaptation of larvae transferred to T. majus. Midgut proteolytic activities were inhibited to different extents by serine protease inhibitors, including putative trypsin inhibitors isolated from herbivore-attacked and herbivore-free leaves of cauliflower (CfTI) and T. majus (TpTI). Transfer of larvae to T. majus significantly influenced feeding parameters but not necessarily when transferred to different tissues of the same host. Results obtained are relevant for devising sustainable pest management strategies, including transgenic approaches using genes encoding plant protease inhibitors.

Revisiting rubisco as a protein substrate for insect midgut proteases.

Gene fragments encoding the large subunit (LS) of Rubisco (RBCL) were cloned from various species of host plants of phytophagous Lepidoptera and expressed as recombinant proteins in Escherichia coli. Recombinant RBCLs were compared among each other along with casein and native Rubisco as proteinaceous substrates for measuring total midgut protease activities of fourth instar larvae of Helicoverpa armigera feeding on casein, Pieris brassicae feeding on cauliflower, and Antheraea assamensis feeding on Litsea monopetala and Persea bombycina. Cognate rRBCL (from the pertinent host plant species) substrates performed similar to noncognate rRBCL reflecting the conserved nature of encoding genes and the versatile use of these recombinant proteins. Casein and recombinant RBCL generally outperformed native Rubisco as substrates, except where inclusion of a reducing agent in the enzyme assay likely unfolded the plant proteins. Levels of total midgut protease activities detected in A. assamensis larvae feeding on two primary host species were similar, suggesting that the suite(s) of digestive enzymes in these insects could hydrolyze a plant protein efficiently. Protease activities detected in the presence of protease inhibitors and the reducing agent dithiothreitol (DTT) suggested that recombinant RBCL was a suitable protein substrate for studying insect proteases using in vitro enzyme assays and substrate zymography.

Genetic variation within native populations of endemic silkmoth Antheraea assamensis (Helfer) from Northeast India indicates need for in situ conservation.

A. assamensis is a phytophagous Lepidoptera from Northeast India reared on host trees of Lauraceae family for its characteristic cocoon silk. Source of these cocoons are domesticated farm stocks that crash frequently and/or wild insect populations that provide new cultures. The need to reduce dependence on wild populations for cocoons necessitates assessment of genetic diversity in cultivated and wild populations. Molecular markers based on PCR of Inter-simple sequence repeats (ISSR) and simple sequence repeats (SSR) were used with four populations of wild insects and eleven populations of cultivated insects. Wild populations had high genetic diversity estimates (H(i) = 0.25; H(S) = 0.28; H(E) = 0.42) and at least one population contained private alleles. Both marker systems indicated that genetic variability within populations examined was significantly high. Among cultivated populations, insects of the Upper Assam region (H(i) = 0.19; H(S) = 0.18; H(E) = 0) were genetically distinct (F(ST) = 0.38 with both marker systems) from insects of Lower Assam (H(i) =0.24; H(S) =0.25; H(E) = 0.3). Sequencing of polymorphic amplicons suggested transposition as a mechanism for maintaining genomic diversity. Implications for conservation of native populations in the wild and preserving in-farm diversity are discussed.

The critical role of N- and C-terminal contact in protein stability and folding of a family 10 xylanase under extreme conditions.

BACKGROUND: Stabilization strategies adopted by proteins under extreme conditions are very complex and involve various kinds of interactions. Recent studies have shown that a large proportion of proteins have their N- and C-terminal elements in close contact and suggested they play a role in protein folding and stability. However, the biological significance of this contact remains elusive. METHODOLOGY: In the present study, we investigate the role of N- and C-terminal residue interaction using a family 10 xylanase (BSX) with a TIM-barrel structure that shows stability under high temperature, alkali pH, and protease and SDS treatment. Based on crystal structure, an aromatic cluster was identified that involves Phe4, Trp6 and Tyr343 holding the N- and C-terminus together; this is a unique and important feature of this protein that might be crucial for folding and stability under poly-extreme conditions. CONCLUSION: A series of mutants was created to disrupt this aromatic cluster formation and study the loss of stability and function under given conditions. While the deletions of Phe4 resulted in loss of stability, removal of Trp6 and Tyr343 affected in vivo folding and activity. Alanine substitution with Phe4, Trp6 and Tyr343 drastically decreased stability under all parameters studied. Importantly, substitution of Phe4 with Trp increased stability in SDS treatment. Mass spectrometry results of limited proteolysis further demonstrated that the Arg344 residue is highly susceptible to trypsin digestion in sensitive mutants such as DeltaF4, W6A and Y343A, suggesting again that disruption of the Phe4-Trp6-Tyr343 (F-W-Y) cluster destabilizes the N- and C-terminal interaction. Our results underscore the importance of N- and C-terminal contact through aromatic interactions in protein folding and stability under extreme conditions, and these results may be useful to improve the stability of other proteins under suboptimal conditions.

The critical role of partially exposed N-terminal valine residue in stabilizing GH10 xylanase from Bacillus sp.NG-27 under poly-extreme conditions.

BACKGROUND: Understanding the mechanisms that govern protein stability under poly-extreme conditions continues to be a major challenge. Xylanase (BSX) from Bacillus sp. NG-27, which has a TIM-barrel structure, shows optimum activity at high temperature and alkaline pH, and is resistant to denaturation by SDS and degradation by proteinase K. A comparative circular dichroism analysis was performed on native BSX and a recombinant BSX (R-BSX) with just one additional methionine resulting from the start codon. The results of this analysis revealed the role of the partially exposed N-terminus in the unfolding of BSX in response to an increase in temperature. METHODOLOGY: We investigated the poly-extremophilicity of BSX to deduce the structural features responsible for its stability under one set of conditions, in order to gain information about its stability in other extreme conditions. To systematically address the role of the partially exposed N-terminus in BSX stability, a series of mutants was generated in which the first hydrophobic residue, valine (Val1), was either deleted or substituted with various amino acids. Each mutant was subsequently analyzed for its thermal, SDS and proteinase K stability in comparison to native BSX. CONCLUSIONS: A single conversion of Val1 to glycine (Gly) changed R-BSX from being thermo- and alkali- stable and proteinase K and SDS resistant, to being thermolabile and proteinase K-, alkali- and SDS- sensitive. This result provided insight into the structure-function relationships of BSX under poly-extreme conditions. Molecular, biochemical and structural data revealed that the poly-extremophilicity of BSX is governed by a partially exposed N-terminus through hydrophobic interactions. Such hitherto unidentified N-terminal hydrophobic interactions may play a similar role in other proteins, especially those with TIM-barrel structures. The results of the present study are therefore of major significance for protein folding and protein engineering.

Bioinsecticidal activity of Archidendron ellipticum trypsin inhibitor on growth and serine digestive enzymes during larval development of Spodoptera litura.

The roles of serine proteases involved in the digestion mechanism of the cutworm Spodoptera litura (Lepidoptera: Noctuidae) were examined (in vitro and in vivo) following feeding of plant protease inhibitors. A trypsin inhibitor from Archidendron ellipticum (AeTI) was purified by ammonium sulfate fractionation, ion-exchange chromatography and size-exclusion chromatography (HPLC) and its bioinsecticidal properties against S. litura were compared with Soybean Kunitz trypsin inhibitor (SBTI). AeTI inhibited the trypsin-like activities of the midgut proteases of fifth instar larvae of S. litura by over 70%. Dixon plot analysis revealed competitive inhibition of larval midgut trypsin and chymotrypsin by AeTI, with an inhibition constant (K(i)) of 3.5x10(-9) M and 1.5x10(-9) M, respectively. However, inhibitor kinetics using double reciprocal plots for both trypsin and chymotrypsin inhibitions demonstrated a mixed inhibition pattern. Feeding experiments conducted on different (neonate to ultimate) instars suggested a dose-dependent decrease for both the larval body weight as well as % survival of larva fed on diet containing 50, 100 and 150 microM AeTI. Influence of AeTI on the larval gut physiology indicated a 7-fold decrease of trypsin-like protease activity and a 5-fold increase of chymotrypsin-like protease activity, after being fed with a diet supplemented with 150 microM AeTI. This study suggests that although the early (1st to 3rd) larval instars of S. litura are susceptible to the trypsin inhibitory action of AeTI, the later instars may facilitate the development of new serine proteases, insensitive to the inhibitor.

Incidence, Spatial Patterns, and Associations Among Viruses in Snap Bean and Alfalfa in New York.

Recent epidemics in snap bean (Phaseolus vulgaris) characterized by virus-like symptoms prompted a survey of commercial fields for Alfalfa mosaic virus (AMV), Cucumber mosaic virus (CMV), and the Bean yellow mosaic virus (BYMV)/Clover yellow vein virus (ClYVV) complex in 2002 and 2003. Snap bean fields were either remote from or adjacent to alfalfa (Medicago sativa), a putative source of these viruses. Bean fields were sampled at the bloom stage in both years. Model-adjusted mean incidences of infection by AMV, BYMV/ClYVV, and CMV were 41.96, 6.56, and 6.69%, respectively, in alfalfa, and 6.66, 6.38, and 17.20% in snap bean. In 2002, 25.9% of snap bean plants were infected by more than one virus; <1% had more than one virus in 2003. Virus incidences did not differ between snap bean adjacent to or remote from alfalfa, but incidence of infection by AMV and BYMV/ClYVV was significantly higher in snap bean planted later in the season rather than earlier. In 2002, there was a positive association between AMV and CMV in the tendency to find both viruses in the same snap bean plant. In some years, infection by aphid-transmitted viruses can become widespread in snap bean in New York.